Latest revision as of 18:28, 21 October 2019

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Team Aboa Notebook

On this page, our workflow is described in a more detailed manner

Work with Cell lines

Week 1

April 29th

Present: Fanni, Pyry

To get started with our iGEM project, the ordered cell lines described below, were cultured. E. coli cells containing pEVOL-pAzF (Chloramphenicol selection) and cell line C321.ΔA.exp (Zeocin selection) were plated to LA-plates containing 0.5 % Glucose, and 35 µg/mL chloramphenicol or 50 µg/mL zeocin. Controls with tetracycline, ampicillin and a plate without antibiotics were also prepared for the detection of contamination. Plates were incubated overnight at 30 °C. In addition, extra zeocin and chloramphenicol plates were made for later use.

Figure 1. C321.ΔA.exp cells growing on LA plate containing zeosin for selection.

Figure 2. pEVOL-pAzF cells growing on LA plate containing chloramphenicol for selection.

As predicted, there were no colonies in tetracycline plates after overnight culturing. Plates were stored in our laboratory’s fridge.

Week 2

Testing the amber suppression of E.coli 321A and pEVOL- pAzF with BBa_K567011 (GFP-tag-RFP)

May 9th

Present: Pyry, Suski, Jutta, Robin, Janniina, Riku

Following LA-plates were made:
MM1:
LA-plates containing: chloramphenicol, 1 mM pAzF, 0.2 % arabinose, 0.5 mM IPTG
LA-plates containing: tetracycline, 1 mM pAzF 0.2 % arabinose, 0.5 mM IPTG
LA-plates containing: zeocin, 1 mM pAzF 0.2 % arabinose, 0.5 mM IPTG
LA-plates containing: chloramphenicol, 1 mM pAzF, 0.2 % arabinose
LA-plates containing: chloramphenicol, 1 mM pAzF, 0.5 mM IPTG

MM2:
LA-plates containing: chloramphenicol, 0.2 % arabinose, 0.5 mM IPTG
LA-plates containing: tetracycline, 0.2 % arabinose, 0.5 mM IPTG
LA-plates containing: zeocin, 0.2 % arabinose, 0.5 mM IPTG

25 µL cells and 1 µL BBa_K567011 (GFP-tag-RFP) was electroporated in 1 mm electroporation cuvette with 1.8 kV voltage in BioRad Gene Pulser Xcell. After electroporation cells were put into 5 mL SOC medium in 37 °C for 40 min.
After incubation cells were plated as follows: 50 µL SOC, 50 µL 1/10 and 50 µL 1/100 dilutions were plated as follows:

Antibiotic	Chloramphenicol plate	Chloramphenicol	Chloramphenicol	Zeocin for 321.A, Tetracycline for XL-1
Plasmid	BBa (GFP-RFP)	pAzF-tRNA plasmid	Both	None (Control)
321.A strain	Red/Green < 1	None	Red/Green =1	None
XL1 Blue strain	Green	None	Green	None

XL1-Blue	321.deltaA.exp
Cmp, IPTG	Cmp, IPTG
Cmp, Ara	Cmp, Ara

Plates were left in 37 °C o/n

May 10th

No fluorescence in any of the plates.

Week 3

Preparing 321.A + pAzF for glycerol preps and doing glycerol preps

May 16th

Present: Janniina, Oskari, Fanny, Riku

A colony from a 321.A + pAzF plate was replated onto an MM2C plate and incubated o/n at 37 °C.

May 17th

A single colony was picked and used to inoculate 4 mL LB medium containing 33 µg/mL chloramphenicol o/n at 30 °C and 150 rpm.

May 18th

20 % glycerol preps were made by adding 500 µL of growth to 167 µL of an 80 % glycerol stock. The glycerol preps were stored at -80 °C.

Week 4

May 24th

Present: Janniina

5 mL of LB containing 5 µL of 25 mg/mL chloramphenicol was inoculated with a colony picker from a plate containing 321.A (BBa_K567018) and put in Infors Multitron (200 rpm, 26 °C) at 10:10 O'clock.
Raised the temperature from 26 °C to 37 °C at 13 O'clock.
Replated 3 colonies in MM2 Chloramphenicol plates at 14:49 O'clock

May 25th

Present: Riku

Glyserol preps 321.A (BBa_K567018)
Four glycerol preps were made from yesterdays cultures. 500 µL LB of E.coli 321.A with BBa_K567018 plasmid culture was mixed with 170 µL 80 % glyserol. Preps were put in -70 °C freezer.

Week 5

May 27th

Present: Janniina, Riku, Pyry

Our 321.A and XL-1 Blue E.coli strains do not express T7 polymerase so they were unable to express the original BBa_K567018 BioBrick.

Digestion of BBa_K567018 and pUC19-plasmid with EcoRI
Digestion was made as follows:

	BBa_K567018 (µL)	pUC19 (µL)
H2O	12	16
10x buffer	2	2
DNA	5	1
EcoRI (fermentas, expired 8/2019)	1	1
V. Tot.	20	20

incubation at 37 °C started: 15:40, stopped: 16:09
Samples stored in 4 °C.

May 28th

Present: Janniina, Riku

Yesterday’s EcoRI restrictions were mixed with 0.5 µL MidoriGreen Direct (Nippon Genetics) and 1 µL Midori Green Direct was added in 5 µL 1kb DNA Ladder (nippon Genetics).
The ladder, samples and the sample from entry "Making of pLK04R-Histag" is loaded into the 1 % gel as follows:

1 kb ladder

pLK04R (SfiI + HindIII)

pUC19 (EcoRI)

pSB1C3(BBa_K567018) (EcoRI)

Gel run: 1h with 70 V
Results:

The faint longer bands are probably undigested plasmids in lanes 2, 3 and 5. pLK04 is cut from around 4 kb from the lanes 2 and 3. The linearized pUC19 (lane 4, marked with red) is cut from the gel around 3 kb. The GFP- TAG-RFP fragment (lane 5, marked with red) is cut from the gel close to 2 kb even though it is supposed to be 1569 bp long and should be closer to 1.5 kb strands.
The DNA was purified with Nucleospin Gel and PCR Clean- up kit, according to the manufacturer's protocol.
Purified DNA concentrations were measured with ThermoScientific NanoDrop ND-1000
Results:
pUC19: 1.2 ng/µL
GFP-tag-RFP: 14.8 ng/µL
pLK04R: 2.7 ng/µL

May 29th

Present: Riku, Janniina, Pyry

Due to DNA purification low yields digestion was done again similarly as yesterday.
Digestion incubation at 37 °C started at 10:09. Stopped at 10:41 and put on ice to wait. The reaction tubes were put in 65 °C at 11:28 to inactivate the EcoRI enzyme and taken from inactivation at 11:50.
After digestion each reaction mix was mixed with 0.5 µL Midori Green direct and 1 µL Midori green Direct was added to 5 µL 1kb DNA ladder. Samples were loaded in 1% agarose gel as follows:

5 µL 1 kb ladder + 1 µL midori direct

20 µL pUC19 (EcoRI) + 0.5 µL Midori direct

20 µL BBa_K567018 (EcoRI) + 0.5 µL midori direct

20 µL pLK04 (SfiI + HindIII)+ 0.5 µL midori direct

Starting gel electrophoresis at 12:00 using 70 V.
After gel run, gel was removed from the tray and imaged under blue light with Xiaomi Redmi Note 5 Pro:

pUC19 backbone (red box on the left) and BBa_K567018 (red box on the right) were cut from the gel and purified with PCR clean-up Gel extraction kit with 2 15 µL eluation in different tubes.

	1st eluation	2nd eluation
pLK04R Sfil+Hindll	7.5 ng/µL	7.7 ng/µL
pUC19 EcoRI	5.4 ng/µL	2.7 ng/µL
GFP-tag-RFP (BBa_K567018)	5.0 ng/µL	3.4 ng/µL

GFP-tag-RFP-gene was ligated on pUC19 -backbone in a following ligation reaction:

milli-Q	5.8
pUC19	4
GFP-tag-RFP	8
T4 ligase buffer	2
T4 Ligase	0.2
Total volume (µL)	20

Enzyme and buffer was made by ThermoFischer.
Ligation started 15:40. Ended: 15:50.
1 µL ligation mix was mixed with 35 µL electrocompetent E.coli 321.A cells. Electroporation was done with 1mm electroporation cuvette using BioRad Gene Pulsen Xcell electroporation device with 1.8 kV voltage.
After electroporation cells were put into 5 mL prewarmed (37 °C) LB-medium and put into 37 °C, 250 rpm shaking for 40 minutes. Then cells were plated on a LA+chloramphenicol plate and the plate was put to 37 °C o/n.

Multiplying pUC19 and pLK04RCH-MLCR plasmids

Electrocompetent 321.A cells were electroporated with pUC19 or pLK04R plasmid. The cell mixture was transferred to LB medium and incubated on Multritron shaker at 37 °C, 300 rpm for 45 minutes.

May 30th

Present: Janniina

Lot’s of colonies in o/n plate, but no fluorescence. This might be due to wrong gene. In the agarose gel, the gene was too big for what it should have been.

Multiplying pUC19 and pLK04RCH-MLCR plasmids

One colony from each plate was used to inoculate 5 mL of LB with 100 µg/mL ampicillin and incubated on Multitron shaker at 37 °C, 300 rpm o/n.

May 31st

Multiplying pUC19 and pLK04RCH-MLCR plasmids

Present: Janniina, Riku, Fanni, Pyry

The pLK04RCH+MLCR and pUC19 plasmids were purified from the cells using NucleoSpin Plasmid easyPure kit (Macherey-Nagel) according to the manufacturer’s instructions . DNA concentrations were measured with Nanodrop.

DNA concentrations:
pUC19: 380.5 ng/µL
pLK04 original: 115.7 ng/µL

Glycerol preps were also made from both pLK04RCH+MLCR and pUC19 cultures. 167 µL of 80 % glycerol stock was added to 500 µL of cell suspension. The glycerol preps were stored at -80 °C.

Week 6

June 4th

Present: Janniina, Pyry

The plasmid pLK04RCHTag was extracted using NucleoSpin Plasmid easyPure kit. The DNA-concentration measured with Nanodrop was 280.7 ng/µL.

321.A electrocompetent cells were electroporated with pLK04RCHTag(MLCR). 1 µL of DNA (stock concentration 104 ng/µL) was added to 30 µL of cell suspension and the mixture was electroporated. As a negative control we added MQ H2O instead of DNA to the cell mixture. The cell mixture was transferred to LB medium and incubated at 37 °C at 300 rpm for 1 hour. The cell mixture was then plated onto selective plates and incubated o/n at 37 °C.

June 5th

Present: Pyry

5 colonies were inoculated in LB medium and incubated o/n at 37 °C at 300 rpm.

June 6th

Present: Janniina, Riku, Pyry, Linnea

The shaker had stopped during the night so only 3 culture tubes had visible growth in them. Plasmids were extracted from the overnight cultures with visible growth using NucleoSpin Plasmid easyPure kit. The DNA concentration was measured with Nanodrop.

DNA concentration of the extracted pLK04RCHTag-MLCR:
pLK04RCHTag-MLCR 1: 103.9 ng/µL
pLK04RCHTag-MLCR 2: 98.4 ng/µL
pLK04RCHTag-MLCR 3: 88 ng/µL

We digested the pLK04RCHTag-MLCR plasmid with SfiI restriction enzyme:

SfiI digestion mixture:

	µL
MQ	12
Purified DNA	5
G Buffer	2
SfiI	1
Total volume	20

The reaction mixture was incubated at 50 °C for 15 minutes. The samples were run on an agarose gel at 70 V for 1 hour.

Week 7

June 11th

Present: Riku, Fanni, Pyry

We got a new pUC19 plasmid because we were unsure what was in our old tube. We transformed the vector into XL-1electrocompetent cells, incubated on a shaker at 300 rpm, 37 °C for 1 hour and then plated the cell mixture onto selective plates and incubated o/n at 37 °C.

June 12th

A single colony was picked and inoculated with 3 mL of LB with ampicillin and incubated o/n at 37 °C, 300 rpm.

June 13th

Present: Janniina

A modified GFP-tag-RFP was ordered from IDT. Original part was BBa_K567018, but we ordered it without the T7 promoter.

BamHI and AvaI digestion of pUC19 and the ordered GFP-tag-RFP:

pUC19 and GFP-tag-RFP were digested with ThermoFischer BamHI and AvaI digestion ezymes. Reaction mixtures were as follows:

	pUC19	GFP-TAG-RFP
MQ	15	7
DNA	2	10
10x Tango buffer	2	2
BamHI	0.5	0.5
AvaI	0.5	0.5
V. tot. (µL)	20	20

The reactions are put on 37 °C at 11:18 and stopped at 12:16.

0.5 µL Midori Green Direct was put on digestion reaction mixes and 1 µL Midori Green Direct was added to 5 µL 1kb DNA ladder. Samples were loaded on 1 % agarose gel. as follows:

5 µL 1 kb ladder + 1 µL midori direct

20 µL pUC19 (BamHI % AvaI) + 0.5 µL Midori direct

20 µL GFP-TAG-RFP (BamHI % AvaI) + 0.5 µL Midori direct

Gel run:
Voltage:100 V
Start at 12:27
Stop at 13:15.

Fragments marked with red boxes were cut from gel and purified with PCR clean-up Gel extraction kit, according to manufacturer's guide.
Concentrations were measured with Thermo Fischer NanoDrop ND-1000:

	ng/µL
pUC19	9.1
GFP-TAG-RFP	2.6

Ligation of pUC19 backbone and GFP-tag-RFP insert

Purified fragments were ligated with ThermoFischer T4 ligase in following ligation reaction:

	µL
MQ	1.8
pUC19	3
GFP-TAG-RFP	13
10x ligase buffer	2
Ligase	0.2
V. tot.	20

The ligation reaction was incubated for 10 min.

Transformation

3 µL of ligation mix were mixed with 100 µL chemically competent E.coli XL-1 Blue cells. Transformation was incubated 2 min in ice and then put to 45 seconds in 42 °C heat block. After heatshock cells were put on ice for 2 mins and then they were transferreed to 5 mL LB medium.
Cells were incubated in 37 °C 300 rpm shaker for 40 min and then transferred to two LA+ampicillin plates. 200 µL LB growth in first one and 1 mL to second one. Plates were incubated 37 °C overnight.

Multiplying pUC19 and pLK04RCH-MLCR plasmids

The pUC19 plasmid was extracted from the XL-1 electrocompetent cells using the Nucleospin plasmid Easypure kit. The measured DNA concentration was 271.9 ng/µL.

Week 8

June 17th

There were colonies in LA+ampicillin plates, but no GFP fluorescence. We forgot to do ligation and transformation control for work done on Thursday 13th of June, so we had no idea why GFP-tag-RFP gene did not work.
We started pUC19 and GFP-tag-RFP ligation all over again, but this time with ligation control. Ligation was done with ThermoFischer T4 ligase enzyme as follows:

	pUC19 + GFP-tag-RFP	pUC19 transformation control
MQ	1.8	14.8
pUC19	3	3
GFP-TAG-RFP	13	0
10x ligase buffer	2	2
Ligase	0.2	0.2
V. tot. (µL)	20	20

Incubation started at 11:35 and ended at 11:45

Electroporation mix was done by adding 1 µL ligation mix in 35 µL E.coli XL-1 blue electrocompetent in 1 mm electroporation cuvette (BioRad). Electroporation was done with BioRad Gene Pulser Xcell with 1.8 kV voltage

Transformations were put to prewarmed (37 °C) LB medium and 300 rpm shaking in 37 °C.
Start: 12:06
End: 12:55

Both transformations were centrifuged 2000 g, 5 min. Supernatant was thrown away and cells were resuspended in 300 µL LB medium. Medium was divided both 1 mM and 0.5 mM IPTG plates (LA plates, with ampicillin) and incubated in 37 °C o/n

June 18th

Colonies from all the plates were counted, but still no fluorescence.

	pUC19 + GFP-tag- RFP	pUC19 control
1 mM	32	7
0.5 mM	68	6

Starting cultures for plasmid extraction

2 colonies were picked from 0.5 mM pUC19+GRP-tag-RFP plate and put in LB with ampicillin. Tubes were put in 37 °C 300 Rpm for o/n.

June 19th

Plasmids were extracted from o/n cultures with Nucleospin Plasmid Easypure kit, according to manufacturer's protocol.

Yields were measured with NanoDrop ND-1000 and are as follows:

	Yield (ng/µL)
1/2	77.8
2/2	62.5

June 20th

Present: Riku

GFP-tag-RFP Transformation in E.coli 321.A with pEVOL-pAzF

Transformation was done with electroporation in electrocompetent E.coli 321.A. Each transformation mix contained either 1 or 2 plasmids as follows:

35 µL E.coli 321A cells with:

pEVOL- pAzF (µL)	pUC19 - GFP-tag-RFP (µL)
0.5	0.5
0.5	0
0	0.5

Transformation was done in 1 mm electroporation cuvette (BioRad) with 1,85 kV voltage (BioRad Gene Pulser Xcell)

After electroporation cells were transferred in 5 mL LB medium and incubated in 37 °C 250 rpm shaking. Incubation started: 11:25 and ended: 12:35

Transformed cells was plated as follows:

	LA + Ampicillin, Cloramphenicol, Zeocin	LA + ampicillin
pEVOL, pAzF+pUC19, GFPtagRFP	200 µL and 1/100 200 µL
pEVOL, pAzF	200 µL
pUC19, GFPtagRFP	200 µL	50 µL

At this point, we noticed that the pUC19 that was given us by University of Turku was actually pUC18. That might explain quite a lot of struggles we have had with it. We acquired a new pUC19 from University of Turku.

Transforming confirmed pUC19 in E.coli XL-1 Blue strain

Before we could start work with pUC19 it had to be cloned as we got only 2 µL of commercial pUC19.

Transforming mixes as follows:

	XL-1 pUC19	XL-1 mQ control
MQ	0 µL	1 µL
pUC19 (100 pg/µL)	1 µL	0 µL
Cells	35 µL	35 µL

Transformation was done in 1 mm electroporation cuvette using 1.8 kV with BioRad Gene Pulser Xcell.
XL-1 Blue pUC19: T: 4.8
XL-1 Blue mQ control T: 5.0

After electroporation, cells were transferred to 5 mL LB medium and incubation in 37 °C, 250 rpm shaking was started: 16:20 and ended: 17:10.

3 plates were plated:

1: 200 µL E.coli XL-1 Blue pUC19 transformant
2: 100 µL E.coli XL-1 Blue pUC19 transformant
3: 100 µL E.coli XL-1 Blue Control/background

Plates were put to 37 °C o/n

June 21st

Colonies from the o/n plates were counted as follows:

1: 25
2: 14
3: 0

2 colonies from plate 1 and 1 colony from plate 2 was used to start cultures in 5 mL LB medium with 100 µg/mL ampicillin.

Cultures were put in 30C 300 rpm for o/n incubation at 11:20.

June 22nd

O/n cultures were taken out at 10:30 and and glyserol preps were done as follows:

500 µL growth
167 µL 80 % glycerol

Preps were mixed and put to -70 freezer.
The rest of the cells were centrifuged in 2000g for 10 min.

Plasmids were extracted with Nucleospin EasyPure kit, according to manufacturer’s protocol. Yield was measured with Nanodrop ND-1000

Results:

Culture (Plate/colony)	DNA concentration (ng/µL)
1/1	190.6
1/2	138.7
2/1	156.8

Week 9

June 24th

AvaI + BamHI digestion of pUC19 and GFP-tag-RFP

Digestion reactions as follows ( ThermoFisher ): pUC19 reaction was done for 1/1, 1/2 and 2/1 pUC19 vectors:

	GFP-tag-RFP (in pUC18) (µL)	pUC19 (µL)
MQ	13	7
DNA	4	10
10xTango buffer	2	2
BamHI	0.5	0.5
AvaI	0.5	0.5
V. tot.	20	20

Incubation in 37 °C started: 11:31 end: 11:30.

0.5µL Midori Green Direct was added to all the digestion reactions and 1 µL Midori Green Direct was added to 1 kb DNA Ladder (Nippon Genetics).

All 7 samples were loaded on 1% agarose gel as follows:

Ladder

pLk04RCH SfiI digested

pUC19 1/1 BamHI + AvaI digested

pUC19 2/1 BamHI + AvaI

pUC19 BAmHI + AvaI

GFP-TAG-RFP BamHI + AvaI

pLK04 Fab0

Gel run: Voltage: 70 V. Start: 12:56. End:?.

No picture was saved from this gel run due to smartphone malfunction. Correct bands were cut from gel and DNA was purified with PCR clean-up Gel extraction kit, according to manufacturer’s protocol. All pUC19 containing gel pieces were pooled together.

DNA concentrations were measured with ThermoFischer NanoDrop ND-1000.

June 25th

Following ligation reactions were made with ThermoFischer T4 ligase:

MQ	14.3µL	17.3µL
GFP-tag-RFP insert (17.1 ng/µL)	3	0
pUC19 backbone (59.2 ng/µL)	0.5	0.5
10x T4 buffer	2	2
T4 ligase	0.2	0.2
V. tot. (µL)	20	20

Ligation started: 13:56.
Ended: 14:07.

Control ligation started: 14:22.
Ended: 14:34.

Transformation of pUC19-GFP-tag-RFP in E.coli XL-1 Blue

Chemically competent XL-1 Blue tube was put on ice to melt. When it was melted 50 µL cells were put in 2 centrifugetubes that rested on ice. 5 µL previously ligated (pUC19 + GFP-tag-RFP and pUC19 + nothing) reactions was mixed in with cells.

Reaction mixes were incubated on ice for 20-30 min.
Start: 14:45.
End: 14:10.

Cells were put on 42 °C heatblock for 45 s and then put back into ice.

After approx. 2 mins cells were put on 5 mL prewarmed to 37 °C LB medium and put into shaking for 250 rpm in 37 °C.

Incubation started: 15:15.
End: 15:45.

Cells were centrifuged with 2000 g for 7 min. Supernatant was thrown away and cells were resuspensed in residual medium.

Cells were plated on 4 different LA+ampicillin (100 µg/mL) dishes as follows:

Amount (µL)	Ligation transform.
200	pUC19 + GFP-tag-RFP
100 (1/20)	pUC19 + GFP-tag-RFP
100 (1/400)	pUC19 + GFP-tag-RFP
100	pUC19 + no insert

Plates were left o/n in 37 °C

June 26th

Colonies in o/n plates were counted. Results as follows:

Amount plated (µL)	Plate	Colonies
200	pUC19 + GFP-tag-RFP	121
100 (1/20)	pUC19 + GFP-tag-RFP	2
100 (1/400)	pUC19 + GFP-tag-RFP	0
100	pUC19 + no insert	6

No fluorescence was observed. This might be due to XL-1 Blue poor expression combined with pUC19 being poor expression vector.

Incubation in 37 °C overnight.

June 27th

The 1/20 plate has 2 clearly GFP expressing colonies:

In plate 2 there are 2 colonies with fluorescence. 1 is picked in 5 mL liquid culture (LB + ampicillin 100µg/ml) for o/n incubation in 37C 300 rpm.

The reason why only the small plate had GFP expression is due to larger plate containing also glucose, which inhibits the lacZ operator.

June 28th

o/n culture was centrifuged in 2000 g for 10 min and supernatant was thrown away. New liquid culture for glycerol stock was started by inoculating from pellet into 5 mL LB+ampicillin.

Rest of the pellet was used to extract plasmid with NucleoSpin Plasmid -kit. Elution was done 2x with 25 µL Elution buffer and 5 min 70 °C incubation.

Concentration was measured with ND-1000:
pUC19 + GFP-TAG-RFP: 579,5 ng/µL

Week 10

July 2nd

Present: Janniina, Ossi

Transformation of E.coli 321.A with pUC19+GFP-tag-RFP and pEVOl+pAzF

321.A was transformed with 0.5 µL of pEVOL-pAzF and pUC19+GFP-tag-RFP plasmids.

	Actual transformation	Control 1	Control 2
321.A cells	35	35	35
pAzF-pEVOL	0.5	0.5	0
pUC19-GFP-tag-RFP	0.5	0	0.5
MQ	0	0.5	0.5

Electroporation was done using 1 mm electroporation cuvette (BioRad) in BioRad Gene Pulser Xcell.

After electroporation cells were put into LB medium and shaking 300 rm in 37 °C for 40 min.

The transformations were plated on LA plates containing zeocin, ampicillin and chloramphenicol.

July 3rd

No green, red nor yellow fluorescent proteins detected on the plates.

2 growths from o/n LA plates were each used to inoculate 4 mL LB with ampicillin, chloramphenicol, zeocin and glucose. They were then put to grow o/n in 37°C 300rpm.

July 4th

No growth observed.

321.A with pEVOL-pAzF and pUC19(GFP-TAG-RFP) was re-plated in order to get separate colonies. Plates with 1/100 (large plate) and 1/1000 (small plate) dilutions were incubated o/n at 37 °C.

July 5th

Present: Ossi, Riku

From 321.A with pEVOL-pAzF and pUC19(GFP-TAG-RFP) LA plate, 3 colonies were picked to inoculate 4 mL LB with ampicillin, chloramphenicol, zeocin and 0.5 % glucose to grow o/n in 37 °C and 300 rpm.

XL-1 Blue was transformed with pEVOL-pAzF and pUC19-GFP-tag-RFP as follows:

	E.coli XL-1 Blue cells (µL)	pEVOL-pAzF (µL)	pUC19-GFP-tag-RFP (µL)
Actual	35	0.5	0.5
Control 1	35	0.5
Control 2	35		0.5

Transformation was done via electroporation in 1 mm electroporation cuvette (BioRad) by 1.8 kV with BioRad Gene Pulser Xcell.
All transformation T's were 5.0.
After transformation cells were put into 5 mL LB in 37 °C, 300 rpm shaking for 1 hour.

After incubation cells were plated in LA+cloramphenicol+ampicillin+ Zeocin dishes as follows:

Transformation	No dilution (µL)	1/100 (µL)
pUC19 + pEVOL	200	100
pEVOL	100
pUC19	100

100 µL XL-1 blue + pUC19-GFP-tag-RFP transformation was put into LA+ampicillin+tetracyclin+IPTG dish.

All dished were put into 37 °C o/n.

July 6th

No growth was observed in A+cloramphenicol+ampicillin+ Zeocin dishes, due to zeocin that killed XL-1.

Nice green glow was observed in XL-1 Blue + pUC19-GFP-tag-RFP plate:

July 7th

2 colonies 321.A pEVOL+pAzF - pUC19+GFP-tag-RFP and 2 colonies XL-1 GFP-tag-RFP was inoculated into LB culture with ampicillin, chloramphenicol and zeocin (321.A strain) or tetracycline (XL-1 Blue strain) in 30 °C 300 rpm for o/n.

Week 11

July 8th

Present: Riku, Janniina

OD600 was measured from 1/10 dilutions of o/n cultures and then multiplying measured OD600 by 10. Results were:

Strain and plasmids	OD600
XL-1 + pUC19-GFP-tag-RFP	2.5
321.A + pUC19-GFP-tag-RFP + pEVOL-pAzF	2.21

To make 0.1 OD600 cultures from o/n cultures, 160 µL of 321.A culture and 180 µL of XL-1 Blue culture were transferred to 4 mL LB containing ampicillin, 0.5 % glucose and tetracycline (for XL-1 Blue) or chloramphenicol (for 321.A). Glucose was added to inhibit Lac-Promoter.

The growths were put in 37 °C 300 rpom at 10:25.

Cultures OD600 was measured at 14:30 o’clock like previously and results were:

Strain and plasmids	OD600
XL-1 + pUC19-GFP-tag-RFP	2
321.A + pUC19-GFP-tag-RFP + pEVOL-pAzF	1.3

1.9 mL of XL-1 culture and 1.5 mL of 321.A culture was put in 2 separate tubes to start new test and control cultures so that in total there were 4 tubes. Cultures were centrifuged 2000g for 15 min and pellets were resuspended into 4 mL of growth medium that contained: LB, ampicillin and tetracycline (for XL-1 Blue) or chloramphenicol (for 321.A). In addition to antibiotics on tube containing XL-1 Blue and One tube containing 321.A had 0.1 mM pAzF and 1 % arabinose in medium. After about 10 min of 26 °C, 300 rpm incubation 0.1 M IPTG was added to test cultures to total IPTG concentration of 0.1 mM.

July 9th

The OD was measured from the o/n growths by making 1/10 dilutions and measuring dilutions OD600 and multiplying results by 10.

	OD in 1:1
321.A test	3.24
321.A control	3.64
XL-1 test	4.06
XL-1 control	4.03

The cells were centrifuged (16100 g, 2 min) and supernatant was transferred to other microcentrifuge tube. Pellets and supernatants were stored at -20 °C.

Producing Digoxigenin Fabs

Cloning Digoxigenin Fabs into pLK04RCHTag

Digoxigenin-Fab-TAG cloning of ordered variants

May 20th

Present: Janniina, Riku, Ville, Linnea, Pyry and Oskari

Gel electrophoresis

We made 500 mL of 1 x TBS (Medicago AB, Uppsala) and 1 L of 1 x TBE (Medicago).

We made 1 % and 2 % agarose gels into 60 mL of 1 x TBE. 3,6 µL of Midori Green Advance DNA Stain was added into each 60 mL of agarose gel.

Restrictions and ligations

The DNA was centrifuged to get the pellet into the bottom. 20 µL of MQ was added into each tube containing the ordered DNA.

Ordered DNA:

1.45 kb fragments:
SfiI restriction (1 % agarose gel)

Digox CL C-terminal TAG
Digox VL Lys 108->TAG
Digox Fab 0
Digox CL Lys 191->TAG

0.1 kb fragments:
SfiI and HindIII restriction (2 % agarose gel)

pEB04 Histag TAG2
pEB04 Histag Cys-TAG
pEB04 Histag TAG

	SfiI (µL)	SfiI + HindIII (µL)
Water	17	15
10 x FastDigest Buffer	3	3
DNA	10	10
FastDigest Enzyme (SfiI)	1	1
FastDigest Enzyme (HindIII)	0	2
Total volume	31	31

Done according the instructions of manufacturer:
Single digestion for 1.45kb fragments:
15 min at 50 °C for SfiI
Stored at RT until gel electrophoresis

Double digestion for 0.1kb fragments:
15 min at 50 °C for SfiI
1h at 37 °C for HindIII
20 min at 80 °C to inactivate HindIII

Thermo Scientific FastDigest SfiI #FD1824
Thermo Scientific HindIII #ER0505

Electrophoresis:
1.45 kb fragments with 5 µL Loading dye (Thermo Fisher)
Ladder: FastGene 50bp DNA Ladder (NIPPON Genetics Europe GmbH, lot:10 1028 2807 01)
70 V
1x TBE buffer

1 % gel	1.45kb	DNA Ladder	Digox CL Lys 191->TAG (gel sample run order number 1)	Digox VL Lys 108->TAG (2)	Digox Fab 0 (3)	Digox CL C-terminal TAG (4)
2 % gel	0.1kb	DNA Ladder	pEB04 Histag TAG2	pEB04 Histag Cys-TAG	pEB04 Histag TAG

Results

Agarose gel 1 %. Midori green stain. Samples: Ladder, 1, 2, 3, 4

Agarose gel 2 %. Sample order (50 bp ladder, 1, 2, 3)

May 21st

Present: Janniina, Riku, Ville, Linnea

Insert purification from gel
DNA bands from gel 1 at 1.5kb and 2 at 0.1kb were purified with Macherey-Nagel NucleoSpin Gel and PCR Clean-up -kit with manufacturer’s guide. Elution was done twice for samples 1-4 from 1% agarose gel, first time using 30 µL and second time using 15 µL elution buffer. For samples from 2 % agarose gel elution was done only once with 30 µL elution buffer.

Preparing 1% agarose gel for plasmid
0.6 g agarose
0.6 mL TBE
6 µL MidoriGreen

Plasmid restriction

Restriction reactions as follows:

	Sfil	Sfil + HindIII
Water	22,5 µL	19.5 µL
10 x Tango buffer	3 µL	3 µL
Plasmid DNA (350 ng/µL)	3 µL	3 µL
Sfil	1.5 µL	1.5 µL
Hindll	0 µL	3 µL
Total volume	30 µL	30 µL

Sfil and Sfil + Hindlll incubated at 50 °C, for 15 min (start: 09:49, end: 10:04) Sfil+Hindll incubated 1h 10 min (start: 10:04, end: 11:14)

Plasmid purification
Agarose gel sample preparation:
5 µL of loading dye was added to restriction reaction mixes

Agarose gel was loaded as follows:

5 µL 1 kb Ladder (nippon genetics)

20 µL Sfil pEBO4CH (1.)

15 µL Sfil pEBO4CH (2.)

20 µL Sfil + HindIII pEBO4CH (3.)

15 µL Sfil + HindIII pEBO4CH (4.)

Electrophoresis:
Voltage: 70 V
Start: 11:24
Stop: 12:42

Imaging with invitrogen Safe Imager(tm)

Purifying with Macherey-Nagel NucleoSpin Gel and PCR Clean-up -kit, with manufacturer’s guide with 2 eluation steps using 30 µL elution buffer both times.

Tubes 1 and 2 were pooled as well as tubes 3 and 4

May 22nd

Riku, Janniina, Ville, Linnea, Pyry

DNA sample concentration measurement ThermoFischer NanoDrop 1000

Sample	ng/µL
pEB04 Sfil	8.0
pEB04 Sfil + Hindlll	8.4
Digox 191TAG	9.9
Digox Lys108 -> TAG	6.9
Digox Fab0	7.3
Digox C-term TAG	7.0
pEB04 Histag TAG2	7.3
pEB04 Histag TAG	9.5

Ligation

	1.5 kb insert	0.1 kb insert (µL)
Vector DNA	2.5 µL	2.5 µL
Insert DNA	3 µL	1 µL
10 x ligase buffer	2 µL	2 µL
ligase	0.5 µL	0.5 µL
water	12 µL	14 µL
total	20 µL	20 µL

nebiocalculator and
Thermo T4 DNA ligase protocol are used as ligation protocols

Name	Insert	pEB04CH (SfiI)	pEB04CH (SfiI + HindIII)	reaction mix
Lig1.23.05.-19/ABOA	Digox 191 TAG	x		using 1.5 kb column in previous table
Lig2.23.5.-19/ABOA	Digox Lys 108	x		using 1.5 kb column in previous table
Lig3.23.5.-19/ABOA	Digox Fab 0	x		using 1.5 kb column in previous table
Lig4.23.5.-19/ABOA	Digox C-term TAG	x		using 1.5 kb column in previous table
Lig5.23.5.-19/ABOA	pEB04 Histag TAG2		x	using 0.1 kb column in previous table
Lig6.23.5.-19/ABOA	pEB04 Histag TAG		x	using 0.1 kb column in previous table
Lig7.23.5.-19/ABOA	hrFab SfiI DIGD12 harm	x		using 1.5 kb column in previous table

Ligation reaction mixtures are left to incubate for approximately an hour. We got a plasmid pLK04R which has AmpR gene in it. Our plan is to use that for production of our Fab fragments:
We will make following constructs for inserts pLK04RCH, a plasmid with a histidine tag; pLK04RCH-TAG, a plasmid with a histidine tag and a TAG codon for insertion of ncAA's and pLK04RCH-TAG2 with a Histidine tag and a TAG in a slightly different position.

Restriction of pLK04R
Reaction mix as follows:

	Sfil + Hindlll
Water	12.5 µL
10x Tango Buffer	3 µL
Plasmid DNA (104 ng/µL)	10 µL
Sfil	1.5 µL
Hindll	3 µL
Total volume	30 µL

Restriction incubation:
50 °C: started 11:50 ended 12:50
37 °C: started 12:54 ended

Gel purification according to Macherey-Nagel gel purification kit protocol.

Ligation

We will insert ~100 bp ordered "pEB04-Histag" fragments (SfiI/HindIII restriction) to the pLK04CH backbone. At the same ligation reaction we can add SfiI restricted Fab gene into the correct backbones to get Histidine tagged TAG-Fabs and Histidine-TAg tagged unmodified Fabs.

Ligation according to Thermo T4 DNA ligase protocol.

One-Pot Triple fragment cloning of pEB04CH SfiI/SfiI and SfiI/HindIII fragments to replace existing pLK04 Histag

May 25th

Present: Ville, Linnea, Pyry

Introduction

The idea is to clone our Fab fragments into Amp (ampicillin) resistant pLK04 vector. However, the pLK04 vector has a histidine tail that contains a double stop codon of TAG-TAA, so we need to replace this Histidine tail from a pEB04CH where the Histidine tail ends only on one TAA stop. We can do this cloning in a single reaction without gel purifying our insert because the acceptor backbone has an Amp resistance and the donor backbone Cmp (chloramphenicol). The resulting construct can only be assembled correctly in one way to the pLK04 vector. All transformed pEB04 constructs will die in Amp.

Procedure

12,5 µL	MQ
2 µL	Buffer Tango
3 µL	DNA (pLK04)
2.5 µL	Enzyme (1 µL SfiI + 1.5 HindIII)
20 µL	Total vol

0 µL	MQ
6 µL	Buffer Tango
30 µL	DNA (pEB04) (8ng/µL)
3 µL	Enzyme (SfiI)
39 µL	Total vol

Gel Run at 100V 50mA:
Sample order:
1kb ladder (Nippon); pEB04CH HindIII SfiI; pLk04 HindIII SfiI; pEB04CH Hind SfiIII; pLk04 HindIII SfiI

Purifying with NucleoSpin Gel and PCR Clean-up -kit, according to the manufacturer’s guide with 1 eluation step using 30 µL elution buffer.

Concentration of the samples were:
pEB04CH HindIII SfiI (done on saturday) 0.5 ng/µL
pLk04 HindIII SfiI (done on saturday) 0.8 ng/µL
pEB04CH HindIII SfiI (done on friday) 0.9 ng/µL
pLk04 HindIII SfiI (done on friday) 1.6 ng/µL

Ligation

Digestion mix was added 4.5 µL to 5 µL of vector. Control without digestion mix also done. Thermo T4 DNA ligase instructions were followed.

Afterwards, the ligation reaction was electroporated.

Ligation reactions (1-4 and Bg) that were left over were stored to freezer after incubation for 1h

May 27th

Present: Pyry

Plates 1 and 3 has significantly more growth than 2, 4 or bg.

5 mL LB culturing (LB medium with 100 µg/ml ampicillin) were started from growth plates. 3 colonies were picked from plate 3 and 1 colony from plate 1. Cultures were incubated o/n 37 °C 200 rpm incubator.

May 28th

Present: Janniina, Riku

o/n cultures were fuged in 3000 rpm for 5 minutes. Supernatant was tossed away.

New 5 mL cultures were started by inoculating pellet into a new 5 mL LB+ampicillin (100 µg/ml) medium. Cultures were set to incubate in 37 °C 200 rpm shaking.

Rest of the pellet was used to extract plasmid from the cells with NucleoSpin Plasmid EasyPure -kit with manufacturer’s instruction.

DNA concentrations are measured with Nanodrop ND-1000 (elution buffer as blank) after the purification.

	c (µg/mL)
1/1	98.5
3/1	172.9
3/2	74
3/3	202.1

May 29th

Present: Pyry

20% Glycerol preps were made from o/n cultures.
500 µL culture medium
80% 170 µL Glycerol
Tubes were vortexed gently and put to -70 °C freezer

Cloning Digoxigenin fabs into pLK04RCHTag

June 3rd

Digestion and purification of plasmid backbone of pLK04RCHTag+MCLR

Present: Janniina, Linnea

Extraction of the plasmid backbone

pLK04RCHTag plasmid containing gene for microcystin LR (MCLR) (BTEK/Tuomas) was purified from C321.ΔA.exp-cells with NucleoSpin Plasmid EasyPure kit and its DNA concentration was measured with nanodrop.

	ng/µL
1st tube	61.7
2nd tube	183.7

Purified plasmids were digested with SfiI according to manufacturer's protocol. Digested plasmid backbones were separated from MCLR-genes on 1 % agarose gel using Mupid-One 70 V.

The desired fragment (pLK04RCHTag) should be 3753 bp long, but in the 2nd tube there was a contamination of chromosomal DNA and anyway so little DNA that no clear bands were seen, only a greenish line.

Designing ligation reactions

Digoxigenin fabs were purified and digested with SfiI elsewhere, with following concentrations:

Sample	ng/µL
DigoxFab191TAG	9,9
DigoxFab108 -> TAG	6,9
DigoxFab0	7.3
Digox C-term TAG	7.0

June 4th

Present: Janniina, Riku, Fanni, Pyry

The pLK04RCHTag plasmid was digested with SfiI in order to ligate the digoxigenin fabs into it. The DNA concentration measured to be 66.19 ng/µL (but 280 ng/µL in reality due to an error) and SfiI digestion was done according to manufacturer's protocol. Since the digestion was made with smaller concentrations than in reality, it resulted in some extra amounts of DNA.

Digested plasmids were separated on a 1 % agarose gel:

Results were not that good, lowest band (4 kb) was over twice the size it should have been (1.7 kb, MCLR Fab).

Gel run was repeated but with success this time.

June 6th

Present: Janniina, Linnea, Oskari, Pyry, Riku

pLK04RCHTag band from the successful gel run was purified with the Gel and PCR cleanup-kit. The extracted fragment had a concentration of 4.1 ng/µL. Purified pLK04RCHTag and DigoxFab0 were ligated together using T4 ligase, with MQ water and normal Digoxigenin as controls. Most of the purified vector was used for the DigoxFab0 sample so that only 0.5 µL was left for MQ and normal Digoxigenin controls.

Ligated plasmids were transformed in to 30 µL CaCl competent XL-1 Blue cells by incubating 20 min on ice following with 45 s at 42 °C and a further 2 min on ice. 1 mL of LB was added to each mixture and the mixtures were left to grown at 37 °C, 300 rpm Multitron. After 1 h of incubation cells were plated and left to grow o/n at 37 °C.

June 7th

Present: Oskari, Pyry

Colonies from yesterday’s plates were counted:
Background (MQ): 21 colonies
pLK04RCHTag + Digox: 70 colonies
pLK04RCHTag + DigoxFab0: 30 colonies

Five colonies from DigoxFAb0 were picked giving a 15 % chance of picking a background colony.
For normal Digox the chance was 0.02 %.

June 10th

Present: Janniina, Jutta, Linnea

10 colonies from the plate containing XL-1 with pLK04RCHTag+DigoxFab0 were used to inoculate 5 mL of LB with ampicillin. Same was done to 3 colonies from the plate containing 100 µL of XL-1 with pLK04RCHTag+normal Digoxigenin and 2 colonies from the plate containing 900 µL of the same transformant. All of the inoculations were left to grow at 37 °C, 300 rpm Multitron. Mistakenly chloramphenicol was added instead of ampicillin resulting in no growth.

New try with 10 colonies from the plate containing XL-1 with pLK04RCDTag+Digox Fab0, 5 colonies from the plate containing 100 µL of XL-1 with pLK04RCHTag+harmonized digox Fab and 1 colony from the plate containing 900 µL of the same transformant. All of the inoculations were left to grow at 37 °C, 300 rpm Multitron.

June 11th

Present: Janniina, Pyry

The plasmids were extracted from the cells using the EasyPure kit. The DNA-concentrations were measured with nanodrop.

sample of DigoxFab0	ng/µL
1/10	26.3
2/10	20.6
3/10	19.7
4/10	12.9
5/10	12.9
6/10	9.7
7/10	29.2
8/10	15.6
9/10	12.5
10/10	29.2

sample of normal Digox	ng/µL
1/5	19.1
2/5	16
3/5	10.5
4/5	57.6
5/5	78.2

To pretest the presence of a 1.5 kb fragment before sequencing, a SfiI digestion and gel run (1 % agarose, 135 V) were made to separate the DNA fragments:

Since only the fifth of the pLK04RCHTag+harm Digox Fab had a band at 1.5 kb, it was saved and others discarded. 5 colonies from the plate containing
pLK04RCHTag+DigoxFab0 were used to inoculate 3.5 mL of LB with ampicillin and left to grow at 37 °C. The plate was also left in 37 °C for further growth.

June 12th

Present: Janniina, Pyry, Riku

The OD600 was measured from the most opaque growth (1/4) and it was about 4 (1:10 dilution). 3/4 was the clearest, when visually observed. After taking glycerol preps (20 %), the plasmids were extracted using the EasyPure kit and the concentration was measured with Nanodrop.

sample	ng/µL
pLK04RCHTag+Fab0 Digox 1/4	13.3
pLK04RCHTag+Fab0 Digox 2/4	17.0
pLK04RCHTag+Fab0 Digox 3/4	217.5
pLK04RCHTag+Fab0 Digox 4/4	49.4

As it is seen from the table above, the 3rd colony yielded the highest amount of plasmid, which may be due to the smaller amount of cells.

The presence of a 1.5 kb was tested with SfiI digestion and gel electrophoresis. The gel showed that sample 3/4 yielded some plasmid, but the insert was longer than DigoxFab0 would be, so it was unsure if it really was DigoxFab0. It could also have been due to 135 V. The 3/4 colony was taken to be sequenced.

June 17th

Present: Janniina, Jutta, Pyry

The sequencing results came and since the reverse primer was actually forward, we only checked the forward sequence. The normal Digoxigenin had some mutations in the recognition site, so the transformant was unusable. We thought have 20 glycerol preps.

All 10 glycerol preps made on 11th June were used to inoculate 3 mL of LB with ampicillin, 1 % glucose and left to grow at 37 °C and 300 rpm.

June 18th

Present: Pyry, Riku

Plasmid extraction was done for 2 mL o/n cultures with the NucleoSpin Plasmid -kit.
Yields were as follows (measured with ND-1000 with elution buffer as blank):

Sample	ng/µL
1	8.7
2	10.1
3	8.8
4	9.1
5	5.6
6	8.4
7	8.3
8	8.6
9	7.7
10	10.6

Yields showed no DNA from extractions. 3 mL of LB and 3 µL of ampicillin were added to every culture and growth was continued for a further 3 h. Plasmids were extracted as before and concentrations measured with nanodrop.

Sample nr.	ng/µL
1	6.2
5	3.1

Again no DNA in samples.

New cultures (3 mL of LB) were started from glycerol preps.

June 19th

Present: Fanny, Janniina, Linnea, Oskari, Pyry, Riku, Susanna

2 mL of each culture was used to extract plasmid from cells. Extraction was done with NucleoSpin Plasmid Kit. Plasmid concentrations were measured with Nanodrop.

Sample	ng/µL
1	7.5
2	8.7
3	7.0
4	7.0
5	8.6
6	8.8
7	9.6
8	8.4
9	8.4
10	9.6

Again no good yield in any of the growths. This might be due to DigoxFabs being toxic to cells.

June 25th

Present: Linnea, Pyry, Riku

pLK04RCHTag was digested with SfiI to allow for ligation with Digoxigenin fabs and the digested fragments were separated with agarose gel electrophoresis (1 % agarose, 70 V). Separated fragments were purified with NucleoSpin Gel and PCR Clean-up kit. Concentration of purified pLK04RCHTag fragments was measured to be 18.2 ng/µL (NanoDrop ND-1000).

June 26th

Present: Janniina, Pyry

Purified DigoxFab0 was ligated to the purified pLK04RCHTag. Ligation was done with T4 ligase according to manufacturer’s protocol. Ligated fragments were transformed into XL-1 Blue (chemical competent) cells according to the protocol from Addgene.org . Transformed cells were plated and grown o/n at 37 °C, 300 rpm.

June 28th

Present: Riku

Colonies from plates were counted but the control plate had more colonies so transformation was done again as before. New plates were left to grow o/n at 37 °C, 300 rpm.

June 29th

Present: Jutta, Fanni

A colony from yesterday’s plates was used to inoculate 4 mL of LB with ampicillin to grow o/n at 37 °C, 300 rpm.

June 30th

Present: Janniina, Susanna

pLK04RCHTag was extracted from the cells using NucleoSpin Plasmid kit. Concentration of purified DNA was 343.8 ng/µL. Extracted plasmids were digested with SfiI and separated on a 1 % agarose gel (70 V) together with pEB04RCH ligations.

July 4th

Present: Jutta, Linnea, Oskari, Pyry

Re-digested the vector out of pLK04RCHTag+DigoxFab0 (30.6.) with SfiI and separated it on a gel together with SfiI digested pEB04RCH+DigoxFab0. SfiI digested DigoxFab0 from pEB04RCH and SfiI digested pLK04RCHTag were cut from the gel and stored in -20 °C.

June 5th

Present: Oskari, Ossi

DigoxFab0 and pLK04RCHTag were extracted with NucleoSpin Plasmid kit and measured with Nanodrop resulting in 3.5 ng/µL and 37.7 ng/µL respectively.

Extracted DigoxFab0 was ligated to pLK04RCH and pLK04RCHTag with T4 ligase.

XL-1 Blue cells (35 µL) were electroporated with 1 µL pLK04RCHTag+DigoxFab0. Transformed cells were incubated in 5 mL of LB for 1 h at 37 °C, 300 rpm shaking. After incubation cells were plated onto LA plates with ampicillin and stored o/n at 37 °C.

July 6th

Present: Riku

The transformed cells had grown nicely overnight. 3 colonies from an undiluted plate and 1 colony form 1/100 diluted plate were incubated o/n in LB+ampicillin in 37 °C, 300 rpm.

July 7th

Present: Riku

Glycerol preps were made from 500 µL of o/n cultures and the rest of the medium was centrifuged (2000 g, 10 min) to collect plasmids.

Centrifuged supernatant was discarded and the plasmids were extracted with NucleoSpin EasyPure-kit. Elution was made twice with 25 µL of 70 °C prewarmed Elution buffer.

Concentrations were measured with NanoDrop ND-1000:

Sample	ng/µL
1/1	89.4
1/2	75.3
1/3	80.6
100/1	59.1

Cloning Synthesised Digox Fabs into pLK04RCH

June 21st

Present: Janniina, Linnea

A colony was picked from glycerol prep (321.a with pLK04RCH*) and inoculated to 4 mL of LB with 100 µg/mL ampicillin. It was then put to Multitron overnight in 37°C, 300 rpm. This vector has been checked by sequencing, it’s from colony 1/1.

June 22nd

Present: Riku, Linnea

We noticed that yesterday's growth had been in RT over night.

pLK04RCH was purified from the growth with NucleoSpin Plasmid EasyPure -kit according to manufacturer's instructions.

Measured concentration: 16.2 ng/µL

Due to low yield, a new growth was started in 5 mL LB with 100 µg/ml ampicillin.

June 23rd

Present: Riku, Linnea

Growth was taken out and fuged at 2000 g for 10 minutes to pellet cells. Plasmids were extracted with NucleoSpin Plasmid EasyPure -kit according to manufacturer’s instructions. Yield was measured with Nanodrop ND-1000.

Results: 75.9 ng/µL
260/280: 1.99
260/230: 1.33

June 24th

Present: Janniina, Riku

Digestion of PLK04RCH was made with ThermoFisher FastDigest SfiI according to manufacturers instructions and run on gel.

Since there is no DNA visible in the second lane a, new SfiI digestion was made according to the following table.

	µL
MQ	7
FastDigest Buffer	2
DNA	10
SfiI FastDigest	1
Total volume	20

June 25th

Present: Riku, Linnea

5 µL 1 kb ladder	pLK04RCH + Sfi1
1 µL Midori Direct	0.5 µL Midori Direct

Samples A and B were loaded to 1 % agarose gel and run with 70 V.

pLK04RCH doesn't show up on the gel because the endonucleases in 321.a have disintegrated it completely. Conclusion: we need to produce the vector in XL-1 instead of 321.a.

35 µL of electrocompetent XL-1 cells and 1 µL of pLK04RCH (from 22.6.) were used for transformation. Transformants were put in LB-medium. Incubation was started in 37 °C, 300 rpm at 13:05 and stopped at 13:50.

20 and 200 µL of growth were spread on LA plates with ampicillin and incubated in 37 °C overnight.

New 5 mL culture of 321.A with pLK04RCH was started in 37 °C 250 rpm.

June 26th

Present: Janniina, Riku, Linnea

Cells in previous days culture was centrifuged in 2000 g for 10 min. Supernatant was thrown away and plasmids were extracted with NucleoSpin Plasmid -kit according to manufacturers instructions with additional AW-buffer wash. Concentration was measured with ND-1000.

Result: 13.4 ng/µL

Digestion was made with ThermoFisher FastDigest SfiI according to manufacturer’s instructions. Digested plasmid was run on gel.

Results: Vector has dimerized and cannot be used.

pLK04RCH from tubes 3/2 and 3/1 from 28.5.-19 were transformed into XL-1 according to this protocol from Addgene

After transformation, cells were put into 5 mL LB for 45 min 37 °C 250 rpm shaking. After shaking cells were put into LA + ampicillin plate.

A colony from previous days transformation plate (20 µL) was used to inoculate 4 mL SB medium containing ampicillin and put on 37 °C 300 rpm overnight at 13:40.

June 27th

Present: Janniina, Riku

There was a good growth in all transformation plates except mQ-control. 2 colonies were picked from each transformation (3/1: 1/100 dilution & 3/2: no dilution) plate. and put into 5 mL LB 100µg/ml ampicillin. Cultures were put into 37 °C 250 rpm shaking overnight.

Since the 1/1 was forgotten, a new overnight growth was made with 1 colony into 4 mL of LB with ampicillin. Put in 37 °C at 17.

June 28th

Present: Janniina, Riku, Ossi

Overnight cultures (1/1, 3/1.1, 3/1.2, 3/2.1 & 3/2.2) were centrifuged in 2000 g for 10 min and supernatant was thrown away. New liquid cultures were started by inoculating from pellet.

Rest of the pellet was used to extract plasmid with NucleoSpin Plasmid -kit, according to manufacturer's instructions.

Concentrations were measured with ND-1000.

Results:

1/1	333.8
3/1.1	652.5
3/1.2	538.0
3/2.1	390
3/2.2	484.5

Plasmids (1 µg) were SfiI digested for around 30 minutes at + 50°C.

	1/1	3/1.1	3/1.2	3/2.1	3/2.2
Vol. (DNA) µL	3	1.53	1.86	2.56	2.06
MQ	17	18.47	18.14	17.44	17.94
FD buffer	2	2	2	2	2
FastDigest SfiI	1	1	1	1	1
Total volume	20	20	20	20	20

After digestion, reactions were purified in 1% agarose gel.

Gel loaded as follows:

1 kb Ladder (5 µL) + midori green (1 µL)

pLK04RCH 1/1 (20 µL) + 0.5 µL Midori Green

pLK04RCH 3/1.1 (20 µL) + 0.5 µL Midori Green

pLK04RCH 3/1.2 (20 µL) + 0.5 µL Midori Green

pLK04RCH 3/2.1 (20µL) + 0.5 µL Midori Green

pLK04RCH 3/2.2 (20 µL) + 0.5 µL Midori Green

2nd highest band matches the size of pLK04RCH - no fab, so they were cut out from gel and put into freezer.

June 30th

Present: Janniina, Susanna

According to the sequencing results the pLK04RCH 3/1 is also good to use. Since we are not sure yet whether or not 1/1 has suffered from the transformation and growing in 321.A, we'll start using 3/1.

The vector backbone is needed for 5 different digoxigenin Fab ligations and since it will be purified from the gel, the yield will decrease. Assuming that 20 ng of backbone is needed for each reaction, at least 1000 ng has to be recovered from the gel. Let's say we need at least 10 µg of the backbone. Only 1 µg is recommended per 20 µL reaction, but we have used up to 2 µg with fine results. Only 1 well is free, so we'll SfiI digest only 2 µg of the 3/1 backbone.

MQ	14 µL
DNA	3 µL
FD buffer	2 µL
FD SfiI	1 µL
Total volume	20 µL

Incubation at 50 °C started at 11:27. Stopped at 11:56.
0.5 µL of Midori direct is added to the sample and separated on 1 % agarose gel.

1 µL Midori Direct + 5 µL 1 kb Ladder

0.5 µL Midori Direct + 20 µL pLK04RCH 3/1

0.5 µL Midori Direct + 10 µL pEB04RCH+lig 1/1

0.5 µL Midori Direct + 10 µL pEB04RCH+lig ½

0.5 µL Midori Direct + 10 µL pEB04RCH+lig 2/1

0.5 µL Midori Direct + 10 µL pEB04RCH+lig 2/2

0.5 µL Midori Direct + 10 µL pEB04RCH+lig 3/1

0.5 µL Midori Direct + 10 µL pEB04RCH+lig 3/2

0.5 µL Midori Direct + 10 µL pEB04RCH+lig 4/1

0.5 µL Midori Direct + 10 µL pEB04RCH+lig 4/2

0.5 µL Midori Direct + 10 µL pEB04RCH+lig 7/1

0.5 µL Midori Direct + 10 µL pLK04RCHTag+Fab0

Gel run was started using 70 V at 12:18. Stopped at 13:25.
Gel run stopped and started again 13:10.
Gel run stopped at 13:20.

Empty Eppendorf weighs 1.03 g.

2nd well cut out and put to -20 °C.

The bands didn't move accordingly to their size, apparently, which may be due to active SfiI. The 2nd more clear band was cut and stored in - 20 °C.

July 1st

Present: Linnea, Janniina, Ossi

Samples made with 3/1 and 3/2 from gel were combined and purified using NucleoSpin Gel and PCR Clean-up -kit, according to manufacturer’s instructions. Concentration measured with ND-1000 was 44,8 ng/µL.

All the Fabs were ligated into pLK04RCH (SfiI digested and purified from gel) according to the following reaction.

	µL
Vector DNA	0.5
Insert DNA	3
10x ligase buffer	2
Ligase	0.2
H2O	14.3
Total volume	20

7 reactions of a master mix that does not have insert were made.

MQ	100.1
10x buffer	14
ligase	1.4
Total volume (µL)	115.5

16.5 µL of above prepared mix to all tubes.

1 µL of each ligation reaction and ligation vector control plus MQ transformation control were electroporated to 40 µL of electrocompetent XL-1 Blue cells.

1 and digd2 exploded in an arc during electroporation. All else were fine with time constants 4.8 – 5.0.

July 2nd

Present: Pyry

1 and digd2 were retried along with a new background control as previously. 1 exploded again twice.

July 3rd

Present: Linnea, Ossi

3 growths from plate containing XL-1 Digox Lys 191 TAG (number 1) were each used to inoculate 4 mL LB with ampicillin and tetracycline and put to Multitron to grow overnight in 37°C and 300 rpm.

Three separate colonies from 2 (Digox Lys108), 3 (Digox Fab0) and 4 (Digox C-term TAG) were each separately inoculated to 5 mL of LB with ampicillin and incubated overnight in Multitron at 37 °C and 300 rpm.

Digd2 (Digox harmonized) was re-plated as the growth was too dense and grown overnight at 37 °C.

July 4th

Janniina, Linnea, Riku, Ossi, Jutta

Overnight cultures OD was measured.

Most dense culture was chosen for od measurement:

Digd2 (Digox harmonized) was re-plated as the growth was again too dense. Cells were suspensed with 300µL of LB and 1/100 and 1/1000 dilutions were re-plated. Cells were grown overnight at 37°C.

Plasmids were extracted from 1-4 ligations (3 colonies each) with plasmid EasyPure kit according to manufacturer’s instructions.

The DNA concentration was measured with Nanodrop 1000.

Culture	OD600
Lig 1	3.23
2	2.90
3	4.15
4	2.87

pLK04RCHTag was digested with Sfi1 according to manufacturer’s instructions and incubated for 2 hours at 50 °C.

sample name	concentration
pLK04RCH +lig 1/1	340.4
pLK04RCH +lig 1/2	309.9
pLK04RCH +lig 1/3	28.2
pLK04RCH +lig 2/1	368.7
pLK04RCH +lig 2/2	57.4
pLK04RCH +lig 2/3	337.2
pLK04RCH +lig 3/1	61.8
pLK04RCH +lig 3/2	53
pLK04RCH +lig 3/3	51.6
pLK04RCH +lig 4/1	42.7
pLK04RCH +lig 4/2	58.1
pLK04RCH +lig 4/3	431.4

Sample	1/1	1/2	1/3	2/1	2/2	2/3	3/1	3/2	3/3	4/1	4/2	4/3
MQ	17	17	17	17	17	17	15	15	15	14	14	14
DNA	1	1	1	1	1	1	3	3	3	4	4	4
Buffer (FD-green)	1	1	1	1	1	1	1	1	1	1	1	1
FD-SfiI	1	1	1	1	1	1	1	1	1	1	1	1
Total volume (µL)	20	20	20	20	20	20	20	20	20	20	20	20

0.5 µL Midori Direct was added to 19 µL of samples and 5 µL of ladder.

1/1

1st gel

1/2

1/3

2/1

2/2

2/3

ladder

3/1

3/2

3/3

4/1

4/2

4/3

2nd gel

ladder

Fab0

pLK04RCHTag Sfi1

Gel run started at 13:56 and stopped 14:50

SfiI digested Fab0 from pEB04RCH vector (3/1 colony) and SfiI-digested pLK04RCHTag were cut from gel and stored at -20 °C.

July 5th

Present: Ossi, Oskari

pLK04RCH with Digox harmonized (digd2) was re-plated as the growth was again too dense. Cells were suspensed with 600 µL of LB and 1/10000 and 1/100000 dilutions were re-plated. Cells were grown overnight at 37 °C.

Miniprepkit extraction and Nanodrop measurements were made for Fab0 and plk04RCHTag: 3.5 and 37.7 ng/µL respectively.

Ligation:

	ng/µL	V(20ng)	Fab0	Ligase	Buffer	MQ >20µL
pLK04RCHTag	37.7	0.5305039788	6.4142857143	0.2	2	10.8552103069
pLK04RCHTag ctrl	37.7	0.5305039788		0.2	2	17.2694960212
pLK04RCH	44.8	0.4464285714	6.4142857143	0.2	2	10.9392857143
pLK04RCH ctrl	44.8	0.4464285714		0.2	2	17.3535714286

nebiocalculator was used for calculations.

Ligation controls didn’t include the Fab0 sequence.

35 µL of XL-1 blue cells were transformed with 1 µL of pLK04RCH + Fab0 ligation reaction with electroporation.

Electroporated cells were incubated in 5 mL LB at 37°C and in 300 rpm shaking for 1 hour.

Cells were plated to LA plates with ampicillin and incubated overnight at 37 °C.

July 6th

Present: Riku

Digid2 1/100000 was washed with 500 µL LB medium and cells were put into another container. 1/100 and 1/10000 dilutions were made and 100 µL of dilutions was put into LB+ampicillin dish. Dishes were put 37 °C for overnight.

July 7th

Present: Riku

Glycerol preps were made from 500 µL overnight cultures and the rest of the medium was centrifuged in 2000g 10 minutes.

Supernatant was thrown away and plasmid was extracted with NucleoSpin EasyPure -kit, according to manufacturer’s instructions. Elution was made twice with 25 µL 70C prewarmed Elution buffer.

Concentrations were measured with NanoDrop ND-1000. Results as follows:

Sample	ng/µL
1/1	86.0
1/2	74.4
1/3	65.4
100/1	86.7

3 LB + ampicillin cultures were started and put into 30 °C 300 rpm overnight.

July 8th

Present: Riku

Cells were centrifuged in 16 000 g for 3 mins and plasmids were extracted with NucleoSpin PlasmidEasyPure -kit according to manufacturer’s instructions. Buffers A1-A3 were used 1.5 x amount.

Concentrations were measured with NanoDrop ND-1000. Result as follows:

Sample	ng/µL
1	15.4
2	16.4
3	0.4

Production of Digoxigenin Fabs

Production of pLK04RCHTag-harmonized digoxigenin Fab

June 17th

Present: Janniina, Jutta, Pyry

50 µL 321.A with pEVOL-pAZF and/or pLK04RCHTag-harm were transformed. Digox Fab according to the following table + one control transformation with 1 µL of MQ.

pEVOL-pAzF	pLK04RCHTag-harm. digox fab
x (0.5 µL)	x (0.5 µL)
x (0.5 µL)
	x (0.5 µL)

Reactions were put to 37 °C at 15:17, taken out at 16:00. The cells were centrifuged to the bottom, the supernatant was removed and the pellet was resuspended to 100 µL LB before dispensing it on the plates containing zeocin, chloramphenicol and ampicillin. The plates were put to 37 °C overnight.

June 18th

Present: Janniina

Previous day’s growths were replated, since the cells were really dense.

June 19th

Present: Janniina

50 mL of LB with 25 µL of zeocin (1 x), 75 µL ampicillin (1.5 x), 50 µL chloramphenicol (1 x) and 1 % glucose was made. A colony was taken from a plate containing 321.A with pLK04RCHTag+harm digox Fab and pEVOL-pAzF and put in 4 mL of above mentioned LB to grow overnight.

June 20th

Present: Janniina, Linnea

The OD 600 from the overnight precultured 4 mL growth was measured from 1:10 dilution yielding 0.145 which is 1.45 undiluted at 9:21. The growth was diluted into OD of 0.1 by adding 280 µL of the growth into 3,7 mL of LB with glucose, zeocin, 1,5 x ampicillin and chloramphenicol and put into 37 °C 230 rpm at 9:30.

OD was measured at 11:34, (200 µL of growth into 800 µL LB): 0.04 in dilution, 0.2 undiluted.

At 13:36 OD was 0.021 diluted and 0.21 undiluted.

June 24th

Janniina Jutta Pyry

4.5 mL of above prepared LB was inoculated from a glycerol prep made on 20th June and put in 37 °C.

June 25th

Janniina RIku Pyry

Calculated amount for 5 mL, OD 0.1:

	A	B	C	D
1	C1	V1	C2	V2
2	2.3	0.2260869565	0.1	5.2

Culture was diluted into 0.1 OD600 by adding 225 µL into 5 mL LB medium with 1% glucose, 25 µg/mL cloramphenicol, 100 µg/mL ampicillin, 50 µg/mL zeocin.
Culture was put into 37 °C 300 rpm.

OD was measured as follows:
1/10 dilution is made with 100 µL growth and 900 µL LB medium.

Time	OD600	*calculated
09:34	~0.1	*
10:00	0.12
10:50	0.14
11:45	0.27

LB medium was changed to SB medium. Cells were centrifuged in bottom of Falcon tube and supernatant was thrown away. Cells were resuspensed in 5 mL SB medium (1% glucose, 25 µg/mL cloramphenicol, 100 µg/mL ampicillin, 50 µg/mL zeocin).

Time	OD600
13:06	0.42
13:20	0.51
13:40	0.72

Growth curve until induction:

After OD had reached 0.72 cells were centrifuged in 2000 g for 10 min. Supernatant was thrown away and cells were resuspensed in SB medium (25 µg/mL cloramphenicol, 100 µg/mL ampicillin, 50 µg/mL zeocin, 0.1 % arabinose).

Cells were incubated 26 ℃ 200 rpm for 10 min.
After incubation P-azidophenylalanine and IPTG was added in growth medium to final concentration 1mM
After 4h from induction, a 1 mL sample was taken and centrifuged to pellet cells.
After 4h incubation 1 mL sample was taken, centrifuged (16.1 g, 1 min) and the pellet put into freezer.

June 26th

Present: Linnea

5 µL of glycerol prep (321.A) was used to inoculate 5 mL of LB with 25 µg/mL chloramphenicol and 0.5 % glucose and put into Multitron (300 rpm, 37 °C) at 10:53.

Two XL1 colonies with the biobrick were used to inoculate 5 mL of LB with 25 µg/ml chloramphenicol and 0.5 % glucose and put into Multitron (300 rpm, 37 °C) at 12:20.

June 27th

The cell pellets were sonicated with biotechnology sonicator. Cells were resuspended into 1 mL of Assay Buffer per 1 mL of culture before sonication.

Sonication procedure:

1 minute, low setting, power level 40, cycle 0.5 seconds. Cells were held on ice while sonicating.

Assay procedure:

The cell lysate was diluted 1/10 and 1/100 and 200 µL was incubated 30 minutes at low shake on Goat Anti Human 96-well plate.

Plate was washed 4x with Wash Buffer (kaivogen).

2A11-Europium labeled anti human antibody was used to detect Fabs. Assay concentration was 0.25 ng/µL/well. Assay volume 200 µL Plate was washed 4x with WAsh Buffer (kaivogen) Delfia Enhancement Solution was used to develop signal for 10 min. Hidex multilabel reader was used for signal detection

Results:

Production of DigoxFab0, DigoxFab108 and DigoxFab191 in pLK04RCH

July 9th

Present: Linnea, Riku

To produce the modified Digox antibody fragments with TAG-codons in positions 0, 108 or 191 the corresponding genes in pLK04RCH vectors and a pEVOL-pAzF-vector were electroporated into C321.ΔA.exp-cells.

Electroporation reactions were mixed as follows:

Digox Fab	Digox Fab Plasmid (µL)	pEVOL-pAzF (µL)	MQ H₂O (µL)	Electrocompetent C321.ΔA.exp cells (µL)
DigoxFab108	0.5	0	0.5	35
DigoxFab108	0.5	0.5	0	35
DigoxFab191	0.5	0	0.5	35
DigoxFab191	0.5	0.5	0	35
DigoxFab0	0.5	0	0.5	35
DigoxFab0	0.5	0.5	0	35
NoFabControl	0	0.5	0.5	35

Electroporation was done with 1.8 kV in 1 mm electroporation cuvette (Bio-Rad Gene Pulser Cuvette, 165-2089).

Time constants were 5.0 ms for all samples.

Electroporated cells were put into 5 mL of LB for 45 min in 37 °C and 250 rpm shaking.

Plating was done as follows:

Dilution	1/1 (µL)	1/100 (µL)
DigoxFab108	50
DigoxFab108 + pEVOL-pAzF	50	50
DigoxFab191	50
DigoxFab191 + pEVOL-pAzF	50	50
DigoxFab0	50
DigoxFab0 + pEVOL-pAzF	50	50
NoFabControl	50

Plate cultures were grown o/n at 37 °C.

July 10th

Present: Linnea

Two colonies from each 1/100 diluted plate (DigoxFab108 + pEVOL-pAzF, DigoxFab191 + pEVOL-pAzF, DigoxFab0 + pEVOL-pAzF) were used to inoculate 4 mL LB with ampicillin, chloramphenicol, zeocin and glucose. Tubes were grown o/n at 37 °C, 300 rpm.

July 11th

Present: Janniina, Oskari

OD600 was measured from each of the two replicate growths. Measurements were made from a 1:10 diluted sample. The OD600 was used to calculate the growth volume needed for inoculation of 4 mL of SB.

Growth	OD600 (1:10)	OD600 (1:1)	Volume need for dilution to OD600 of 0.1 in 4ml of SB (µL)
DigoxFab191 + pEVOL-pAzF	0.219	2.19	183
DigoxFab108 + pEVOL-pAzF	0.303	3.03	132
DigoxFab0 + pEVOL-pAzF)	0.229	2.29	175

Antibiotics and 0.05 % glucose were added to the growths for selection and increased growth. The cells were grown until OD600 of 0.6-0.8 at 37 °C, 300 rpm.

OD600 was measured again after 3 h.

DigoxFab191 + pEVOL-pAzF	DigoxFab108 + pEVOL-pAzF	DigoxFab0 + pEVOL-pAzF)
0.784	0.276	0.27

Arabinose (final concentration = 5 %) and pAzF (1 mM) was added to DigoxFab191 + pEVOL-pAzF test growth and after 10 min incubation in 37 °C 0.1 mM IPTG was added to induce production of DigoxFab191. Once induced the growth was left to grow at 26 °C, while a 20 % glycerol prep was made from the control growth.

The OD600 was measured again from DigoxFab108 + pEVOL-pAzF and DigoxFab0 + pEVOL-pAzF test growths from 1:10 dilutions (SB) resulting in unexpectedly low OD compared to the previous OD600 but the growths were induced anyway. Arabinose (5 %) and pAzF (1 mM) were added after 4.5 h. After 10 min incubation in RT 0.1 mM IPTG was added. After adding IPTG, growths were left to grow o/n at 26 °C, 300 rpm (multitron).

All growths were wrapped in tinfoil to prevent photolysis of pAzF and zeocin.

DigoxFab191 + pEVOL-pAzF	DigoxFab108 + pEVOL-pAzF	DigoxFab0 + pEVOL-pAzF)
Already induced	0.102	0.097

July 12th

Present: Fanny, Janniina, Linnea, Oskari, Pyry

An aliquot of 1 mL of overnight growths was harvested by centrifugation and resuspended to 1 mL of assay buffer. Rest of the growth was stored in -20 °C. The cells were sonicated with Braun Labsonic'u for a minute (duty cycle 0.5; output -053).

After lysis the cells were pelleted by centrifugation to collect and dilute supernatant to 1:10 ratio in AB. 200 µL of the dilution was added to each well (goat anti-human plate) with three parallel samples of each.

	Wells 1-3	Wells 4-6	Wells 7-9	Wells 10-12
A	AB control	DigoxFab108 test	DigoxFab108 control	AB control
B	DigoxFab191 control	DigoxFab0 test	DigoxFab191 test	DigoxFab0 control
C	Harmonized DigoxFab

The 96-well plate was incubated for 1 h on a shaker. Europium labeled 2A11 antibody was diluted with AB to 0.25 ng/µL from stock (390 µg/mL). The dilution was filtered through ⌀ 0.22 µm filter. The plate was washed using Delfia platewasher (Wallac) 4 times. 200 µL of 2A11 (0.25 ng/µL) is added into each well. The dispenser dispensed some of the liquid that was supposed to be in well C1 (AB control) into D1 (DigoxFab108 test). So DigoxFab108 test should give lower results than what might be expected. The plate is incubated on a shaker for 30 min after which the plate was washed again 4 times. 200 µL of Delfia Enhancement Solution was dispensed into each well. After incubating for 10 minutes, the fluorescence was measured with Hidex Sense.

To transform the plasmids (pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1) into cells (C321.ΔA.exp) for production; 0.5 µL of each plasmid was electroporated with 30 µL of cells. Three transformation controls were made; only pEVOL-pAzF, only pLK04RCHTag+DigoxFab0 1/1 (ligation date?) and MQ water only. After electroporation the cells were moved to a round-bottomed culture tube and grown for 1 h in 3 mL of SB at 37 °C.

Transformation cultures were diluted to 1/50 and plated with tetracycline, ampicillin and zeocine.

July 13th

Present: Pyry

Main plate had too many colonies to count with a couple of individual colonies at the edges. Decided to do dilutions of 1/100 or higher in future. No growth on any control plates.

July 14th

Present: Janniina

Two colonies from the plate were used to inoculate 4 mL of SB containing zeocin, ampicillin and chloramphenicol and grown at 37 °C, 300 rpm o/n. Since the plate was dense from colonies, a couple were used to restreak a new plate, which was grown at 37 °C o/n.

July 15th

Present: Janniina

The o/n cultures did not grow.

July 16th

Present: Jutta

Colonies from the plates were used to inoculate again 100 mL of SB containing zeocin, ampicillin and chloramphenicol and grown o/n at 37 °C, 300 rpm.

July 17th

Present: Janniina, Jutta, Linnea, Oskari, Pyry, Robin

The growths were diluted to 1:10 in 1 mL of SB for measurement of OD600.

OD600 of 1:10 dilutions	1:10	1:1
DigoxFab0	0.508	5.08
DigoxFab191	0.358	3.58
DigoxFab108	0.244	2.44

Growths were diluted to OD600 of 0.1 in 400 mL of SB and antibiotics in working concentrations. These growths were left to grown at 37 °C, 230 rpm. RPM was increased to 300 rpm after 2h. OD600 was measured again after 4h; giving 0.104 for DigoxFab0, 0.086 for DigoxFab191 and 0.088 forDigoxFab108. After 5h 0.05 % glucose was added to each culture to accelerate growth. OD600 measured again after 8h were: DigoxFab108: 1.01; DigoxFab191: 0.69; DigoxFab0: 0.65.

0.2 % arabinose was added to all growths. After 10 min of incubation in RT growths were induced with 0.4 mM IPTG and 1 mM pAzF. Growths were covered in foil and left to grown o/n at 26 °C and 300 rpm shaking.

Again no growth observed in the preculture. Transformed pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1 into C321.ΔA.exp. After electroporation the cells were moved to a round-bottomed culture tube and grown for 1 h in 3 mL of SB at 37 °C. After incubation the cultures were diluted to 1/100 and plated with tetracycline, ampicillin and zeocine and grown o/n at 37 °C, 300 rpm.

July 21st

Present: Janniina

Two cultures of 20 mL of SB containing zeocin, ampicillin, chloraphenicol and 0.5 % glucose were made, with cells inoculated from the plate containing C321.ΔA.exp transformed with pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1. Cultures grown o/n at 37 °C, 300 rpm.

July 22nd

Present: Janniina, Jutta

Glycerol prep was made of the cultures.

Cultures were diluted to maximum OD600 of 0.1 in 400 mL of SB (antibiotics: ZCA, 0.05 % glucose). When OD600 was around 0.7 added 1 % arabinose and 1 mM p-azido-L-phenylalanine (pAzF). Cultures were covered with aluminium foil and after 10 min 1 mM of IPTG was added to induce production of DigoxFab0. Cultures were grown o/m at 26 °C.

July 23rd

Present: Janniina, Ossi

Prepared histag column prior to purifications. A sample (500 µL) was taken from the growth. Cells were washed and lysed according to protocol. Weights of the two lysate pellets were 6,46 g and 6,28 g. Second pellet was resuspended into 15 mL NPI-lysis buffer. Another 15 mL was added since the pellet did not completely dissolve. Once resuspended the second tube was poured to the first one. Lysate (25 mL) were purified according to protocol and collected in 5 fractions of 1 mL.

ANTIBODY PRODUCTION AND PURIFICATION

Protein concentration was determined with Nanodrop 280 nm.

Fraction 1	1.26
Fraction 2	1.34
Fraction 3	1.11
Fraction 4	0.16
Fraction 5	0.06
Lysate	28.75

July 30th

Present: Janniina, Ossi, Pyry

Fractions 1-3 from earlier purification were further purified by buffer exchange with centripure P10 according to the manufacturer's protocol.

Last Production of Azido-Modified Fabs

August 22nd

Present: Janniina

Precultures for production were started by inoculating pLK04RCHTag (DigoxFab108Tag, DigoxFab191Tag and DigoxFab0HistagTag) and pEVOL-pAzF containing C321.ΔA.exp-cells from a glycerol prep into 20 mL of SB. Inoculated cells were incubated o/n at 37 °C, 250 rpm.

August 23rd

Present: Ossi, Pyry

Yesterdays growths were inoculated inoculated into the main culture (400 mL) so that the main culture’s OD600 was 0.1 with chloramphenicol, ampicillin and 0.05 % glucose. Inoculated main growth was left to grow at 37 °C, 250 rpm until OD600 was 0.6 - 0.8. Once ready, cells were induced with 1 mM pAzF, 0.5 mM IPTG and 0.5 % arabinose and incubated o/n 26 °C, 250 rpm.

August 24th

Present: Pyry

Cells were washed according to the protocol, with the modification of using ice cold PBS for washing. After washing the pellet was stored in -70 °C.

August 26th

Present: Pyry

Cells were lysed and Fabs purified according to our protocol.

NiNTA-column was equilibrated with NPI-10. Lysate was added to the column and the column was then capped. The resin lysate mixture was then on rotation for 1 h at 4 °C.

All the supernatant was combined to a 50ml bottle and the mixing with resin continued for 30 min.

The fractions were eluted in 1ml batches. 5 each. Preliminary results showed good yields of DigoxFab0Histag and control DigoxFab0 (Histag fraction 2 2.9mg/ml and Fab0 fraction 1 0.90 mg/ml). Other fractions had lower yields and were interfered by imidazole from elution buffer. Buffer was changed into PBS, except for the negative control. Added preservatives (0.05 % 2-methyl-4-isothiazolin-3-one) and stored at +4 °C.

August 27th

Present: Pyry

Negative control was purified with SEC and SDS-PAGE was done to determine the purity and incorporation-rate of pAzF.

Producing Dengue Fabs

Cloning DengueFabs to pLK04RCH/Tag

In this lab, we attempted to clone the antigen-binding fragment (Fab) from a Dengue virus antibody into our pLK04RCH and pLK04RCH-Tag vectors.

July 18th

Present: Oskari, Robin, Janniina

The Dengue-Fab sequences from Integrated DNA Technologies were suspended in 20 µL MQ-H2O to a final concentration of 50 ng/µL. We have four different Dengue-Fab fragments to work with, three of which have had an amino acid replaced with an amber stop codon (TAG):
DengueFab Ser221 > TAG (DengueFab221),
DengueFab Thr196 > TAG (DengueFab196)
and DengueFab Asp152 > TAG (DengueFab152).
The last one is an unmodified Dengue-Fab fragment (DengueFab0). The concentration of the previously SfiI-digested vector backbones were:

pLK04RCH: 44.8 ng/µL
pLK04RCH-Tag: 37.7 ng/µL

The Dengue-Fabs were digested with SfiI:

	volume (µL)
MQ-H₂O	7
10x FastDigest buffer	2
DNA	10
SfiI (FastDigest)	1
total volume	20

The reactions were incubated for 15 minutes at 50°C. SfiI was inactivated by addition of 20 mM EDTA.

We then attempted ligation of the Dengue-Fab inserts into the vector backbones. DengueFab0 was cloned into the pLK04RCH and the pLK04RCH-Tag vector. The DengueFab152, DengueFab196 and DengueFab221 were cloned into the pLK04RCH vector:

	pLK04RCH	pLK04RCH-Tag	pLK04RCH ligation ctrl	pLK04RCH-Tag ligation ctrl
Vector	0.45	0.5	0.4	0.5
Insert	0.95	1	0
10x ligase buffer	2	2	2	2
ligase	0.5	0.5	0.5	0.5
MQ-H₂O	16.1	16	17.1	17
total volume (µL)	20	20	20	20

The reactions were incubated for 1 hour at room temperature. Then 1 µL of the ligated product was electroporated into 40 µL of XL-1 electrocompetent cells, according to our electroporation protocol. Electroporation was followed by a 45-minute incubation at 37°C and 300rpm. 200 µL of the mixture was plated onto selective LA plates containing tetracyclin and ampicillin and incubated o/n at 37°C.

July 19th

Present: Oskari

Colonies were counted:

Vector + insert	number of colonies
pLK04RCHTag + DengueFab0	108
pLK04RCH + DengueFab0	0
pLK04RCH + DengueFab152	110
pLK04RCH + DengueFab196	24
pLK04RCH + DengueFab221	10
pLK04RCHTag – ligation ctrl	9
pLK04RCH - ligation ctrl	2
Transformation ctrl	0

July 21st

Present: Janniina, Jutta

40 mL of SB medium, containing ampicillin and 0.5 % glucose, was prepared. Two colonies were picked from the pLK04RCH-Tag+DengueFab0, pLK04RCH+DengueFab152, -196 and -221, and inoculated to 2x4 mL of SB medium. The growths were incubated o/n at 37°C.

July 22nd

Present: Jutta

Glycerol preps were made from the overnight cultures according to our protocol for making glycerol preps. Plasmids were extracted according to our miniprep protocol.

The DNA concentration was measured with Nanodrop:

	miniprep 1	miniprep 2
pLK04RCH-DengueFab152	136.4 ng/µL	124.5 ng/µL
pLK04RCH-DengueFab221	120.7 ng/µL	130.1 ng/µL
pLK04RCH-DengueFab196	129 ng/µL	431.3 ng/µL
pLK04RCHTag-DengueFab0	448.1 ng/µL	137.9 ng/µL

To verify that the ligation worked, the vectors were re-digested with SfiI:

	volume (µL)
MQ-H₂O	7.5
DNA	1
FastDigest buffer	1
SfiI	0.5
total volume	20

The reaction was incubated for 30 minutes at 50 °C. 0.5 µL Midori Green Direct (Nippon Genetics) was added to each digestion reaction and 1 µL to 1 kB ladder.

The digested vectors and inserts were separated on a 1 % agarose gel:

From left to right: ladder, pLK04RCH-DengueFab152 (miniprep 1), pLK04RCH-DengueFab152 (miniprep 2), pLK04RCH-DengueFab196 (miniprep 1), pLK04RCH-DengueFab196 (miniprep 2), pLK04RCH-DengueFab221 (miniprep 1), pLK04RCH-DengueFab221 (miniprep2), pLK04RCHTag-DengueFab0 (miniprep 1), pLK04RCHTag-DengueFab0 (miniprep 2).

We repeat the ligation of DengueFab0 and the pLK04RCH vector (vector concentration: 18.8 ng/µL):

	pLK04RCH	pLK04RCH ligation ctrl
vector	1.1	1.1
insert	1	0
10x ligase buffer	2	2
ligase	0.5	0.5
MQ-H₂O	15.4	16.4
total volume (µL)	20	20

Reaction was incubated for 1 hour at room temperature. XL-1 electrocompetent cells were transformed with the ligation mixture according to our electroporation protocol, and then incubated for 45 minutes at 37 °C and 300 rpm, plated onto selective plates and incubated o/n at 37 °C.

July 23rd

Present: Ossi

Colonies from the plates were inoculated into 4 mL SB cultures containing ampicillin and 0.5 % glucose. The growths were incubated o/n at 37 °C and 300 rpm.

The pLK04RCH-DengueFab196, pLK04RCH-DengueFab221, pLK04RCH-DengueFab152 and pLK04RCH-Tag-DengueFab0 were sent for sequencing.

July 24th

Present: Ossi

Glycerol preps and minipreps were made from the o/n cultures, according to our protocols. The DNA concentration as measured with Nanodrop:

pLK04RCH-DengueFab0 (prep 1): 518.8 ng/µL
pLK04RCH-DengueFab0 (prep 2): 471.9 ng/µL

To verify if the cloning worked we re-digested the vectors with SfiI:

MQ-H₂O	7.5
vector	1
FastDigest buffer	1
SfiI	0.5
total volume (µL)	10

The reaction was incubated for 10 minutes at 50 °C. The digestion products were separated on a 1 % agarose gel:

A sample of pLK04RCH-DengueFab0 was sent for sequencing.

According to the results from the previous sequencing, we decided to not use the pLK04RCH-DengueFab221 vector. The rest were OK.

321.A cells were transformed with the pEVOL-pAzF vector and one of the pLK04RCH-DengueFab196, -152 or pLK04RCH-Tag-DengueFab0 vectors. Transformation was done with electroporation according to our electroporation protocol. The cell mixtures were afterward plated onto selective LA plates containing chloramphenicol, zeocin, ampicillin and 0.5 % glucose.

Comment:
we attempted test production on a small scale of the DengueFab0 in the 321.A strain, but we soon realized that it has a mutation in a stop codon (TAA > TAG), leading to a nonsense peptide that is not able to bind anything.

July 30th

New 4 mL SB growths containing ampicillin and 0.5 % glucose were inoculated with pLK04RCH-Tag-DengueFab0 and incubated o/n at 37°C.

July 31st

Glycerol preps and minipreps were made from the o/n cultures according to our protocols. The DNA concentration in the minipreps:

pLK04RCH-Tag-DengueFab0 (prep 1): 27.9 ng/µL
pLK04RCH-Tag-DengueFab0 (prep 2): 7.9 ng/µL
pLK04RCH-Tag-DengueFab0 (prep 3): 40.8 ng/µL

The vector (prep 1 and 3) was re-digested with SfiI:

MQ-H₂O	7.5
vector	1
FastDigest buffer	1
SfiI	0.5
total volume (µL)	10

The reaction was incubated for 10 minutes at 50°C, and the digestion fragments separated on a 1 % agarose gel.

There was a 8 kB band visible in the gel, which is too large to be the vector.

August 5th

Present: Janniina

2 new colonies from the plate containing XL-1 with pLK04RCHTag-Dengue Fab0 were picked and inoculated to 4 mL of SB with ampicillin and 0.5 % glucose and incubated o/n at 37 °C, 300 rpm.

August 6th

Present: Janniina

Glycerol preps and minipreps were made from the o/n cultures according to our protocols. The DNA concentration in the minipreps:

pLK04RCH-Tag-DengueFab0 (prep 1): 413.6 ng/µL
pLK04RCH-Tag-DengueFab0 (prep 2): 512.7 ng/µL

SfiI digestion:

MQ-H₂O	7.5
vector	1
FastDigest buffer	1
SfiI	0.5
total volume (µL)	10

The reaction was incubated for 10 minutes at 50 °C, and the digestion fragments separated on a 1 % agarose gel.

Comment: We now had bands of the correct size in the gel.Therefore, pLK04RCH-Tag-DengueFab0 vector was sent for sequencing.

Test production of dengue Fabs

July 25th

E.coli 321.A was transformed with pEVOL-pAzF and pLK04RCH-AntiDengueFab plasmids according to electroporation protocol.

After transformation, cells were plated on LA+ampicillin+cloramphenicol plates. Plates were incubated o/n in 37 °C.

Friday July 26th

After o/n incubation, plates were put on +4 °C.

Sunday July 28th

4 mL precultures or test production was started. Precultures were done on each AntiDengueFab. Preculture was done in SB-medium containing 0.5 % glucose ampicillin, cloramphenicol and zeocin.

All precultures were put on 250 rpm shaking 37 °C for o/n.

July 28th

Each preculture was diluted to OD 0.1 with similar SB medium as before, so that the final volume of cultures were 4 mL. Cultures were put to grow in 37 °C 250 rpm shaking.

When OD reached 0.6-0.8, 20 µL of 20 % arabinose and 80 µL of 50 mM pAzF was added to growth medium. Cultures were protected from the light from this point on due to light sensitivity of p-Azidophenylalanine.

After 10 min of incubation 4 µL 0.1 M IPTG was added to growth medium and temperature was lowered to 26 °C and cultures were incubated o/n.

July 29th

Sequencing results came and revealed that one of colonies used to start preculture contained a mutation. As a result we abandoned all precultures.

Troubleshooting

Testing all previously restricted plasmids

June 4th

Riku, Linnea, Pyry, Janniina

We came to suspect, that at some point we have mixed at least the plasmids pUC19 and the original pLK04RCH we had been multiplying and purifying ourselves. Therefore, we decided to SfiI digest and separate on a 1 % agarose gel everything we thought might have become compromised in addition to the constructs we had just cloned and purified.

SfiI digestions were done using the enzyme from Thermo Fisher according to the manufacturer’s protocol. The digested constructs were:
pLK04RTagCH+harm. (5 colonies: ⅕-5/5)
pLK04RCH (1 colony from a plate (1/1) and 3 from another plate (3/1, 3/2 & 3/3)).
Also, two copies of the plasmid we had been multiplying in 321.A, that was thought to be the pLK04RCHTag from professor Huovinen was also SfiI digested. In the last well 1 µL of the vector gotten from Huovinen was loaded undigested by adding 10 µL of MQ.

The samples and 1 kb ladder (Nippon Genetics) were loaded with MidoriDirect (BioRad) according to the manufacturer (0.5 µL in a sample and 1 µL to a DNA ladder) to visualize the bands under blue light and orange plastic filter. After the gel electrophoresis we discovered, that the pLK04RTagCH+harm. ⅕ - ⅘ transformants as well as the pLK04RCH 3/3 did not show the supposed 3.7 and 1.5 kb bands (figure X). The 5/5, 1/1, 3/1-3/3 were chosen to be sequenced.

Figure 1. Gel electrophoresis of the vectors constructed and plasmids multiplied.

From the gel, it can also be seen that the original vector was possibly present as a double plasmid. More disturbingly, the gel run also revealed that the original pLK04RCHTag plasmid that we have thought we have been multiplying did not create the correct sized bands indicating that it was in fact some other similar sized plasmid we have been working with. We had been multiplying pUC19 at the same time to be able to clone the biobrick into it. We have also plated the GFP-TAG-RFP gene from the biobrick gotten from the iGEM plate that was cloned into pUC19.

Since both pUC19 and the pLK04 vectors we had, also had XbaI site, we decided to digest the suspicious plasmids with it (FastDigest XbaI, ThermoFisher) and run them on a gel. However, we later figured out, that all our strains have the native methylation enzymes, which create methylations sites blocking XbaI (see ____).

Since either the plasmid or MidoriDirect was forgotten from the digestion sample, there was nothing to see in the gel. Regardless, it was quite clear that the plasmids had been mixed up at some point, so we got rid of all the plasmids we thought were possibly mixed. Since we still needed pUC19 and pLK04RCH backbones, we transformed the original pLK04RCH plasmid achieved from Tuomas and the pUC19 (2nd try) we got from professor Käpylä.

Changing signal sequence to pLK04RCHTag+Fab0 digox

Janniina & Pyry

We were encountering some difficulties with extracting pLK04-based vectors with our Fabs containing PelB signal sequence; the colonies with Fab0 we were able to pick from the plate and grow in liquid medium seemed to have disturbingly often a deletion in the PelB signal sequence according to our sequencing services purchased from Eurofins. However, the sequences containing amber stop codons showed at least one good sequence per cloning with XL-1 as the host. This could be due to the toxicity of Fabs to E. coli, since XL-1 is not able to translate the complete Fab sequence due to the amber stop codon and the absence of corresponding tRNA synthetase.

So we decided to use a sledgehammer to crack a nut and change the signal sequence with restriction enzymes XbaI and XhoI. To maximize the probability to succeed, we tried a pLK04 vector with harmonized Fab and a signal sequence obtained from utu (not shown) as the “donor”.

Today, we transformed the donor from utu into XL-1 and digested the recipient vector using the enzymes from Thermo Scientific. The sizes of the bands were not checked with Benchling’s virtual digestion, but a band close to the size of the vector was cut from the 1 % agarose gel after electrophoresis. A colony from the transformation today was picked to inoculate 4 mL of SB containing ampicillin and grown over night for plasmid extraction.

The following day, the signal sequence donor plasmid was extracted and 1394 ng of it was digested with FastDigest XhoI and XbaI. While the digestion was incubating, using the virtual digestion attribute of benchling I checked the sizes of the insert and the vector to calculate the ligation reactions. Based on the virtual digestion we learned that instead of one XhoI and two XbaI sites there were actually two of both of them in the pLK04RCHTag+Fab0 with deletion making it too difficult to change the signal sequence by digesting and ligating. Also this meant that the fragment cut from the gel yesterday was missing all of the Fab sequence and little extra making our customary SfiI digestion just as useful. Therefore we did not continue to change the signal sequence, but to try to pick new colonies and to try to clone the correct Fab0 sequence from vector to the pLK04RCHTag backbone by SfiI digestion.

Immunoassays

Fab activity determination on Goat Anti-Human plates

16th of August
Niemelä P., Karinsalo J. & Leppänen O.

Goat-anti-human plates were used in determination of Fab amount in samples. Fab standard was made so that there was 10, 50, 100, 150, 300 ng (rhFab in TSA, 3,62 mg/mL) of fab in the wells.
Fabs and lysates were diluted 1/10, 1/100 and 1/1000 before adding to the wells in the volume or 200 µL. Each sample was added to the plate as triplicates. The samples were incubated for 30 minutes in shaking before washing of the unbounds.

200 µL of 250 ng/µL Eu-labeled antibody solution was put into each well and plate incubated in shaking for 30 minutes. Plate was washed and 200 µL of Delfia Enhancement Solution per well was added with delfia plate dispenser. After 10 min incubation, time-resolved fluorescence was measured with Hidex. As a result, it was noticed that there were no dengue fabs in the reaction.

28th of August
Karinsalo J. & Leppänen O.

SEC- Purified Fab fractions were diluted into 1/500, 1/5000 and 1/1000 in AB

Table 1. Following dilutions were made from each fab.

amount of standard Fab (ng)	ng/µL	µL of 2400 ng/µL stock	STOCK (mg/mL)	µL AB	V_tot (µL)	Add µL of standard Fab	µL AB
2400	12	1.32	3,62	399	400	1.3	399
120	0.6	50	2.40	950	1000	50	950
100	0.5	41.6	2.40	958	1000	42	958
70	0.35	29.2	2.40	971	1000	29	971
50	0.25	20.8	2.40	979	1000	21	979
20	0.1	8.3	2.40	992	1000	8.3	992
10	0.05	4.2	2.40	996	1000	4.2	996

200 µL of each fab dilution was incubated on GAH plates in triplicates for 30 minutes.

17 mL of Eu-2A11 0.25 ng/mL dilution was made from 20 µg/ml stock to Assay Buffer and the solution was filtered. Add 200 µL of the 2A11 dilution to each well, The tracer was incubated for 30 minutes. Unbounds were removed by washing the wells four times. 200 µL of Delfia Enhancement solution was added to each well and incubated 10 min in shaking. Time-resolved fluorescence was measured with Hidex.

Digoxigenin binding capacity

Fabs were incubated on plates for 30 min as previously. The wells were washed twice afterwards.

15 mL of biotinylated digoxigenin (0.1 ng/µL concentration) was made. 200 µL of the 0.1 ng/µL bio-dig dilution was added to each well and incubated for 30. 15 mL of 0.125 ng/µL dilution of Eu-streptavidin (stock concentration 0.1 mg/mL) was made by adding 18.8 µL of Eu-Streptavidin to 15 mL of Assay Buffer.

The wells were washed twice and 200 µL of the 0.125 ng/µL Eu-streptavidin dilution was added to each well. The tracer was incubated for 30 minutes. Unbounds were removed by washing the wells four times. 200 µL of Delfia Enhancement solution was added to each well and incubated 10 min in shaking. Time-resolved fluorescence was measured with Hidex.

Coating DBCO beads with NHS-containing Fab molecules

15th of August
Niemelä P.

The produced Fabs were immobilized via azide groups onto magnetic beads (MB) with dibenzocyclo-octyne (DBCO). The DBCO-MBs were obtained from Jena Bioscience and the assumed amount of DBCO per 1 mg of beads was 40 nmol. 30 to 50 µg of DBCO-MBs were coated with two fold molar excess of digoxigenin Fabs 191, 108 and histag as well as Fab without any azide groups (Fab0) and Fabs with randomly attached azide groups (Fab NHS-AZ).

The total volume of the reactions was not adjusted to keep it as low as possible. Strain promoted azide-alkyne cycloaddition (SPAAC) was allowed to take place for an hour in RT while covered with aluminum foil to block light. After incubation, unbound Fabs were washed away with first 1000 µL and then 500 µL of PBS prior to washing the MBs twice with 250 µL of washing buffer (Kaivogen). The MBs were washed again with 1000 µL and then with 500 µL of PBS before resuspending into final concentration of 0.5 µg/µL of MB in PBS. The oated MBs were stored in +4 °C.

Materials

2nd of September
Karinsalo J., Leppänen O., Niemelä P.

Since the Fabs had started to decay, a new set of MBswas coated with digoxigenin Fabs 191, 108 and Histag as well as Fab NHS-AZ and Fab. 17.1 µg of MBs was used in each reaction, but the MBs were pipetted by accident in the protein stocks instead of the reaction tubes. After 5 minutes the mistake was noticed and the Fab stocks were pipetted into new tubes while holding the MBs with magnets. Assuming that there was 40 nmol of DBCO on 1 mg of MBs, 1.5 molar excess of digoxigenin Fabs was used to coat the 17.1 µg of MBs. PBS was added to the total volume of 250 µL, after which the SPAAC was incubated for 16 hours in +4 °C covered with aluminium foil. However the reaction with Fab 191 was forgotten in RT and disposed due to that.

3rd of September
Karinsalo J.

The MBs coated with digoxigenin Fab 108, Histag and NHS-AZ in addition to the MBs with Fab0 were washed first with 1000 µL of PBS. Since it took a long time for the MBs to aggregate in such a volume, the MBs were next washed with only 350 µL of PBS (500 µL last time). The MBs were washed twice with 250 µL of washing buffer (Kaivogen) before resuspending the coated MBs into 0.5 µg/µL of MBs in PBS with 0.05% 2-methyl-4-isothiazolin-3-one to preserve the Fabs. However, it was visually observed that the size of the MB precipitates varied between the coated MBs. The coated MBs were stored in +4 °C for later use.

Relative Fab activity determination on DBCO beads

21th of August
Karinsalo J. & Lindfors J.

This is continuation to “Coating DBCO beads with NHS-containing Fab molecules”.

0.5 ng/µL working solution of Eu-2A11 was made into Assay Buffer. The solution was filtered before use. 100 µL of 2A11 mAb working solution was added into wells. Therefore, there was 50 ng of Eu-2A11 mAb in each well.

10, 30, 60, and 100 ng of DBCO magnetic beads was added to each well in three replicates. In addition, non-coated DBCO bead control was used. 100 µL of Eu-2A11 mAb was added to wells and reaction was incubated for 1 hour in shaking.

The beads were washed three times and 200 µL of DELFIA Enhancement Solution. Enhancement solution was incubated for 10 minutes in shaking and time-resolved fluorescence was measured with Hidex.

Optimization of click immunomagetic bead (CIMB) concentration in assay

21th of August
Karinsalo J. & Lindfors J.

1 µg of beads was added to each well/tube in 100 µL. 10 µg/mL stock from empty MB was made by adding 180 µL of the 100 µg/ml stock to 1620 µL of Assay Buffer.

Because there wasn’t enough of 10 µg/mL stock 15 µg/mL stock was diluted by adding 600 µL of beads to 300 µL of Assay Buffer. 0.01, 0.1, 1, 10, 50 ng of Eu-2A11 was added to each well in the volume of 100 µL in Assay Buffer. The Eu-226A2 solution was filtered before use to reduce %CV. The tracer was incubated at shaking for 1h.

The wells were washed three times with Kaivogen wash buffer on a magnetic plate holder. (Invitrogen). 200 µL of Delfia Enhancement solution was added to wells and incubated for 10 minutes in shaking. Time-resolved fluorescence was measured with Hidex

Table 1. Results

Biotinylation of Fabs with DBCO Biotin

8th of August
Niemelä P.

1 mg of DBCO-PEG4-Biotin was dissolved to 1000 µL of DMSO

1000 µL of azido derivatized Dengue Fab0 (stock concentration 0.09 mg/mL) and Digox fab 0 (0.58 mg/mL) were biotinylated. Additionally, 500 µL of HistagTAG Digox Fab (stock concentration 0.80 mg/mL) was biotinylated with three-fold molar excess of DBCO-PEG4-Biotin.

Slide-A-Lyser 10 kDa MWCO (Thermo Scientific) Dialysis Cassette was used according to instructions to remove unreacted labels. First, biotinylated Fabs were dialysed for 2 hours and Dialyse buffer was changed to fresh. This was done twice and samples were retrieved afterwards.

2nd of September
Karinsalo K., Leppänen O.

250 µL of Fab0 was incubated with NHS-PEG4-azide (stock concentration 0.23 mg/mL).

Table 1. Contents of biotinylation reactions.

	protein (mg/mL)	Protein (mL)	PBS (mL)	DBCO-PEG4-biotin (µL)
Fab0	0.81	0.035	0	12.4
191	0.49	0.1173	0.1326	0.26
108	0.29	0.1982	0.0517	0.26
TAG	2.47	0.0232	0.2267	0.26

Buffer exchange was done with Nanosep 10k Omega to remove excess DBCO label: The column was washed four times with 500 µL of PBS buffer. Fabs were eluted with two times 20 µL of PBS buffer. The total volume was around 70 µL since excess liquid was still present in the column. Concentration of the biotinylated fabs was measured with Nanobrop and 0.05% of preservative was added for storage at + 4°C protected from light.

Table 2. Protein concentrations after reaction.

	mg/mL
Bio-Fab0	0.33
Bio-108	0.36
Bio-191	0.41
Bio-TAG	0.65

Determination of Streptavidin plate binding capacity with Fabs biotinylated with different biotinylation strategies

12th of August
Niemelä P.

This is a continuation from work "Biotinylation with DBCO-Biotin"

Biotinylated Fab was inserted into wells with increasing amounts to determine SA binding capacity. The tested Fabs were Fab0 (without any pAzF) and Fab0 Histag-TAG (where pAzF is located after 6x His tail).

Capacity determination of streptavidin plates with Fabs biotinylated with different strategies protocol was used in this immunoassay with Fab amounts of 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30 ng per well.

Table 1. Analysis of signals.

	Fab0-NHS-Azide-DBCO-Biotin
ng/well	CPS			average	std. Dev	CV%
0	282	331	293	302	20.99206	7 %
2	8490	8228	9278	8665.333	446.2296	5 %
4	22290	22513	27353	24052	2335.934	10 %
6	43705	43632	47703	45013.33	1902.115	4 %
8	99658	97701	110325	102561.3	5547.573	5 %
10	124563	133717	136137	131472.3	4984.527	4 %
12	179571	207073	176129	187591	13847.34	7 %
14	209872	240720	235876	228822.7	13545.28	6 %
16	278056	274925	287992	280324.3	5570.495	2 %
18	315992	287959	321476	308475.7	14679.21	5 %
20	355686	342806	347386	348626	5330.841	2 %
30	489319	534859	509428	511202	18633.9	4 %

	FAb0-Histag-TAG-DBCO-Biotin
ng/well	CPS			average	std. Dev	CV%
0	460	459	404	441	26.16614	6 %
2	3655	3331	3377	3454.333	143.1301	4 %
4	6865	7504	7579	7316	320.3717	4 %
6	11920	11807	11048	11591.67	387.1884	3 %
8	16215	17734	18364	17437.67	902.0016	5 %
10	20272	21460	21218	20983.33	512.5995	2 %
12	28444	33457	29546	30482.33	2150.982	7 %
14	41178	42606	33716'	41892	714	2 %
16	55522	47123	55440	52695	3940.141	7 %
18	63181	72166	69026	68124.33	3723.109	5 %
20	71606	79041	72873	74506.67	3247.713	4 %
30	121981	132264	141396	131880.3	7930.782	6 %

Figure 1. Signal-to-background plotted standard curves for Fab0-NHS-DBCO and Fab0-Histag-TAG.

Table 2. The assay was repeated with broader range of Fab per well. A couple of measurement points were removed from plotted image because at 45 ng/well only protein stock was used instead of diluting Fabs first to 1000 ng/mL of AB. This caused bias in measurement points when sub-micromolar volumes of stock were used. Removed points are in bold.

ng/well	CPS			Average	stdev	CV%
1	472	547	509	509.3333	30.61953	6 %
2	1126	1178	1287	1197	67.08701	6 %
5	3912	4077	3867	3952	90.27735	2 %
10	17437	12638	13673	14582.67	2062.073	14 %
20	45686	48325	44913	46308	1460.73	3 %
25	70606	90006	75487	78699.67	8239.374	10 %
30	108469	99911	88514	98964.67	8174.031	8 %
35	154547	138559	133625	142243.7	8929.916	6 %
40	186262	213886	195294	198480.7	11500.36	6 %
45	200182	182486	195448	192705.3	7480.141	4 %
50	241520	224263	239563	235115.3	7715.237	3 %
60	310825	300665	318143	309877.7	7166.738	2 %
70	418202	449384	449434	439006.7	14711.14	3 %
100	649618	652603	624289	642170	12702.37	2 %
150	807853	820781	852370	827001.3	18698.67	2 %

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                      </h1>
                      <p>
-                         Site is under construction, please wait until group of pinguins and a polar bear
+                         On this page, our workflow is described in a more detailed manner
-                        has done their work. Thank you!
                      </p>
                  </div>
@@ Line 30: / Line 29: @@
                  <div class="toc-card" id="tocbox">
                      <h2>
-                         Table of content
+                         Table of contents
                      </h2>
                      <ul>
@@ Line 41: / Line 40: @@
                          <li>
                              <a class="toc-link" href="#Producing_dengue_fab"> Producing Dengue-NS1 Fabs</a>
+                        </li>
+						 <li>
+                            <a class="toc-link" href="#Troubleshooting">Troubleshooting</a>
                          </li>
 						 <li>
@@ Line 86: / Line 88: @@
                                          To get started with our iGEM project, the ordered cell lines described below, were
                                          cultured. E. coli cells containing pEVOL-pAzF (Chloramphenicol selection) and cell
-                                         line C321.ΔA.exp (Zeocin selection) were plated to LA-plates containing 0,5 % Glucose,
+                                         line C321.ΔA.exp (Zeocin selection) were plated to LA-plates containing 0.5 % Glucose,
-                                         and 35µg/ml chloramphenicol or 50 µg/ml zeocin. Controls with tetracycline, ampicillin
+                                         and 35 µg/mL chloramphenicol or 50 µg/mL zeocin. Controls with tetracycline, ampicillin
                                          and a plate without antibiotics were also prepared for the detection of contamination.
                                          Plates were incubated overnight at 30 °C. In addition, extra zeocin and chloramphenicol
@@ Line 145: / Line 147: @@
                                          MM1:
                                          <br>
-                                         LA-plates containing: chloramphenicol , 1 mM pAzF, 0,2 % arabinose, 0,5 mM IPTG
+                                         LA-plates containing: chloramphenicol, 1 mM pAzF, 0.2 % arabinose, 0.5 mM IPTG
                                          <br>
-                                         LA-plates containing: tetracycline, 1mM pAzF 0,2 % arabinose, 0,5 mM IPTG
+                                         LA-plates containing: tetracycline, 1 mM pAzF 0.2 % arabinose, 0.5 mM IPTG
                                          <br>
-                                         LA-plates containing: zeocin, , 1mM pAzF 0,2 % arabinose, 0,5 mM IPTG
+                                         LA-plates containing: zeocin, 1 mM pAzF 0.2 % arabinose, 0.5 mM IPTG
                                          <br>
-                                         LA-plates containing: chloramphenicol , 1 mM pAzF, 0,2 % arabinose
+                                         LA-plates containing: chloramphenicol, 1 mM pAzF, 0.2 % arabinose
                                          <br>
-                                         LA-plates containing: chloramphenicol, 1 mM pAzF, 0,5 mM IPTG
+                                         LA-plates containing: chloramphenicol, 1 mM pAzF, 0.5 mM IPTG
                                          <br>
                                      </p>
@@ Line 159: / Line 161: @@
                                          MM2:
                                          <br>
-                                         LA-plates containing: chloramphenicol, 0,2 % arabinose, 0,5 mM IPTG
+                                         LA-plates containing: chloramphenicol, 0.2 % arabinose, 0.5 mM IPTG
                                          <br>
-                                         LA-plates containing: tetracycline, 0,2 % arabinose, 0,5 mM IPTG
+                                         LA-plates containing: tetracycline, 0.2 % arabinose, 0.5 mM IPTG
                                          <br>
-                                         LA-plates containing: zeocin, 0,2 % arabinose, 0,5 mM IPTG
+                                         LA-plates containing: zeocin, 0.2 % arabinose, 0.5 mM IPTG
                                          <br>
                                      </p>
                                      <p>
-ul cells and 1 ul BBa_K567011 (GFP-tag-RFP) was electroporated in 1 mm electroporation
+µL cells and 1 µL BBa_K567011 (GFP-tag-RFP) was electroporated in 1 mm electroporation
-                                         cuvette with 1,8kV voltage in BioRad Gene Pulser Xcell. After electroporation cells
+                                         cuvette with 1.8 kV voltage in BioRad Gene Pulser Xcell. After electroporation cells
-                                         were put into 5 ml SOC medium in 37C for 40 min.
+                                         were put into 5 mL SOC medium in 37 °C for 40 min.
                                          <br>
-                                         After incubation cells were plated as follows: 50 ul SOC, 50 ul 1/10 and 50 ul 1/100
+                                         After incubation cells were plated as follows: 50 µL SOC, 50 µL 1/10 and 50 µL 1/100
                                          dilutions were plated as follows:
                                      </p>
@@ Line 283: / Line 285: @@
                                      </p>
                                      <p>
-                                         Plates were left in 37 C o/n
+                                         Plates were left in 37 °C o/n
                                      </p>
                                      <p>
@@ Line 316: / Line 318: @@
 <p> <b> May 16th  </b> </p>
 <p> Present: Janniina, Oskari, Fanny, Riku </p>
-<p> A colony from a 321.A + pAzF plate was replated onto an MM2C plate and incubated o/n at 37 C.
+<p> A colony from a 321.A + pAzF plate was replated onto an MM2C plate and incubated o/n at 37 °C.
 <p> <b> May 17th </b> </p>
-<p> A single colony was picked and used to inoculate 4 mL LB medium containing 33 µg/mL chloramphenicol o/n at 30 C and 150 rpm. </p>
+<p> A single colony was picked and used to inoculate 4 mL LB medium containing 33 µg/mL chloramphenicol o/n at 30 °C and 150 rpm. </p>
 <p> <b> May 18th </b> </p>
-<p> 20 % glycerol preps were made by adding 500 µl of growth to 167 µl of an 80 % glycerol stock. The glycerol preps were stored at -80 C. </p>
+<p> 20 % glycerol preps were made by adding 500 µL of growth to 167 µL of an 80 % glycerol stock. The glycerol preps were stored at -80 °C. </p>
                                   </p>
@@ Line 354: / Line 356: @@
                                      </p>
                                      <p>
-ml of LB containing 5 µl of 25 mg/ml chloramphenicol was inoculated with a colony
+mL of LB containing 5 µL of 25 mg/mL chloramphenicol was inoculated with a colony
-                                         picked from a plate containing 321.A (BBa_K567018) and put in Infors Multitron (200
+                                         picker from a plate containing 321.A (BBa_K567018) and put in Infors Multitron (200
                                          rpm, 26 °C) at 10:10 O'clock.
                                          <br>
@@ Line 375: / Line 377: @@
                                          </b>
                                          <br>
-                                         Four glycerol preps were made from yesterdays cultures. 500 ul LB of E.coli 321.A
+                                         Four glycerol preps were made from yesterdays cultures. 500 µL LB of E.coli 321.A
-                                         with BBa_K567018 plasmid culture was mixed with 170 ul 80 % glyserol. Preps were
+                                         with BBa_K567018 plasmid culture was mixed with 170 µL 80 % glyserol. Preps were
-                                         put in -70C freezer.
+                                         put in -70 °C freezer.
                                  </div>
                              </div>
@@ Line 421: / Line 423: @@
                                                  <td>
                                                      <b>
-                                                         BBa_K567018 (µl)
+                                                         BBa_K567018 (µL)
                                                      </b>
                                                  </td>
                                                  <td>
                                                      <b>
-                                                         pUC19 (µl)
+                                                         pUC19 (µL)
                                                      </b>
                                                  </td>
@@ Line 494: / Line 496: @@
                                      </p>
                                      <p>
-                                         incubation at 37 C started: 15:40, stopped: 16:09
+                                         incubation at 37 °C started: 15:40, stopped: 16:09
                                          <br>
-                                         Samples stored in 4 C.
+                                         Samples stored in 4 °C.
                                      </p>
                                      <p>
@@ Line 507: / Line 509: @@
                                      </p>
                                      <p>
-                                         Yesterday’s EcoRI restrictions were mixed with 0,5 ul MidoriGreen Direct (Nippon
+                                         Yesterday’s EcoRI restrictions were mixed with 0.5 µL MidoriGreen Direct (Nippon
-                                         Genetics) and 1 ul Midori Green Direct was added in 5 ul 1kb DNA Ladder (nippon Genetics).
+                                         Genetics) and 1 µL Midori Green Direct was added in 5 µL 1kb DNA Ladder (nippon Genetics).
                                          <br>
                                          The ladder, samples and the sample from entry "Making of pLK04R-Histag" is loaded
@@ Line 546: / Line 548: @@
                                                  with red) is cut from the gel around 3 kb. The GFP- TAG-RFP fragment (lane 5, marked
                                                  with red) is cut from the gel close to 2 kb even though it is supposed to be 1569
-                                                 bp long and should be closer to 1,5 kb strands.
+                                                 bp long and should be closer to 1.5 kb strands.
                                                  <br>
                                                  The DNA was purified with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> Nucleospin Gel and PCR Clean- up kit, </a>  according
@@ Line 555: / Line 557: @@
                                                  Results:
                                                  <br>
-                                                 pUC19: 1,2 ng/ul
+                                                 pUC19: 1.2 ng/µL
                                                  <br>
-                                                 GFP-tag-RFP: 14,8 ng/ul
+                                                 GFP-tag-RFP: 14.8 ng/µL
                                                  <br>
-                                                 pLK04R: 2,7 ng/ul
+                                                 pLK04R: 2.7 ng/µL
                                              </p>
                                              <p>
@@ Line 576: / Line 578: @@
                                                  and taken from inactivation at 11:50.
                                                  <br>
-                                                 After digestion each reaction mix was mixed with 0,5 ul Midori Green direct and
+                                                 After digestion each reaction mix was mixed with 0.5 µL Midori Green direct and
-ul Midori green Direct was added to 5 ul 1kb DNA ladder. Samples were loaded in
+µL Midori green Direct was added to 5 µL 1kb DNA ladder. Samples were loaded in
 % agarose gel as follows:
                                              </p>
@@ Line 584: / Line 586: @@
                                                      <tr>
                                                          <td>
-ul 1 kb ladder + 1 ul midori direct
+µL 1 kb ladder + 1 µL midori direct
                                                          </td>
                                                          <td>
-µl pUC19 (EcoRI) + 0,5 µl Midori direct
+µL pUC19 (EcoRI) + 0.5 µL Midori direct
                                                          </td>
                                                          <td>
-µl BBa_K567018 (EcoRI) + 0,5 µl midori direct
+µL BBa_K567018 (EcoRI) + 0.5 µL midori direct
                                                          </td>
                                                          <td>
-µl pLK04 (SfiI + HindIII)+ 0,5 µl midori direct
+µL pLK04 (SfiI + HindIII)+ 0.5 µL midori direct
                                                          </td>
                                                      </tr>
@@ Line 611: / Line 613: @@
                                                  pUC19 backbone (red box on the left) and BBa_K567018 (red box on the right) were
                                                  cut from the gel and purified with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> PCR clean-up Gel extraction kit </a>
-                                                 with 2 15 ul eluation in different tubes.
+                                                 with 2 15 µL eluation in different tubes.
                                              </p>
                                              <p>
@@ Line 634: / Line 636: @@
                                                          </td>
                                                          <td>
-,5 ng/ul
+.5 ng/µL
                                                          </td>
                                                          <td>
-,7 ng/ul
+.7 ng/µL
                                                          </td>
                                                      </tr>
@@ Line 645: / Line 647: @@
                                                          </td>
                                                          <td>
-,4 ng/ul
+.4 ng/µL
                                                          </td>
                                                          <td>
-,7 ng/ul
+.7 ng/µL
                                                          </td>
                                                      </tr>
@@ Line 656: / Line 658: @@
                                                          </td>
                                                          <td>
-,0 ng/ul
+.0 ng/µL
                                                          </td>
                                                          <td>
-,4 ng/ul
+.4 ng/µL
                                                          </td>
                                                      </tr>
@@ Line 711: / Line 713: @@
                                                          <td>
                                                              <b>
-                                                                 Total volume (ul)
+                                                                 Total volume (µL)
                                                              </b>
                                                          </td>
@@ Line 727: / Line 729: @@
                                                  Ligation started 15:40. Ended: 15:50.
                                                  <br>
-ul ligation mix was mixed with 35 ul electrocompetent E.coli 321.A cells. Electroporation
+µL ligation mix was mixed with 35 µL electrocompetent E.coli 321.A cells. Electroporation
                                                  was done with 1mm electroporation cuvette using BioRad Gene Pulsen Xcell electroporation
                                                  device with 1.8 kV voltage.
                                                  <br>
-                                                 After electroporation cells were put into 5 ml prewarmed (37 C) LB-medium and put
+                                                 After electroporation cells were put into 5 mL prewarmed (37 °C) LB-medium and put
-                                                 into 37 C, 250 rpm shaking for 40 minutes. Then cells were plated on a LA+chloramphenicol
+                                                 into 37 °C, 250 rpm shaking for 40 minutes. Then cells were plated on a LA+chloramphenicol
-                                                 plate and the plate was put to 37 C o/n.
+                                                 plate and the plate was put to 37 °C o/n.
                                              </p>
 <p> <b> Multiplying pUC19 and pLK04RCH-MLCR plasmids </b> </p>
-<p> Electrocompetent 321.A cells were electroporated with pUC19 or pLK04R plasmid. The cell mixture was transferred to LB medium and incubated on Multritron shaker at 37 oC, 300 rpm for 45 minutes.</p>
+<p> Electrocompetent 321.A cells were electroporated with pUC19 or pLK04R plasmid. The cell mixture was transferred to LB medium and incubated on Multritron shaker at 37 °C, 300 rpm for 45 minutes.</p>
-<p 200 µl of the cell mixture was plated onto LB-AIX plates.  </p>
+<p 200 µL of the cell mixture was plated onto LB-AIX plates.  </p>
                                              <p>
@@ Line 753: / Line 755: @@
                                              </p>
 <p> <b> Multiplying pUC19 and pLK04RCH-MLCR plasmids </b> </p>
-<p> One colony from each plate was used to inoculate 5 ml of LB with 100 µg/ml ampicillin and incubated on Multitron shaker at 37 °C, 300 rpm o/n. </p>
+<p> One colony from each plate was used to inoculate 5 mL of LB with 100 µg/mL ampicillin and incubated on Multitron shaker at 37 °C, 300 rpm o/n. </p>
 <p> <b> May 31st </b> </p>
 <p> <b> Multiplying pUC19 and pLK04RCH-MLCR plasmids </b> </p>
@@ Line 762: / Line 764: @@
 <p>DNA concentrations:
-<br> pUC19: 380,5 ng/µl
+<br> pUC19: 380.5 ng/µL
-<br> pLK04 original: 115,7 ng/µl </p>
+<br> pLK04 original: 115.7 ng/µL </p>
-<p>Glycerol preps were also made from both pLK04RCH+MLCR and pUC19 cultures. 167 µl of 80 % glycerol stock was added to 500 µl of cell suspension. The glycerol preps were stored at -80 oC.</p>
+<p>Glycerol preps were also made from both pLK04RCH+MLCR and pUC19 cultures. 167 µL of 80 % glycerol stock was added to 500 µL of cell suspension. The glycerol preps were stored at -80 °C.</p>
                                  </div>
@@ Line 792: / Line 794: @@
 <p> Present: Janniina, Pyry </p>
-<p> The plasmid pLK04RCHTag was extracted using <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid easyPure kit. </a>  The DNA-concentration measured with Nanodrop was 280,7 ng/µl. </p>
+<p> The plasmid pLK04RCHTag was extracted using <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid easyPure kit. </a>  The DNA-concentration measured with Nanodrop was 280.7 ng/µL. </p>
-<p> 321.A electrocompetent cells were electroporated with pLK04RCHTag(MLCR). 1 µl of DNA (104 ng/µl) was added to 30 µl of cell suspension and the mixture was electroporated. As a negative control we added MQ H2O instead of DNA to the cell mixture. The cell mixture was transferred to LB medium and incubated at 37 oC at 300 rpm for 1 hour. The cell mixture was then plated onto selective plates and incubated o/n at 37 oC. </p>
+<p> 321.A electrocompetent cells were electroporated with pLK04RCHTag(MLCR). 1 µL of DNA (stock concentration 104 ng/µL) was added to 30 µL of cell suspension and the mixture was electroporated. As a negative control we added MQ H2O instead of DNA to the cell mixture. The cell mixture was transferred to LB medium and incubated at 37 °C at 300 rpm for 1 hour. The cell mixture was then plated onto selective plates and incubated o/n at 37 °C. </p>
@@ Line 800: / Line 802: @@
 <p> Present: Pyry </p>
-<p> 5 colonies were inoculated in LB medium and incubated o/n at 37 oC at 300 rpm. </p>
+<p> 5 colonies were inoculated in LB medium and incubated o/n at 37 °C at 300 rpm. </p>
 <p> <b> June 6th </b> </p>
@@ Line 808: / Line 810: @@
 <p> DNA concentration of the extracted pLK04RCHTag-MLCR:
-<br> pLK04RCHTag-MLCR 1: 103.9 ng/µl
+<br> pLK04RCHTag-MLCR 1: 103.9 ng/µL
-<br>pLK04RCHTag-MLCR 2: 98.4 ng/µl
+<br>pLK04RCHTag-MLCR 2: 98.4 ng/µL
-<br>pLK04RCHTag-MLCR 3: 88 ng/µl </p>
+<br>pLK04RCHTag-MLCR 3: 88 ng/µL </p>
 <p> We digested the pLK04RCHTag-MLCR plasmid with SfiI restriction enzyme:</p>
@@ Line 823: / Line 825: @@
 </td>
 <td width="231">
-<p>&micro;l</p>
+<p>µL</p>
 </td>
 </tr>
@@ Line 869: / Line 871: @@
 </table> </p>
-<p> The reaction mixture was incubated at 50 oC for 15 minutes. The samples were run on an agarose gel at 70 V for 1 hour. </p>
+<p> The reaction mixture was incubated at 50 °C for 15 minutes. The samples were run on an agarose gel at 70 V for 1 hour. </p>
 <p>
@@ Line 896: / Line 898: @@
 <p>Present: Riku, Fanni, Pyry </p>
-<p> We got a new pUC19 plasmid because we were unsure what was in our old tube. We transformed the vector into XL-1electrocompetent cells, incubated on a shaker at 300 rpm, 37 oC for 1 hour and then plated the cell mixture onto selective plates and incubated o/n at 37 °C. </p>
+<p> We got a new pUC19 plasmid because we were unsure what was in our old tube. We transformed the vector into XL-1electrocompetent cells, incubated on a shaker at 300 rpm, 37 °C for 1 hour and then plated the cell mixture onto selective plates and incubated o/n at 37 °C. </p>
 <p> <b> June 12th </b> </p>
-<p> A single colony was picked and inoculated with 3 ml of LB with ampicillin and incubated o/n at 37 °C, 300 rpm. </p>
+<p> A single colony was picked and inoculated with 3 mL of LB with ampicillin and incubated o/n at 37 °C, 300 rpm. </p>
                                          <p>
@@ Line 912: / Line 914: @@
                                      <p>
-                                         We ordered a modified GFP-tag-RFP from IDT. Original part was BBa_K567018, but we
+                                         A modified GFP-tag-RFP was ordered from IDT. Original part was BBa_K567018, but we
                                          ordered it without the T7 promoter.
                                      </p>
@@ Line 998: / Line 1,000: @@
                                                  <td>
                                                      <b>
-                                                         V. tot. (ul)
+                                                         V. tot. (µL)
                                                      </b>
                                                  </td>
@@ Line 1,018: / Line 1,020: @@
                                      </p>
                                      <p>
-.5 ul Midori Green Direct was put on digestion reaction mixes and 1 ul Midori Green
+.5 µL Midori Green Direct was put on digestion reaction mixes and 1 µL Midori Green
-                                         Direct was added to 5ul 1kb DNA ladder. Samples were loaded on 1 % agarose gel. as
+                                         Direct was added to 5 µL 1kb DNA ladder. Samples were loaded on 1 % agarose gel. as
                                          follows:
                                      </p>
@@ Line 1,026: / Line 1,028: @@
                                              <tr>
                                                  <td>
-µl 1 kb ladder + 1 µl midori direct
+µL 1 kb ladder + 1 µL midori direct
                                                  </td>
                                                  <td>
-µl pUC19 (BamHI % AvaI) + 0,5 µl Midori direct
+µL pUC19 (BamHI % AvaI) + 0.5 µL Midori direct
                                                  </td>
                                                  <td>
-µl GFP-TAG-RFP (BamHI % AvaI) + 0,5 µl Midori direct
+µL GFP-TAG-RFP (BamHI % AvaI) + 0.5 µL Midori direct
                                                  </td>
                                              </tr>
@@ Line 1,063: / Line 1,065: @@
                                                  <td>
                                                      <b>
-                                                         ng/ul
+                                                         ng/µL
                                                      </b>
                                                  </td>
@@ Line 1,101: / Line 1,103: @@
                                                  <td>
                                                      <b>
-                                                         ul
+                                                         µL
                                                      </b>
                                                  </td>
@@ Line 1,168: / Line 1,170: @@
                                      </p>
                                      <p>
-ul of ligation mix were mixed with 100 µl chemically competent E.coli XL-1 Blue
+µL of ligation mix were mixed with 100 µL chemically competent E.coli XL-1 Blue
                                          cells. Transformation was incubated 2 min in ice and then put to 45 seconds in 42
-                                         C heat block. After heatshock cells were put on ice for 2 mins and then they were
+                                         °C heat block. After heatshock cells were put on ice for 2 mins and then they were
-                                         transferreed to 5 ml LB medium.
+                                         transferreed to 5 mL LB medium.
                                          <br>
-                                         Cells were incubated in 37 C 300 rpm shaker for 40 min and then transferred to two
+                                         Cells were incubated in 37 °C 300 rpm shaker for 40 min and then transferred to two
-                                         LA+ampicillin plates. 200 µl LB growth in first one and 1 ml to second one. Plates
+                                         LA+ampicillin plates. 200 µL LB growth in first one and 1 mL to second one. Plates
-                                         were incubated 37C overnight.
+                                         were incubated 37 °C overnight.
                                      </p>
 <p> <b> Multiplying pUC19 and pLK04RCH-MLCR plasmids </b> </p>
-<p> The pUC19 plasmid was extracted from the XL-1 electrocompetent cells using the <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> Nucleospin plasmid Easypure kit. </a>  The measured DNA concentration was 271,9 ng/µl. </p>
+<p> The pUC19 plasmid was extracted from the XL-1 electrocompetent cells using the <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> Nucleospin plasmid Easypure kit. </a>  The measured DNA concentration was 271.9 ng/µL. </p>
                                  </div>
@@ Line 1,205: / Line 1,207: @@
                                      <p>
                                          There were colonies in LA+ampicillin plates, but no GFP fluorescence. We forgot
-                                         to do ligation and transformation control for work done on Thursday 13 June, so we
+                                         to do ligation and transformation control for work done on Thursday 13th of June, so we
                                          had no idea why GFP-tag-RFP gene did not work.
                                          <br>
@@ Line 1,285: / Line 1,287: @@
                                                  <td>
                                                      <b>
-                                                         V. tot. (ul)
+                                                         V. tot. (µL)
                                                      </b>
                                                  </td>
@@ Line 1,305: / Line 1,307: @@
                                      </p>
                                      <p>
-                                         Electroporation mix was done by adding 1 ul ligation mix in 35 ul E.coli XL-1 blue
+                                         Electroporation mix was done by adding 1 µL ligation mix in 35 µL E.coli XL-1 blue
                                          electrocompetent in 1 mm electroporation cuvette (BioRad). Electroporation was done
-                                         with BioRad Gene Pulser Xcell with 1,8 kV voltage
+                                         with BioRad Gene Pulser Xcell with 1.8 kV voltage
                                      </p>
                                      <p>
-                                         Transformations were put to prewarmed (37C) LB medium and 300 rpm shaking in 37C.
+                                         Transformations were put to prewarmed (37 °C) LB medium and 300 rpm shaking in 37 °C.
                                          <br>
                                          Start: 12:06
@@ Line 1,317: / Line 1,319: @@
                                      </p>
                                      <p>
-                                         Both transformations were centrifuged 2000 g 5 min. Supernatant was thrown away
+                                         Both transformations were centrifuged 2000 g, 5 min. Supernatant was thrown away
-                                         and cells were resuspended in 300 ul LB medium. Medium was divided both 1 mM and
+                                         and cells were resuspended in 300 µL LB medium. Medium was divided both 1 mM and
-,5 mM IPTG plates (LA plates, with ampicillin) and incubated in 37 C o/n
+.5 mM IPTG plates (LA plates, with ampicillin) and incubated in 37 °C o/n
                                      </p>
                                      <p>
@@ Line 1,358: / Line 1,360: @@
                                              <tr>
                                                  <td>
-,5 mM
+.5 mM
                                                  </td>
                                                  <td>
@@ Line 1,375: / Line 1,377: @@
                                      </p>
                                      <p>
-colonies were picked from 0,5 mM pUC19+GRP-tag-RFP plate and put in LB with ampicillin.
+colonies were picked from 0.5 mM pUC19+GRP-tag-RFP plate and put in LB with ampicillin.
-                                         Tubes were put in 37 C 300 Rpm for o/n.
+                                         Tubes were put in 37 °C 300 Rpm for o/n.
                                          <p>
                                              <p>
@@ Line 1,397: / Line 1,399: @@
                                                          <td>
                                                              <b>
-                                                                 Yield (ng/ul)
+                                                                 Yield (ng/µL)
                                                              </b>
                                                          </td>
@@ Line 1,429: / Line 1,431: @@
                                              <p>
                                                  <b>
-                                                     GFP-tag-RFP Transformation in e.coli 321.A with pEVOL-pAzF
+                                                     GFP-tag-RFP Transformation in E.coli 321.A with pEVOL-pAzF
                                                  </b>
                                              </p>
@@ Line 1,437: / Line 1,439: @@
                                              </p>
                                              <p>
-µl E.coli 321A cells with:
+µL E.coli 321A cells with:
                                                  <br>
                                                  <table style="width:5%">
@@ Line 1,443: / Line 1,445: @@
                                                          <td>
                                                              <b>
-                                                                 pEVOL- pAzF (ul)
+                                                                 pEVOL- pAzF (µL)
                                                              </b>
                                                          </td>
                                                          <td>
                                                              <b>
-                                                                 pUC19 - GFP-tag-RFP (ul)
+                                                                 pUC19 - GFP-tag-RFP (µL)
                                                              </b>
                                                          </td>
@@ Line 1,483: / Line 1,485: @@
                                              </p>
                                              <p>
-                                                 After electroporation cells were transferred in 5 ml LB medium and incubated in
+                                                 After electroporation cells were transferred in 5 mL LB medium and incubated in
-C 250 rpm shaking. Incubation started: 11:25 and ended: 12:35
+°C 250 rpm shaking. Incubation started: 11:25 and ended: 12:35
                                              </p>
                                              <p>
@@ Line 1,512: / Line 1,514: @@
                                                          </td>
                                                          <td>
-ul and 1/100 200 ul
+µL and 1/100 200 µL
                                                          </td>
                                                          <td>
@@ Line 1,524: / Line 1,526: @@
                                                          </td>
                                                          <td>
-ul
+µL
                                                          </td>
                                                          <td>
@@ Line 1,536: / Line 1,538: @@
                                                          </td>
                                                          <td>
-ul
+µL
                                                          </td>
                                                          <td>
-ul
+µL
                                                          </td>
                                                      </tr>
@@ Line 1,555: / Line 1,557: @@
                                              </p>
                                              <p>
-                                                 Before we could start work with pUC19 it had to be cloned as we got only 2 ul of
+                                                 Before we could start work with pUC19 it had to be cloned as we got only 2 µL of
                                                  commercial pUC19.
                                              </p>
@@ Line 1,582: / Line 1,584: @@
                                                          </td>
                                                          <td>
-ul
+µL
                                                          </td>
                                                          <td>
-ul
+µL
                                                          </td>
                                                      </tr>
                                                      <tr>
                                                          <td>
-                                                             pUC19 (100pg/ul)
+                                                             pUC19 (100 pg/µL)
                                                          </td>
                                                          <td>
-ul
+µL
                                                          </td>
                                                          <td>
-ul
+µL
                                                          </td>
                                                      </tr>
@@ Line 1,604: / Line 1,606: @@
                                                          </td>
                                                          <td>
-ul
+µL
                                                          </td>
                                                          <td>
-ul
+µL
                                                          </td>
                                                      </tr>
@@ Line 1,613: / Line 1,615: @@
                                              </p>
                                              <p>
-                                                 Transformation was done in 1 mm electroporation cuvette using 1,8 kV with BioRad
+                                                 Transformation was done in 1 mm electroporation cuvette using 1.8 kV with BioRad
                                                  Gene Pulser Xcell.
                                                  <br>
-                                                 XL-1 Blue pUC19: T: 4,8
+                                                 XL-1 Blue pUC19: T: 4.8
                                                  <br>
-                                                 XL-1 Blue mQ control T: 5,0
+                                                 XL-1 Blue mQ control T: 5.0
                                              </p>
                                              <p>
-                                                 After electroporation, cells were transferred to 5 ml LB medium and incubation in
+                                                 After electroporation, cells were transferred to 5 mL LB medium and incubation in
-C, 250 rpm shaking was started: 16:20 and ended: 17:10.
+°C, 250 rpm shaking was started: 16:20 and ended: 17:10.
                                              </p>
                                              <p>
@@ Line 1,628: / Line 1,630: @@
                                              </p>
                                              <p>
-: 200 ul E.coli XL-1 Blue pUC19 transformant
+: 200 µL E.coli XL-1 Blue pUC19 transformant
                                                  <br>
-: 100 ul E.coli XL-1 Blue pUC19 transformant
+: 100 µL E.coli XL-1 Blue pUC19 transformant
                                                  <br>
-: 100 µl E.coli XL-1 Blue Control/background
+: 100 µL E.coli XL-1 Blue Control/background
                                              </p>
                                              <p>
-                                                 Plates were put to 37 C o/n
+                                                 Plates were put to 37 °C o/n
                                              </p>
                                              <p>
@@ Line 1,654: / Line 1,656: @@
                                              <p>
 colonies from plate 1 and 1 colony from plate 2 was used to start cultures in
-ml LB medium with 100 µg/ml ampicillin.
+mL LB medium with 100 µg/mL ampicillin.
                                              </p>
                                              <p>
@@ Line 1,668: / Line 1,670: @@
                                              </p>
                                              <p>
-µl growth
+µL growth
                                                  <br>
-µl 80 % glycerol
+µL 80 % glycerol
                                              </p>
                                              <p>
@@ Line 1,693: / Line 1,695: @@
                                                          <td>
                                                              <b>
-                                                                 DNA concentration (ng/ul)
+                                                                 DNA concentration (ng/µL)
                                                              </b>
                                                          </td>
@@ Line 1,761: / Line 1,763: @@
                                                  <td>
                                                      <b>
-                                                         GFP-tag-RFP (in pUC18) (ul)
+                                                         GFP-tag-RFP (in pUC18) (µL)
                                                      </b>
                                                  </td>
                                                  <td>
                                                      <b>
-                                                         pUC19 (ul)
+                                                         pUC19 (µL)
                                                      </b>
                                                  </td>
@@ Line 1,845: / Line 1,847: @@
                                      </p>
                                      <p>
-                                         Incubation in 37C started: 11:31 end: 11:30.
+                                         Incubation in 37 °C started: 11:31 end: 11:30.
                                      </p>
                                      <p>
-.5 ul Midori Green Direct was added to all the digestion reactions and 1 ul Midori
+.5µL Midori Green Direct was added to all the digestion reactions and 1 µL Midori
                                          Green Direct was added to 1 kb DNA Ladder (Nippon Genetics).
                                      </p>
@@ Line 1,911: / Line 1,913: @@
                                                  </td>
                                                  <td>
-.3 ul
+.3µL
                                                  </td>
                                                  <td>
-.3 ul
+.3µL
                                                  </td>
                                              </tr>
                                              <tr>
                                                  <td>
-                                                     GFP-tag-RFP insert (17,1 ng/µl)
+                                                     GFP-tag-RFP insert (17.1 ng/µL)
                                                  </td>
                                                  <td>
@@ Line 1,930: / Line 1,932: @@
                                              <tr>
                                                  <td>
-                                                     pUC19 backbone (59,2 ng/µl)
+                                                     pUC19 backbone (59.2 ng/µL)
                                                  </td>
                                                  <td>
@@ Line 1,964: / Line 1,966: @@
                                                  <td>
                                                      <b>
-                                                         V. tot. (ul)
+                                                         V. tot. (µL)
                                                      </b>
                                                  </td>
@@ Line 1,997: / Line 1,999: @@
                                      <p>
                                          Chemically competent XL-1 Blue tube was put on ice to melt. When it was melted 50
-                                         µl cells were put in 2 centrifugetubes that rested on ice. 5 µl previously ligated
+                                         µL cells were put in 2 centrifugetubes that rested on ice. 5 µL previously ligated
                                          (pUC19 + GFP-tag-RFP and pUC19 + nothing) reactions was mixed in with cells.
                                      </p>
@@ Line 2,008: / Line 2,010: @@
                                      </p>
                                      <p>
-                                         Cells were put on 42 C heatblock for 45 s and then put back into ice.
+                                         Cells were put on 42 °C heatblock for 45 s and then put back into ice.
                                      </p>
                                      <p>
-                                         After approx. 2 mins cells were put on 5 ml prewarmed to 37C LB medium and put into
+                                         After approx. 2 mins cells were put on 5 mL prewarmed to 37 °C LB medium and put into
-                                         shaking for 250 rpm in 37 C.
+                                         shaking for 250 rpm in 37 °C.
                                      </p>
                                      <p>
@@ Line 2,024: / Line 2,026: @@
                                      </p>
                                      <p>
-                                         Cells were plated on 4 different LA+ampicillin (100 µg/ml) dishes as follows:
+                                         Cells were plated on 4 different LA+ampicillin (100 µg/mL) dishes as follows:
                                      </p>
                                      <p>
@@ Line 2,031: / Line 2,033: @@
                                                  <td>
                                                      <b>
-                                                         Amount (ul)
+                                                         Amount (µL)
                                                      </b>
                                                  </td>
@@ Line 2,075: / Line 2,077: @@
                                      </p>
                                      <p>
-                                         Plates were left o/n in 37 C
+                                         Plates were left o/n in 37 °C
                                      </p>
                                      <p>
@@ Line 2,090: / Line 2,092: @@
                                                  <td>
                                                      <b>
-                                                         Amount plated (ul)
+                                                         Amount plated (µL)
                                                      </b>
                                                  </td>
@@ Line 2,155: / Line 2,157: @@
                                      </p>
                                      <p>
-                                         Incubation in 37 C overnight.
+                                         Incubation in 37 °C overnight.
                                      </p>
                                      <p>
@@ Line 2,169: / Line 2,171: @@
                                      </p>
                                      <p>
-                                         In plate 2 there are 2 colonies with fluorescence. 1 is picked in 5 ml liquid culture
+                                         In plate 2 there are 2 colonies with fluorescence. 1 is picked in 5 mL liquid culture
                                          (LB + ampicillin 100µg/ml) for o/n incubation in 37C 300 rpm.
                                      </p>
@@ Line 2,184: / Line 2,186: @@
                                          o/n culture was centrifuged in 2000 g for 10 min and supernatant was thrown away.
                                          New liquid culture for glycerol stock was started by inoculating from pellet into
-ml LB+ampicillin.
+mL LB+ampicillin.
                                      </p>
                                      <p>
                                          Rest of the pellet was used to extract plasmid with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid
-                                         -kit. </a> Elution was done 2x with 25 µl Elution buffer and 5 min 70 C incubation.
+                                         -kit. </a> Elution was done 2x with 25 µL Elution buffer and 5 min 70 °C incubation.
                                      </p>
                                      <p>
                                          Concentration was measured with ND-1000:
                                          <br>
-                                         pUC19 + GFP-TAG-RFP: 579,5 ng/µl
+                                         pUC19 + GFP-TAG-RFP: 579,5 ng/µL
                                      </p>
                                  </div>
@@ Line 2,225: / Line 2,227: @@
                                      </p>
                                      <p>
-.A was transformed with 0,5 µl of pEVOL-pAzF and pUC19+GFP-tag-RFP plasmids.
+.A was transformed with 0.5 µL of pEVOL-pAzF and pUC19+GFP-tag-RFP plasmids.
                                      </p>
                                      <p>
@@ Line 2,311: / Line 2,313: @@
                                      </p>
                                      <p>
-                                         After electroporation cells were put into LB medium and shaking 300 rm in 37 C for
+                                         After electroporation cells were put into LB medium and shaking 300 rm in 37 °C for
 min.
                                      </p>
@@ Line 2,326: / Line 2,328: @@
                                      </p>
                                      <p>
-growths from o/n LA plates were each used to inoculate 4 ml LB with ampicillin,
+growths from o/n LA plates were each used to inoculate 4 mL LB with ampicillin,
                                          chloramphenicol, zeocin and glucose. They were then put to grow o/n in 37°C 300rpm.
                                      </p>
@@ Line 2,339: / Line 2,341: @@
                                      <p>
 .A with pEVOL-pAzF and pUC19(GFP-TAG-RFP) was re-plated in order to get separate
-                                         colonies. Plates with 1/100 (large plate) and 1/1000 (smal plate) dilutions were
+                                         colonies. Plates with 1/100 (large plate) and 1/1000 (small plate) dilutions were
-                                         incubated o/n at 37°C.
+                                         incubated o/n at 37 °C.
                                      </p>
                                      <p>
@@ Line 2,352: / Line 2,354: @@
                                      <p>
                                          From 321.A with pEVOL-pAzF and pUC19(GFP-TAG-RFP) LA plate, 3 colonies were picked
-                                         to inoculate 4 ml LB with ampicillin, chloramphenicol, zeocin and 0.5% glucose to
+                                         to inoculate 4 mL LB with ampicillin, chloramphenicol, zeocin and 0.5 % glucose to
-                                         grow o/n in 37°C and 300rpm.
+                                         grow o/n in 37 °C and 300 rpm.
                                          <p>
                                              <p>
@@ Line 2,365: / Line 2,367: @@
                                                          <td>
                                                              <b>
-                                                                 E.coli XL-1 Blue cells (ul)
+                                                                 E.coli XL-1 Blue cells (µL)
                                                              </b>
                                                          </td>
                                                          <td>
                                                              <b>
-                                                                 pEVOL-pAzF (ul)
+                                                                 pEVOL-pAzF (µL)
                                                              </b>
                                                          </td>
                                                          <td>
                                                              <b>
-                                                                 pUC19-GFP-tag-RFP (ul)
+                                                                 pUC19-GFP-tag-RFP (µL)
                                                              </b>
                                                          </td>
@@ Line 2,427: / Line 2,429: @@
                                                  All transformation T's were 5.0.
                                                  <br>
-                                                 After transformation cells were put into 5 ml LB in 37 C 300 rpm shaking for 1 hour.
+                                                 After transformation cells were put into 5 mL LB in 37 °C, 300 rpm shaking for 1 hour.
                                              </p>
                                              <p>
@@ Line 2,443: / Line 2,445: @@
                                                          <td>
                                                              <b>
-                                                                 No dilution (ul)
+                                                                 No dilution (µL)
                                                              </b>
                                                          </td>
                                                          <td>
                                                              <b>
-/100 (ul)
+/100 (µL)
                                                              </b>
                                                          </td>
@@ Line 2,486: / Line 2,488: @@
                                              </p>
                                              <p>
-µl XL-1 blue + pUC19-GFP-tag-RFP transformation was put into LA+ampicillin+tetracyclin+IPTG
+µL XL-1 blue + pUC19-GFP-tag-RFP transformation was put into LA+ampicillin+tetracyclin+IPTG
                                                  dish.
                                              </p>
                                              <p>
-                                                 All dished were put into 37 C o/n.
+                                                 All dished were put into 37 °C o/n.
                                              </p>
                                              <p>
@@ Line 2,515: / Line 2,517: @@
 colonies 321.A pEVOL+pAzF - pUC19+GFP-tag-RFP and 2 colonies XL-1 GFP-tag-RFP
                                                  was inoculated into LB culture with ampicillin, chloramphenicol and zeocin (321.A
-                                                 strain) or tetracycline (XL-1 Blue strain) in 30 C 300 rpm for o/n.
+                                                 strain) or tetracycline (XL-1 Blue strain) in 30 °C 300 rpm for o/n.
                                              </p>
                                  </div>
@@ Line 2,579: / Line 2,581: @@
                                      </p>
                                      <p>
-                                         To make 0.1 OD600 cultures from o/n cultures, 160 µl of 321.A culture and 180 µl
+                                         To make 0.1 OD600 cultures from o/n cultures, 160 µL of 321.A culture and 180 µL
-                                         of XL-1 Blue culture were transferred to 4 ml LB containing ampicillin, 0,5 % glucose
+                                         of XL-1 Blue culture were transferred to 4 mL LB containing ampicillin, 0.5 % glucose
                                          and tetracycline (for XL-1 Blue) or chloramphenicol (for 321.A). Glucose was added
                                          to inhibit Lac-Promoter.
@@ Line 2,623: / Line 2,625: @@
                                      </p>
                                      <p>
-,9 ml of XL-1 culture and 1,5 ml of 321.A culture was put in 2 separate tubes to
+.9 mL of XL-1 culture and 1.5 mL of 321.A culture was put in 2 separate tubes to
                                          start new test and control cultures so that in total there were 4 tubes. Cultures
-                                         were centrifuged 2000g for 15 min and pellets were resuspended into 4 ml of growth
+                                         were centrifuged 2000g for 15 min and pellets were resuspended into 4 mL of growth
                                          medium that contained: LB, ampicillin and tetracycline (for XL-1 Blue) or chloramphenicol
                                          (for 321.A). In addition to antibiotics on tube containing XL-1 Blue and One tube
-                                         containing 321.A had 0,1 mM pAzF and 1 % arabinose in medium. After about 10 min
+                                         containing 321.A had 0.1 mM pAzF and 1 % arabinose in medium. After about 10 min
                                          of 26 °C, 300 rpm incubation 0.1 M IPTG was added to test cultures to total IPTG
                                          concentration of 0.1 mM.
@@ Line 2,687: / Line 2,689: @@
                                      </p>
                                      <p>
-                                         The cells were centrifuged (16100g, 2 min) and supernatant was transferred to other
+                                         The cells were centrifuged (16100 g, 2 min) and supernatant was transferred to other
                                          microcentrifuge tube. Pellets and supernatants were stored at -20 °C.
                                      </p>
@@ Line 2,727: / Line 2,729: @@
 <p> <b> Gel electrophoresis </b> </p>
-<p> We made 500 ml of 1 x TBS (Medicago AB, Uppsala) and 1 L of 1 x TBE (Medicago). </p>
+<p> We made 500 mL of 1 x TBS (Medicago AB, Uppsala) and 1 L of 1 x TBE (Medicago). </p>
-<p> We made 1 % and 2 % agarose gels into 60 ml of 1 x TBE. 3,6 µl of Midori Green Advance DNA Stain was added into each 60 ml of agarose gel. </p>
+<p> We made 1 % and 2 % agarose gels into 60 mL of 1 x TBE. 3,6 µL of Midori Green Advance DNA Stain was added into each 60 mL of agarose gel. </p>
 <p> <b> Restrictions and ligations </b> </p>
 <p>The DNA was centrifuged to get the pellet into the bottom.
-µl of MQ was added into each tube containing the ordered DNA. </p>
+µL of MQ was added into each tube containing the ordered DNA. </p>
 <p><b> Ordered DNA: </b> </p>
@@ Line 2,746: / Line 2,748: @@
 <br>Digox CL Lys 191->TAG</p>
-<p> 0.1kb fragments:
+<p> 0.1 kb fragments:
 <br>SfiI and HindIII restriction (2 % agarose gel) </p>
@@ Line 2,760: / Line 2,762: @@
 </td>
 <td width="73">
-<p>SfiI (&micro;l)</p>
+<p>SfiI (µL)</p>
 </td>
 <td width="138">
-<p>SfiI + HindIII (&micro;l)</p>
+<p>SfiI + HindIII (µL)</p>
 </td>
 <td width="22">
@@ Line 2,863: / Line 2,865: @@
 <p>Double digestion for 0.1kb fragments:
 <br>15 min at 50 °C for SfiI
-<br>1h+ at 37 °C for HindIII
+<br>1h at 37 °C for HindIII
 <br>20 min at 80 °C to inactivate HindIII </p>
@@ Line 2,870: / Line 2,872: @@
 <p> <b> Electrophoresis: </b>
-<br>1.45kb fragments with 5 µl Loading dye (Thermo Fisher)
+<br>1.45 kb fragments with 5 µL Loading dye (Thermo Fisher)
 <br>Ladder: FastGene 50bp DNA Ladder (NIPPON Genetics Europe GmbH, lot:10 1028 2807 01)
 <br>70 V
@@ Line 2,940: / Line 2,942: @@
 <p> <b> Insert purification from gel </b>
-<br>DNA bands from gel 1 at 1.5kb and 2 at 0.1kb were purified with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> Macherey-Nagel NucleoSpin Gel and PCR Clean-up -kit </a> with manufacturer’s guide. Elution was done twice for samples 1-4 from 1% agarose gel, first time using 30 µl and second time using 15 µl elution buffer. For samples from 2 % agarose gel elution was done only once with 30 µl elution buffer. </p>
+<br>DNA bands from gel 1 at 1.5kb and 2 at 0.1kb were purified with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> Macherey-Nagel NucleoSpin Gel and PCR Clean-up -kit </a> with manufacturer’s guide. Elution was done twice for samples 1-4 from 1% agarose gel, first time using 30 µL and second time using 15 µL elution buffer. For samples from 2 % agarose gel elution was done only once with 30 µL elution buffer. </p>
 <p> <b> Preparing 1% agarose gel for plasmid </b>
-<br>0,6 g agarose
+<br>0.6 g agarose
-<br>0,6 ml TBE
+<br>0.6 mL TBE
-<br>6 µl MidoriGreen </p>
+<br>6 µL MidoriGreen </p>
@@ Line 2,969: / Line 2,971: @@
 </td>
 <td width="72">
-<p>22,5 &micro;l</p>
+<p>22,5 µL</p>
 </td>
 <td width="141">
-<p>19.5 &micro;l</p>
+<p>19.5 µL</p>
 </td>
 </tr>
@@ Line 2,980: / Line 2,982: @@
 </td>
 <td width="72">
-<p>3 &micro;l</p>
+<p>3 µL</p>
 </td>
 <td width="141">
-<p>3 &micro;l</p>
+<p>3 µL</p>
 </td>
 </tr>
 <tr>
 <td width="186">
-<p>Plasmid DNA (350 ng/ul)</p>
+<p>Plasmid DNA (350 ng/µL)</p>
 </td>
 <td width="72">
-<p>3 &micro;l</p>
+<p>3 µL</p>
 </td>
 <td width="141">
-<p>3 &micro;l</p>
+<p>3 µL</p>
 </td>
 </tr>
@@ Line 3,002: / Line 3,004: @@
 </td>
 <td width="72">
-<p>1.5 &micro;l</p>
+<p>1.5 µL</p>
 </td>
 <td width="141">
-<p>1.5 &micro;l</p>
+<p>1.5 µL</p>
 </td>
 </tr>
@@ Line 3,013: / Line 3,015: @@
 </td>
 <td width="72">
-<p>0 &micro;l</p>
+<p>0 µL</p>
 </td>
 <td width="141">
-<p>3 &micro;l</p>
+<p>3 µL</p>
 </td>
 </tr>
@@ Line 3,024: / Line 3,026: @@
 </td>
 <td width="72">
-<p>30 &micro;l</p>
+<p>30 µL</p>
 </td>
 <td width="141">
-<p>30 &micro;l</p>
+<p>30 µL</p>
 </td>
 </tr>
@@ Line 3,037: / Line 3,039: @@
 <p> <b>Plasmid purification </b>
 <br>Agarose gel sample preparation:
-<br>5 µl of loading dye was added to restriction reaction mixes </p>
+<br>5 µL of loading dye was added to restriction reaction mixes </p>
 <p> Agarose gel was loaded as follows:
@@ Line 3,045: / Line 3,047: @@
 <tr>
 <td width="119">
-<p>5 &micro;l 1 kb Ladder (nippon genetics)</p>
+<p>5 µL 1 kb Ladder (nippon genetics)</p>
 </td>
 <td width="113">
-<p>20 &micro;l Sfil pEBO4CH (1.)</p>
+<p>20 µL Sfil pEBO4CH (1.)</p>
 </td>
 <td width="113">
-<p>15 &micro;l Sfil pEBO4CH (2.)</p>
+<p>15 µL Sfil pEBO4CH (2.)</p>
 </td>
 <td width="129">
-<p>20 &micro;l Sfil + HindIII pEBO4CH (3.)</p>
+<p>20 µL Sfil + HindIII pEBO4CH (3.)</p>
 </td>
 <td width="129">
-<p>15 &micro;l Sfil + HindIII pEBO4CH (4.)</p>
+<p>15 µL Sfil + HindIII pEBO4CH (4.)</p>
 </td>
 </tr>
@@ Line 3,074: / Line 3,076: @@
 <p> Purifying with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> Macherey-Nagel NucleoSpin Gel and PCR Clean-up -kit,</a>
-  with manufacturer’s guide with 2 eluation steps using 30 µl elution buffer both times. </p>
+  with manufacturer’s guide with 2 eluation steps using 30 µL elution buffer both times. </p>
 <p> Tubes 1 and 2 were pooled as well as tubes 3 and 4 </p>
@@ Line 3,092: / Line 3,094: @@
 </td>
 <td width="54">
-<p>ng/ul</p>
+<p>ng/µL</p>
 </td>
 </tr>
@@ Line 3,100: / Line 3,102: @@
 </td>
 <td width="54">
-<p>8,0</p>
+<p>8.0</p>
 </td>
 </tr>
@@ Line 3,108: / Line 3,110: @@
 </td>
 <td width="54">
-<p>8,4</p>
+<p>8.4</p>
 </td>
 </tr>
@@ Line 3,116: / Line 3,118: @@
 </td>
 <td width="54">
-<p>9,9</p>
+<p>9.9</p>
 </td>
 </tr>
@@ Line 3,124: / Line 3,126: @@
 </td>
 <td width="54">
-<p>6,9</p>
+<p>6.9</p>
 </td>
 </tr>
@@ Line 3,132: / Line 3,134: @@
 </td>
 <td width="54">
-<p>7,3</p>
+<p>7.3</p>
 </td>
 </tr>
@@ Line 3,140: / Line 3,142: @@
 </td>
 <td width="54">
-<p>7,0</p>
+<p>7.0</p>
 </td>
 </tr>
@@ Line 3,148: / Line 3,150: @@
 </td>
 <td width="54">
-<p>7,3</p>
+<p>7.3</p>
 </td>
 </tr>
@@ Line 3,156: / Line 3,158: @@
 </td>
 <td width="54">
-<p>9,5</p>
+<p>9.5</p>
 </td>
 </tr>
@@ Line 3,174: / Line 3,176: @@
 </td>
 <td width="125">
-<p>0.1kb insert (&micro;l)</p>
+<p>0.1 kb insert (µL)</p>
 </td>
 </tr>
@@ Line 3,182: / Line 3,184: @@
 </td>
 <td width="129">
-<p>2.5 &micro;l</p>
+<p>2.5 µL</p>
 </td>
 <td width="125">
-<p>2.5 &micro;l</p>
+<p>2.5 µL</p>
 </td>
 </tr>
@@ Line 3,193: / Line 3,195: @@
 </td>
 <td width="129">
-<p>3 &micro;l</p>
+<p>3 µL</p>
 </td>
 <td width="125">
-<p>1 &micro;l</p>
+<p>1 µL</p>
 </td>
 </tr>
@@ Line 3,204: / Line 3,206: @@
 </td>
 <td width="129">
-<p>2 &micro;l</p>
+<p>2 µL</p>
 </td>
 <td width="125">
-<p>2 &micro;l</p>
+<p>2 µL</p>
 </td>
 </tr>
@@ Line 3,215: / Line 3,217: @@
 </td>
 <td width="129">
-<p>0.5 &micro;l</p>
+<p>0.5 µL</p>
 </td>
 <td width="125">
-<p>0.5 &micro;l</p>
+<p>0.5 µL</p>
 </td>
 </tr>
@@ Line 3,226: / Line 3,228: @@
 </td>
 <td width="129">
-<p>12 &micro;l</p>
+<p>12 µL</p>
 </td>
 <td width="125">
-<p>14 &micro;l</p>
+<p>14 µL</p>
 </td>
 </tr>
@@ Line 3,237: / Line 3,239: @@
 </td>
 <td width="129">
-<p>20 &micro;l</p>
+<p>20 µL</p>
 </td>
 <td width="125">
-<p>20 &micro;l</p>
+<p>20 µL</p>
 </td>
 </tr>
@@ Line 3,414: / Line 3,416: @@
 </td>
 <td width="135">
-<p>12.5 &micro;l</p>
+<p>12.5 µL</p>
 </td>
 </tr>
@@ Line 3,422: / Line 3,424: @@
 </td>
 <td width="135">
-<p>3 &micro;l</p>
+<p>3 µL</p>
 </td>
 </tr>
 <tr>
 <td width="186">
-<p>Plasmid DNA (104 ng/ul)</p>
+<p>Plasmid DNA (104 ng/µL)</p>
 </td>
 <td width="135">
-<p>10 &micro;l</p>
+<p>10 µL</p>
 </td>
 </tr>
@@ Line 3,438: / Line 3,440: @@
 </td>
 <td width="135">
-<p>1.5 &micro;l</p>
+<p>1.5 µL</p>
 </td>
 </tr>
@@ Line 3,446: / Line 3,448: @@
 </td>
 <td width="135">
-<p>3 &micro;l</p>
+<p>3 µL</p>
 </td>
 </tr>
@@ Line 3,454: / Line 3,456: @@
 </td>
 <td width="135">
-<p>30 &micro;l</p>
+<p>30 µL</p>
 </td>
 </tr>
@@ Line 3,461: / Line 3,463: @@
 <p>Restriction incubation:
-<br>50 C: started 11:50 ended 12:50
+<br>50 °C: started 11:50 ended 12:50
-<br>37 C: started 12:54 ended </p>
+<br>37 °C: started 12:54 ended </p>
 <p> Gel purification according to <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> Macherey-Nagel gel purification kit protocol. </a>
@@ Line 3,490: / Line 3,492: @@
 <tr>
 <td width="77">
-<p>12,5 &micro;l</p>
+<p>12,5 µL</p>
 </td>
 <td width="234">
@@ Line 3,498: / Line 3,500: @@
 <tr>
 <td width="77">
-<p>2 &micro;l</p>
+<p>2 µL</p>
 </td>
 <td width="234">
@@ Line 3,506: / Line 3,508: @@
 <tr>
 <td width="77">
-<p>3 &micro;l</p>
+<p>3 µL</p>
 </td>
 <td width="234">
@@ Line 3,514: / Line 3,516: @@
 <tr>
 <td width="77">
-<p>2,5 &micro;l</p>
+<p>2.5 µL</p>
 </td>
 <td width="234">
-<p>Enzyme (1 &micro;l SfiI + 1,5 HindIII)</p>
+<p>Enzyme (1 µL SfiI + 1.5 HindIII)</p>
 </td>
 </tr>
 <tr>
 <td width="77">
-<p>20 &micro;l</p>
+<p>20 µL</p>
 </td>
 <td width="234">
@@ Line 3,535: / Line 3,537: @@
 <tr>
 <td width="62">
-<p>0 &micro;l</p>
+<p>0 µL</p>
 </td>
 <td width="249">
@@ Line 3,543: / Line 3,545: @@
 <tr>
 <td width="62">
-<p>6 &micro;l</p>
+<p>6 µL</p>
 </td>
 <td width="249">
@@ Line 3,551: / Line 3,553: @@
 <tr>
 <td width="62">
-<p>30 &micro;l</p>
+<p>30 µL</p>
 </td>
 <td width="249">
-<p>DNA (pEB04) (8ng/&micro;l)</p>
+<p>DNA (pEB04) (8ng/µL)</p>
 </td>
 </tr>
 <tr>
 <td width="62">
-<p>3 &micro;l</p>
+<p>3 µL</p>
 </td>
 <td width="249">
@@ Line 3,567: / Line 3,569: @@
 <tr>
 <td width="62">
-<p>39 &micro;l</p>
+<p>39 µL</p>
 </td>
 <td width="249">
@@ Line 3,580: / Line 3,582: @@
 <br> 1kb ladder (Nippon); pEB04CH HindIII SfiI; pLk04 HindIII SfiI; pEB04CH Hind SfiIII; pLk04 HindIII SfiI </p>
-<p> Purifying with <a href=" https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> NucleoSpin Gel and PCR Clean-up -kit,  </a> according to the manufacturer’s guide with 1 eluation step using 30 ul elution buffer. </p>
+<p> Purifying with <a href=" https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> NucleoSpin Gel and PCR Clean-up -kit,  </a> according to the manufacturer’s guide with 1 eluation step using 30 µL elution buffer. </p>
 <p> <b> Concentration of the samples were: </b>
-<br> pEB04CH HindIII SfiI (done on saturday) 0,5 ng/ul
+<br> pEB04CH HindIII SfiI (done on saturday) 0.5 ng/µL
-<br> pLk04 HindIII SfiI (done on saturday) 0,8 ng/ul
+<br> pLk04 HindIII SfiI (done on saturday) 0.8 ng/µL
-<br> pEB04CH HindIII SfiI (done on friday) 0,9 ng/ul
+<br> pEB04CH HindIII SfiI (done on friday) 0.9 ng/µL
-<br> pLk04 HindIII SfiI (done on friday) 1,6 ng/ul </p>
+<br> pLk04 HindIII SfiI (done on friday) 1.6 ng/µL </p>
 <p> <b> Ligation </b> </p>
-<p> Digestion mix was added 4,5 µl to 5 µl of vector. Control without digestion mix also done. <a href=" https://www.thermofisher.com/order/catalog/product/EL0014#/EL0014 "> Thermo T4 DNA ligase </a> instructions were followed. </p>
+<p> Digestion mix was added 4.5 µL to 5 µL of vector. Control without digestion mix also done. <a href=" https://www.thermofisher.com/order/catalog/product/EL0014#/EL0014 "> Thermo T4 DNA ligase </a> instructions were followed. </p>
 <p> Afterwards, the ligation reaction was electroporated. </p>
@@ Line 3,603: / Line 3,605: @@
 <p> Plates 1 and 3 has significantly more growth than 2, 4 or bg. </p>
-<p> 5 ml LB culturing (LB medium with 100 µg/ml ampicillin) were started from growth plates. 3 colonies were picked from plate 3 and 1 colony from plate 1. Cultures were incubated o/n 37 C 200 rpm incubator. </p>
+<p> 5 mL LB culturing (LB medium with 100 µg/ml ampicillin) were started from growth plates. 3 colonies were picked from plate 3 and 1 colony from plate 1. Cultures were incubated o/n 37 °C 200 rpm incubator. </p>
 <p> <b> May 28th </b> </p>
@@ Line 3,611: / Line 3,613: @@
 <p> o/n cultures were fuged in 3000 rpm for 5 minutes. Supernatant was tossed away. </p>
-<p> New 5 ml cultures were started by inoculating pellet into a new 5 ml LB+ampicillin (100 µg/ml) medium. Cultures were set to incubate in 37C 200 rpm shaking. </p>
+<p> New 5 mL cultures were started by inoculating pellet into a new 5 mL LB+ampicillin (100 µg/ml) medium. Cultures were set to incubate in 37 °C 200 rpm shaking. </p>
 <p> Rest of the pellet was used to extract plasmid from the cells with <a href=" https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf "> NucleoSpin Plasmid EasyPure -kit </a> with manufacturer’s instruction. </p>
@@ Line 3,624: / Line 3,626: @@
 </td>
 <td width="174">
-<p>c (&micro;g/ml)</p>
+<p>c (&micro;g/mL)</p>
 </td>
 </tr>
@@ Line 3,667: / Line 3,669: @@
 <p> 20% Glycerol preps were made from o/n cultures.
-<br>500 µl culture medium
+<br>500 µL culture medium
-<br>80% 170 µl Glycerol
+<br>80% 170 µL Glycerol
-<br>Tubes were vortexed gently and put to -70 C freezer </p>
+<br>Tubes were vortexed gently and put to -70 °C freezer </p>
@@ Line 3,688: / Line 3,690: @@
 </td>
 <td width="58">
-<p>ng/&micro;l</p>
+<p>ng/µL</p>
 </td>
 </tr>
@@ Line 3,711: / Line 3,713: @@
 <p> Purified plasmids were digested with SfiI according to <a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012585_FastDigest_SfiI_UG.pdf"> manufacturer's protocol. </a>
-  Digested plasmid backbones were separated from MCLR-genes on 1 % agarose gel using Mupid-One 70V. </p>
+  Digested plasmid backbones were separated from MCLR-genes on 1 % agarose gel using Mupid-One 70 V. </p>
 <p> The desired fragment (pLK04RCHTag) should be 3753 bp long, but in the 2nd tube there was a contamination of chromosomal DNA and anyway so little DNA that no clear bands were seen, only a greenish line. </p>
@@ Line 3,727: / Line 3,729: @@
 </td>
 <td width="54">
-<p>ng/ul</p>
+<p>ng/µL</p>
 </td>
 </tr>
@@ Line 3,751: / Line 3,753: @@
 </td>
 <td width="54">
-<p>7,3</p>
+<p>7.3</p>
 </td>
 </tr>
@@ Line 3,759: / Line 3,761: @@
 </td>
 <td width="54">
-<p>7,0</p>
+<p>7.0</p>
 </td>
 </tr>
@@ Line 3,767: / Line 3,769: @@
 <p> <b> June 4th </b> </p>
 <p> Present: Janniina, Riku, Fanni, Pyry </p>
-<p> The pLK04RCHTag plasmid was digested with SfiI in order to ligate the digoxigenin fabs into it. The DNA concentration measured to be 66.19 ng/µl (but 280 ng/µl in reality due to an error) and SfiI digestion was done according to <a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012585_FastDigest_SfiI_UG.pdf"> manufacturer's protocol. </a> Since the digestion was made with smaller concentrations than in reality, it resulted in some extra amounts of DNA. </p>
+<p> The pLK04RCHTag plasmid was digested with SfiI in order to ligate the digoxigenin fabs into it. The DNA concentration measured to be 66.19 ng/µL (but 280 ng/µL in reality due to an error) and SfiI digestion was done according to <a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012585_FastDigest_SfiI_UG.pdf"> manufacturer's protocol. </a> Since the digestion was made with smaller concentrations than in reality, it resulted in some extra amounts of DNA. </p>
 <p> Digested plasmids were separated on a 1 % agarose gel:
@@ Line 3,780: / Line 3,782: @@
 <p> Present: Janniina, Linnea, Oskari, Pyry, Riku </p>
-<p> pLK04RCHTag band from the successful gel run was purified with the <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> Gel and PCR cleanup-kit. </a> The extracted fragment had a concentration of 4.1 ng/µl. Purified pLK04RCHTag and DigoxFab0 were ligated together using T4 ligase, with MQ water and normal Digoxigenin as controls. Most of the purified vector was used for the DigoxFab0 sample so that only 0.5 µl was left for MQ and normal Digoxigenin controls. </p>
+<p> pLK04RCHTag band from the successful gel run was purified with the <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> Gel and PCR cleanup-kit. </a> The extracted fragment had a concentration of 4.1 ng/µL. Purified pLK04RCHTag and DigoxFab0 were ligated together using T4 ligase, with MQ water and normal Digoxigenin as controls. Most of the purified vector was used for the DigoxFab0 sample so that only 0.5 µL was left for MQ and normal Digoxigenin controls. </p>
-<p> Ligated plasmids were transformed in to 30 µl CaCl competent XL-1 Blue cells by incubating 20 min on ice following with 45 s at 42 °C and a further 2 min on ice. 1 ml of LB was added to each mixture and the mixtures were left to grown at 37 °C, 300 rpm Multitron. After 1 h of incubation cells were plated and left to grow o/n at 37 °C. </p>
+<p> Ligated plasmids were transformed in to 30 µL CaCl competent XL-1 Blue cells by incubating 20 min on ice following with 45 s at 42 °C and a further 2 min on ice. 1 mL of LB was added to each mixture and the mixtures were left to grown at 37 °C, 300 rpm Multitron. After 1 h of incubation cells were plated and left to grow o/n at 37 °C. </p>
 <p> <b> June 7th </b> </p>
@@ Line 3,796: / Line 3,798: @@
 <p> Present: Janniina, Jutta, Linnea </p>
-<p> 10 colonies from the plate containing XL-1 with pLK04RCHTag+DigoxFab0 were used to inoculate 5 ml of LB with ampicillin. Same was done to 3 colonies from the plate containing 100 µl of XL-1 with pLK04RCHTag+normal Digoxigenin and 2 colonies from the plate containing 900 µl of the same transformant. All of the inoculations were left to grow at 37°C, 300 rpm Multitron. Mistakenly chloramphenicol was added instead of ampicillin resulting in no growth. </p>
+<p> 10 colonies from the plate containing XL-1 with pLK04RCHTag+DigoxFab0 were used to inoculate 5 mL of LB with ampicillin. Same was done to 3 colonies from the plate containing 100 µL of XL-1 with pLK04RCHTag+normal Digoxigenin and 2 colonies from the plate containing 900 µL of the same transformant. All of the inoculations were left to grow at 37 °C, 300 rpm Multitron. Mistakenly chloramphenicol was added instead of ampicillin resulting in no growth. </p>
-<p> New try with 10 colonies from the plate containing XL-1 with pLK04RCDTag+Digox Fab0, 5 colonies from the plate containing 100 µl of XL-1 with pLK04RCHTag+harmonized digox Fab and 1 colony from the plate containing 900 µl of the same transformant. All of the inoculations were left to grown at 37°C, 300 rpm Multitron. </p>
+<p> New try with 10 colonies from the plate containing XL-1 with pLK04RCDTag+Digox Fab0, 5 colonies from the plate containing 100 µL of XL-1 with pLK04RCHTag+harmonized digox Fab and 1 colony from the plate containing 900 µL of the same transformant. All of the inoculations were left to grow at 37 °C, 300 rpm Multitron. </p>
 <p> <b> June 11th </b> </p>
@@ Line 3,813: / Line 3,815: @@
 </td>
 <td width="124">
-<p>ng/&micro;l</p>
+<p>ng/µL</p>
 </td>
 </tr>
@@ Line 3,906: / Line 3,908: @@
 </td>
 <td width="67">
-<p>ng/&micro;l</p>
+<p>ng/µL</p>
 </td>
 </tr>
@@ Line 3,956: / Line 3,958: @@
 <p> <img src="https://2019.igem.org/wiki/images/2/23/T--ABOA--Oskari_notes_img2.png" style="width:420px;height:391px;"> </p>
-<p> Since only the fifth of the pLK04RCHTag+harm Digox Fab had a band at 1.5 kb, it was saved and others discarded. 5 colonies from the plate containing <br> pLK04RCHTag+DigoxFab0 were used to inoculate 3.5 ml of LB with ampicillin and left to grow at 37 °C. The plate was also left in 37 °C for further growth. </p>
+<p> Since only the fifth of the pLK04RCHTag+harm Digox Fab had a band at 1.5 kb, it was saved and others discarded. 5 colonies from the plate containing <br> pLK04RCHTag+DigoxFab0 were used to inoculate 3.5 mL of LB with ampicillin and left to grow at 37 °C. The plate was also left in 37 °C for further growth. </p>
 <p> <b> June 12th </b> </p>
 <p> Present: Janniina, Pyry, Riku </p>
@@ Line 3,969: / Line 3,971: @@
 </td>
 <td width="95">
-<p>ng/&micro;l</p>
+<p>ng/µL</p>
 </td>
 </tr>
@@ Line 4,009: / Line 4,011: @@
 <p> As it is seen from the table above, the 3rd colony yielded the highest amount of plasmid, which may be due to the smaller amount of cells.</p>
-<p>The presence of a 1,5 kb was tested with SfiI digestion and gel electrophoresis.
+<p>The presence of a 1.5 kb was tested with SfiI digestion and gel electrophoresis.
-The gel showed that sample 3/4 yielded some plasmid, but the insert was longer than DigoxFab0 would be, so it was unsure if it really was DigoxFab0. It could also have been due to 135V. The 3/4 colony was taken to be sequenced.</p>
+The gel showed that sample 3/4 yielded some plasmid, but the insert was longer than DigoxFab0 would be, so it was unsure if it really was DigoxFab0. It could also have been due to 135 V. The 3/4 colony was taken to be sequenced.</p>
 <p> <b> June 17th </b> </p>
 <p> Present: Janniina, Jutta, Pyry</p>
-<p>The sequencing results came and since the reverse primer was actually forward, we only checked the forward sequence. The normal Digoxigenin had some mutations in the recognition site, so the transformant was unusable. We though have 20 glycerol preps.</p>
+<p>The sequencing results came and since the reverse primer was actually forward, we only checked the forward sequence. The normal Digoxigenin had some mutations in the recognition site, so the transformant was unusable. We thought have 20 glycerol preps.</p>
-<p>All 10 glycerol preps made on 11th June were used to inoculate 3 ml of LB with ampicillin, 1 % glucose and left to grow at 37 °C and 300 rpm.</p>
+<p>All 10 glycerol preps made on 11th June were used to inoculate 3 mL of LB with ampicillin, 1 % glucose and left to grow at 37 °C and 300 rpm.</p>
@@ Line 4,022: / Line 4,024: @@
 <p> Present: Pyry, Riku </p>
-<p>Plasmid extraction was done for 2 ml o/n cultures with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> the NucleoSpin Plasmid -kit. </a>
+<p>Plasmid extraction was done for 2 mL o/n cultures with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> the NucleoSpin Plasmid -kit. </a>
 <br>Yields were as follows (measured with ND-1000 with elution buffer as blank): </p>
@@ Line 4,033: / Line 4,035: @@
 </td>
 <td width="53">
-<p>ng/&micro;l</p>
+<p>ng/µL</p>
 </td>
 </tr>
@@ Line 4,119: / Line 4,121: @@
 </table> </p>
-<p>Yields showed no DNA from extractions. 3 ml of LB and 3 µl of ampicillin were added to every culture and growth was continued for a further 3 h. Plasmids were extracted as before and concentrations measured with nanodrop.</p>
+<p>Yields showed no DNA from extractions. 3 mL of LB and 3 µL of ampicillin were added to every culture and growth was continued for a further 3 h. Plasmids were extracted as before and concentrations measured with nanodrop.</p>
 <p>
 <table width="0">
@@ Line 4,128: / Line 4,130: @@
 </td>
 <td width="55">
-<p>ng/&micro;l</p>
+<p>ng/µL</p>
 </td>
 </tr>
@@ Line 4,152: / Line 4,154: @@
 <p>Again no DNA in samples. </p>
-<p>New cultures (3 ml of LB) were started from glycerol preps.</p>
+<p>New cultures (3 mL of LB) were started from glycerol preps.</p>
 <p> <b> June 19th </b> </p>
 <p> Present: Fanny, Janniina, Linnea, Oskari, Pyry, Riku, Susanna </p>
-<p>2 ml of each culture was used to extract plasmid from cells. Extraction was done with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid Kit. </a>
+<p>2 mL of each culture was used to extract plasmid from cells. Extraction was done with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid Kit. </a>
   Plasmid concentrations were measured with Nanodrop.</p>
@@ Line 4,168: / Line 4,170: @@
 </td>
 <td width="53">
-<p>ng/ul</p>
+<p>ng/µL</p>
 </td>
 </tr>
@@ Line 4,258: / Line 4,260: @@
 <p>Present:  Linnea, Pyry, Riku</p>
-<p>pLK04RCHTag was digested with SfiI to allow for ligation with Digoxigenin fabs and the digested fragments were separated with agarose gel electrophoresis (1 % agarose, 70 V). Separated fragments were purified with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> NucleoSpin Gel and PCR Clean-up kit. </a> Concentration of purified pLK04RCHTag fragments was measured to be 18.2 ng/µl (NanoDrop ND-1000).</p>
+<p>pLK04RCHTag was digested with SfiI to allow for ligation with Digoxigenin fabs and the digested fragments were separated with agarose gel electrophoresis (1 % agarose, 70 V). Separated fragments were purified with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> NucleoSpin Gel and PCR Clean-up kit. </a> Concentration of purified pLK04RCHTag fragments was measured to be 18.2 ng/µL (NanoDrop ND-1000).</p>
 <p> <b> June 26th </b> </p>
@@ Line 4,273: / Line 4,275: @@
 <p>Present:  Jutta, Fanni</p>
-<p>A colony from yesterday’s plates was used to inoculate 4 ml of LB with ampicillin to grow o/n at 37 °C, 300 rpm. </p>
+<p>A colony from yesterday’s plates was used to inoculate 4 mL of LB with ampicillin to grow o/n at 37 °C, 300 rpm. </p>
 <p> <b> June 30th </b> </p>
 <p> Present:  Janniina, Susanna </p>
-<p> pLK04RCHTag was extracted from the cells using <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid kit. </a> Concentration of purified DNA was 343.8 ng/µl. Extracted plasmids were digested with SfiI and separated on a 1 % agarose gel (70 V) together with pEB04RCH ligations.</p>
+<p> pLK04RCHTag was extracted from the cells using <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid kit. </a> Concentration of purified DNA was 343.8 ng/µL. Extracted plasmids were digested with SfiI and separated on a 1 % agarose gel (70 V) together with pEB04RCH ligations.</p>
 <p> <b> July 4th </b> </p>
@@ Line 4,287: / Line 4,289: @@
 <p> Present:  Oskari, Ossi</p>
-<p>DigoxFab0 and pLK04RCHTag were extracted with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid kit </a> and measured with Nanodrop resulting in 3.5 ng/µl and 37.7 ng/µl respectively.</p>
+<p>DigoxFab0 and pLK04RCHTag were extracted with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid kit </a> and measured with Nanodrop resulting in 3.5 ng/µL and 37.7 ng/µL respectively.</p>
 <p>Extracted DigoxFab0 was ligated to pLK04RCH and pLK04RCHTag <a href="https://nebiocalculator.neb.com/#!/ligation"> with T4 ligase. </a> </p>
-<p>XL-1 Blue cells (35 µl) were electroporated with 1 µl pLK04RCHTag+DigoxFab0. Transformed cells were incubated in 5 ml of LB for 1 h at 37 °C, 300 rpm shaking. After incubation cells were plated onto LA plates with ampicillin and stored o/n at 37 °C.</p>
+<p>XL-1 Blue cells (35 µL) were electroporated with 1 µL pLK04RCHTag+DigoxFab0. Transformed cells were incubated in 5 mL of LB for 1 h at 37 °C, 300 rpm shaking. After incubation cells were plated onto LA plates with ampicillin and stored o/n at 37 °C.</p>
 <p> <b> July 6th </b> </p>
@@ Line 4,301: / Line 4,303: @@
 <p> Present:  Riku </p>
-<p>Glycerol preps were made from 500 µl of o/n cultures and the rest of the medium was centrifuged (2000 g, 10 min) to collect plasmids. </p>
+<p>Glycerol preps were made from 500 µL of o/n cultures and the rest of the medium was centrifuged (2000 g, 10 min) to collect plasmids. </p>
-<p>Centrifuged supernatant was discarded and the plasmids were extracted with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin EasyPure-kit. </a> Elution was made twice with 25 µl of 70 °C prewarmed Elution buffer.</p>
+<p>Centrifuged supernatant was discarded and the plasmids were extracted with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin EasyPure-kit. </a> Elution was made twice with 25 µL of 70 °C prewarmed Elution buffer.</p>
 <p>Concentrations were measured with NanoDrop ND-1000:</p>
@@ Line 4,314: / Line 4,316: @@
 </td>
 <td width="161">
-<p>ng/&micro;l</p>
+<p>ng/µL</p>
 </td>
 </tr>
@@ Line 4,356: / Line 4,358: @@
 <p> <b> June 21st </b> </p>
 <p> Present: Janniina, Linnea </p>
-<p> A colony was picked from glycerol prep (321.a with pLK04RCH*) and inoculated to 4 ml of LB with 100 µg/ml ampicillin. It was then put to Multitron overnight in 37°C, 300 rpm. This vector has been checked by sequencing, it’s from colony 1/1. </p>
+<p> A colony was picked from glycerol prep (321.a with pLK04RCH*) and inoculated to 4 mL of LB with 100 µg/mL ampicillin. It was then put to Multitron overnight in 37°C, 300 rpm. This vector has been checked by sequencing, it’s from colony 1/1. </p>
 <p> <b> June 22nd </b> </p>
 <p> Present: Riku, Linnea </p>
-<p> We noticed that yesterday's growth had been in RT for the night.</p>
+<p> We noticed that yesterday's growth had been in RT over night.</p>
 <p> pLK04RCH was purified from the growth with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid EasyPure -kit </a>
   according to manufacturer's instructions. </p>
-<p> Measured concentration: 16,2 ng/µl </p>
+<p> Measured concentration: 16.2 ng/µL </p>
-<p> Due to low yield, a new growth was started in 5 ml LB with 100 ug/ml ampicillin. </p>
+<p> Due to low yield, a new growth was started in 5 mL LB with 100 µg/ml ampicillin. </p>
 <p> <b> June 23rd </b> </p>
 <p> Present: Riku, Linnea </p>
 <p> Growth was taken out and fuged at 2000 g for 10 minutes to pellet cells. Plasmids were extracted with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid EasyPure -kit </a> according to manufacturer’s instructions. Yield was measured with Nanodrop ND-1000. </p>
-<p> Results: 75,9 ng/ul
+<p> Results: 75.9 ng/µL
-<br> 260/280: 1,99
+<br> 260/280: 1.99
-<br> 260/230: 1,33  </p>
+<br> 260/230: 1.33  </p>
 <p> <b> June 24th </b> </p>
@@ Line 4,380: / Line 4,382: @@
 <p>
 <table style=”width:5%”>
-<tr> <td> </td> <td> <b> ul </b> </td> </tr>
+<tr> <td> </td> <td> <b>µL </b> </td> </tr>
 <tr> <td> MQ </td> <td> 7 </td> </tr>
 <tr> <td> FastDigest Buffer </td> <td> 2 </td> </tr>
@@ Line 4,393: / Line 4,395: @@
 <p>
 <table style=”width:5%”>
-<tr> <td> 5 µl 1 kb ladder </td> <td> pLK04RCH + Sfi1 </td> </tr>
+<tr> <td> 5 µL 1 kb ladder </td> <td> pLK04RCH + Sfi1 </td> </tr>
-<tr> <td> 1 µl Midori Direct </td> <td> 0,5 µl Midori Direct </td> </tr>
+<tr> <td> 1 µL Midori Direct </td> <td> 0.5 µL Midori Direct </td> </tr>
 </table>
 <p> Samples A and B were loaded to 1 % agarose gel and run with 70 V. </p>
 <p> <img src="https://2019.igem.org/wiki/images/f/f2/T--ABOA--Linnea_notes_img2.png" style="width:371px;height:370px;"> </p>
 <p> pLK04RCH doesn't show up on the gel because the endonucleases in 321.a have disintegrated it completely. Conclusion: we need to produce the vector in XL-1 instead of 321.a. </p>
-<p> 35 µl of electrocompetent XL-1 cells and 1 µl of pLK04RCH (from 22.6.) were used for transformation. Transformants were put in LB-medium. Incubation was started in 37 °C, 300 rpm at 13:05 and stopped at 13:50. </p>
+<p> 35 µL of electrocompetent XL-1 cells and 1 µL of pLK04RCH (from 22.6.) were used for transformation. Transformants were put in LB-medium. Incubation was started in 37 °C, 300 rpm at 13:05 and stopped at 13:50. </p>
-<p> 20 and 200 µl of growth were spread on LA plates with ampicillin and incubated in 37 °C overnight. </p>
+<p> 20 and 200 µL of growth were spread on LA plates with ampicillin and incubated in 37 °C overnight. </p>
-<p> New 5 ml culture of 321.A with pLK04RCH was started in 37C 250 rpm.  </p>
+<p> New 5 mL culture of 321.A with pLK04RCH was started in 37 °C 250 rpm.  </p>
 <p> <b> June 26th </b> </p>
 <p> Present: Janniina, Riku, Linnea </p>
 <p> Cells in previous days culture was centrifuged in 2000 g for 10 min. Supernatant was thrown away and plasmids were extracted with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin Plasmid -kit </a> according to manufacturers instructions with additional AW-buffer wash. Concentration was measured with ND-1000. </p>
-<p> Result: 13.4 ng/µl  </p>
+<p> Result: 13.4 ng/µL  </p>
 <p> Digestion was made with ThermoFisher FastDigest SfiI according to <a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012585_FastDigest_SfiI_UG.pdf"> manufacturer’s instructions. </a> Digested plasmid was run on gel.</p>
 <p> Results: Vector has dimerized and cannot be used. </p>
 <p> pLK04RCH from tubes 3/2 and 3/1 from 28.5.-19 were transformed into XL-1 according to <a href="https://www.addgene.org/protocols/bacterial-transformation/"> this protocol from Addgene </a> </p>
-<p> After transformation, cells were put into 5 ml LB for 45 min 37C 250 rpm shaking. After shaking cells were put into LA + ampicillin plate. </p>
+<p> After transformation, cells were put into 5 mL LB for 45 min 37 °C 250 rpm shaking. After shaking cells were put into LA + ampicillin plate. </p>
-<p> A colony from previous days transformation plate (20 µl) was used to inoculate 4 ml SB medium containing ampicillin and put on 37 °C 300 rpm overnight at 13:40.  </p>
+<p> A colony from previous days transformation plate (20 µL) was used to inoculate 4 mL SB medium containing ampicillin and put on 37 °C 300 rpm overnight at 13:40.  </p>
 <p> <b> June 27th </b> </p>
 <p> Present: Janniina, Riku </p>
-<p> There was a good growth in all transformation plates except mQ-control. 2 colonies were picked from each transformation (3/1: 1/100 dilution & 3/2: no dilution) plate. and put into 5 ml LB 100µg/ml ampicillin. Cultures were put into 37 C 250 rpm shaking overnight. </p>
+<p> There was a good growth in all transformation plates except mQ-control. 2 colonies were picked from each transformation (3/1: 1/100 dilution & 3/2: no dilution) plate. and put into 5 mL LB 100µg/ml ampicillin. Cultures were put into 37 °C 250 rpm shaking overnight. </p>
-<p> Since the 1/1 was forgotten, a new overnight growth was made with 1 colony into 4 ml of LB with ampicillin. Put in 37 °C at 17. </p>
+<p> Since the 1/1 was forgotten, a new overnight growth was made with 1 colony into 4 mL of LB with ampicillin. Put in 37 °C at 17. </p>
 <p> <b> June 28th </b> </p>
@@ Line 4,431: / Line 4,433: @@
 <tr> <td> 3/2.2 </td> <td> 484.5 </td> </tr>
 </table> </p>
-<p> Plasmids (1 µg) were SfiI digested for around 30 minutes at +50°C. </p>
+<p> Plasmids (1 µg) were SfiI digested for around 30 minutes at + 50°C. </p>
 <p> <table style=”width:5%”>
 <tr> <td> </td> <td> 1/1 </td> <td> 3/1.1 </td> <td> 3/1.2 </td> <td> 3/2.1 </td> <td> 3/2.2 </td> </tr>
-<tr> <td> Vol. (DNA) µl </td> <td> 3 </td> <td> 1.53 </td> <td> 1.86 </td> <td> 2.56 </td> <td> 2.06 </td> </tr>
+<tr> <td> Vol. (DNA) µL </td> <td> 3 </td> <td> 1.53 </td> <td> 1.86 </td> <td> 2.56 </td> <td> 2.06 </td> </tr>
 <tr> <td> MQ </td> <td> 17 </td> <td> 18.47 </td> <td> 18.14 </td> <td> 17.44 </td> <td> 17.94 </td> </tr>
 <tr> <td> FD buffer </td> <td> 2 </td> <td> 2 </td> <td> 2 </td> <td> 2 </td> <td> 2 </td> </tr>
@@ Line 4,443: / Line 4,445: @@
 <br>
 <table style="width:5%">
-<tr> <td> 1 kb Ladder (5µl) + midori green (1µl) </td> <td> pLK04RCH 1/1 (20µl) + 0.5µl Midori Green </td> <td> pLK04RCH 3/1.1 (20µl) + 0.5µl Midori Green </td> <td> pLK04RCH 3/1.2 (20µl) + 0.5µl Midori Green </td> <td> pLK04RCH 3/2.1 (20µl) + 0.5µl Midori Green </td> <td> pLK04RCH 3/2.2 (20µl) + 0.5µl Midori Green </td> </table> </p>
+<tr> <td> 1 kb Ladder (5 µL) + midori green (1 µL) </td> <td> pLK04RCH 1/1 (20 µL) + 0.5 µL Midori Green </td> <td> pLK04RCH 3/1.1 (20 µL) + 0.5 µL Midori Green </td> <td> pLK04RCH 3/1.2 (20 µL) + 0.5 µL Midori Green </td> <td> pLK04RCH 3/2.1 (20µL) + 0.5 µL Midori Green </td> <td> pLK04RCH 3/2.2 (20 µL) + 0.5 µL Midori Green </td> </table> </p>
 <p> <img src="https://2019.igem.org/wiki/images/f/fe/T--ABOA--Linnea_notes_img3.png" style="width:320px;height:218px;"> </p>
@@ Line 4,450: / Line 4,452: @@
 <p> Present: Janniina, Susanna </p>
 <p> According to the sequencing results the pLK04RCH 3/1 is also good to use. Since we are not sure yet whether or not 1/1 has suffered from the transformation and growing in 321.A, we'll start using 3/1. </p>
-<p> The vector backbone is needed for 5 different digoxigenin Fab ligations and since it will be purified from the gel, the yield will decrease. Assuming that 20 ng of backbone is needed for each reaction, at least 1000 ng has to be recovered from the gel. Let's say we need at least 10 µg of the backbone. Only 1 µg is recommended per 20 µl reaction, but we have used up to 2 µg with fine results. Only 1 well is free, so we'll SfiI digest only 2 µg of the 3/1 backbone. </p>
+<p> The vector backbone is needed for 5 different digoxigenin Fab ligations and since it will be purified from the gel, the yield will decrease. Assuming that 20 ng of backbone is needed for each reaction, at least 1000 ng has to be recovered from the gel. Let's say we need at least 10 µg of the backbone. Only 1 µg is recommended per 20 µL reaction, but we have used up to 2 µg with fine results. Only 1 well is free, so we'll SfiI digest only 2 µg of the 3/1 backbone. </p>
 <p> <table style="width:5%">
-<tr> <td> MQ </td> <td> 14 ul </td> </tr>
+<tr> <td> MQ </td> <td> 14 µL </td> </tr>
-<tr> <td> DNA </td> <td> 3 ul </td> </tr>
+<tr> <td> DNA </td> <td> 3 µL </td> </tr>
-<tr> <td> FD buffer </td> <td> 2 ul </td> </tr>
+<tr> <td> FD buffer </td> <td> 2 µL </td> </tr>
-<tr> <td> FD SfiI </td> <td> 1 ul </td> </tr>
+<tr> <td> FD SfiI </td> <td> 1 µL </td> </tr>
-<tr> <td> <b> Total volume </td> <td> 20 ul </b> </td> </tr>
+<tr> <td> <b> Total volume </td> <td> 20 µL </b> </td> </tr>
 </table> </p>
 <p> Incubation at 50 °C started at 11:27. Stopped at 11:56.
-<br> 0,5 µl of Midori direct is added to the sample and separated on 1 % agarose gel. </p>
+<br> 0.5 µL of Midori direct is added to the sample and separated on 1 % agarose gel. </p>
 <p> <table style="width:5%">
-<tr> <td> 1 µl Midori Direct + 5 µl 1 kb Ladder </td> <td> 0,5 µl Midori Direct + 20 µl pLK04RCH 3/1 </td> <td> 0,5 µl Midori Direct + 10 µl pEB04RCH+lig 1/1 </td> <td> 0,5 µl Midori Direct + 10 µl pEB04RCH+lig ½ </td> <td> 0,5 µl Midori Direct + 10 µl pEB04RCH+lig 2/1 </td> <td> 0,5 µl Midori Direct + 10 µl pEB04RCH+lig 2/2 </td> <td> 0,5 µl Midori Direct + 10 µl pEB04RCH+lig 3/1 </td> <td> 0,5 µl Midori Direct + 10 µl pEB04RCH+lig 3/2 </td> <td> 0,5 µl Midori Direct + 10 µl pEB04RCH+lig 4/1 </td> <td> 0,5 µl Midori Direct + 10 µl pEB04RCH+lig 4/2 </td> <td> 0,5 µl Midori Direct + 10 µl pEB04RCH+lig 7/1 </td> <td> 0,5 µl Midori Direct + 10 µl pEB04RCH+lig 7/1 </td> <td> 0,5 µl Midori Direct + 10 µl pLK04RCHTag+Fab0 </td> </tr> </table> </p>
+<tr> <td> 1 µL Midori Direct + 5 µL 1 kb Ladder </td> <td> 0.5 µL Midori Direct + 20 µL pLK04RCH 3/1 </td> <td> 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 1/1 </td> <td> 0.5 µL Midori Direct + 10 µL pEB04RCH+lig ½ </td> <td> 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 2/1 </td> <td> 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 2/2 </td> <td> 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 3/1 </td> <td> 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 3/2 </td> <td> 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 4/1 </td> <td> 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 4/2 </td> <td> 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 7/1 </td> <td> 0.5 µL Midori Direct + 10 µL pEB04RCH+lig 7/1 </td> <td> 0.5 µL Midori Direct + 10 µL pLK04RCHTag+Fab0 </td> </tr> </table> </p>
 <p> Gel run was started using 70 V at 12:18. Stopped at 13:25.
 <br> Gel run stopped and started again 13:10.
 <br> Gel run stopped at 13:20. </p>
-<p> Empty eppendorf weighs 1,03 g. </p>
+<p> Empty Eppendorf weighs 1.03 g. </p>
-<p> 2nd well cut out and put to -20°C. </p>
+<p> 2nd well cut out and put to -20 °C. </p>
 <p> The bands didn't move accordingly to their size, apparently, which may be due to active SfiI. The 2nd more clear band was cut and stored in - 20 °C. </p>
 <p> <img src="https://2019.igem.org/wiki/images/d/da/T--ABOA--Linnea_notes_img4.png" style="width:800px;height:244px;"> </p>
 <p> <b> July 1st </b> </p>
 <p> Present: Linnea, Janniina, Ossi </p>
-<p> Samples made with 3/1 and 3/2 from gel were combined and purified using <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> NucleoSpin Gel and PCR Clean-up -kit, </a> according to manufacturer’s instructions. Concentration measured with ND-1000 was 44,8 ng/µl. </p>
+<p> Samples made with 3/1 and 3/2 from gel were combined and purified using <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf"> NucleoSpin Gel and PCR Clean-up -kit, </a> according to manufacturer’s instructions. Concentration measured with ND-1000 was 44,8 ng/µL. </p>
 <p> All the Fabs were ligated into pLK04RCH (SfiI digested and purified from gel) according to the following reaction. </p>
 <p> <table style=”width:5%”>
-<tr> <td> </td> <td> ul </td> </tr>
+<tr> <td> </td> <td>µL </td> </tr>
 <tr> <td> Vector DNA </td> <td> 0.5 </td> </tr>
 <tr> <td> Insert DNA </td> <td> 3 </td> </tr>
@@ Line 4,487: / Line 4,489: @@
 <tr> <td> 10x buffer </td> <td> 14 </td> </tr>
 <tr> <td> ligase </td> <td> 1.4 </td> </tr>
-<tr> <td> <b> Total volume (ul) </td> <td> 115.5 </b> </td> </tr>
+<tr> <td> <b> Total volume (µL) </td> <td> 115.5 </b> </td> </tr>
 </table> </p>
-<p> 16,5 µl of above prepared mix to all tubes. </p>
+<p> 16.5 µL of above prepared mix to all tubes. </p>
-<p> 1 µl of each ligation reaction and ligation vector control plus MQ transformation control were electroporated to 40 µl of electrocompetent XL-1 Blue cells. </p>
+<p> 1 µL of each ligation reaction and ligation vector control plus MQ transformation control were electroporated to 40 µL of electrocompetent XL-1 Blue cells. </p>
 <p> 1 and digd2 exploded in an arc during electroporation. All else were fine with time constants 4.8 – 5.0. </p>
 <p> <b> July 2nd </b> </p>
@@ Line 4,498: / Line 4,500: @@
 <p> <b> July 3rd </b> </p>
 <p> Present: Linnea, Ossi </p>
-<p> 3 growths from plate containing XL-1 Digox Lys 191 TAG (number 1) were each used to inoculate 4 ml LB with ampicillin and tetracycline and put to Multitron to grow overnight in 37°C and 300 rpm. </p>
+<p> 3 growths from plate containing XL-1 Digox Lys 191 TAG (number 1) were each used to inoculate 4 mL LB with ampicillin and tetracycline and put to Multitron to grow overnight in 37°C and 300 rpm. </p>
-<p> Three separate colonies from 2 (Digox Lys108), 3 (Digox Fab0) and 4 (Digox C-term TAG) were each separately inoculated to 5 ml of LB with ampicillin and incubated overnight in Multitron at 37°C and 300rpm. </p>
+<p> Three separate colonies from 2 (Digox Lys108), 3 (Digox Fab0) and 4 (Digox C-term TAG) were each separately inoculated to 5 mL of LB with ampicillin and incubated overnight in Multitron at 37 °C and 300 rpm. </p>
-<p> Digd2 (Digox harmonized) was re-plated as the growth was too dense and grown overnight at 37°C. </p>
+<p> Digd2 (Digox harmonized) was re-plated as the growth was too dense and grown overnight at 37 °C. </p>
 <p> <b> July 4th </b> </p>
 <p> Janniina, Linnea, Riku, Ossi, Jutta </p>
@@ Line 4,511: / Line 4,513: @@
 <tr> <td> 3</td> <td> 4.15</td> </tr>
 <tr> <td> 4</td> <td> 2.87</td> </tr>
-<p>Digd2 (Digox harmonized) was re-plated as the growth was again too dense. Cells were suspensed with 300µl of LB and 1/100 and 1/1000 dilutions were re-plated. Cells were grown overnight at 37°C. </p>
+<p>Digd2 (Digox harmonized) was re-plated as the growth was again too dense. Cells were suspensed with 300µL of LB and 1/100 and 1/1000 dilutions were re-plated. Cells were grown overnight at 37°C. </p>
 <p>Plasmids were extracted from 1-4 ligations (3 colonies each) with plasmid EasyPure kit according to manufacturer’s instructions. </p>
 <p>The DNA concentration was measured with Nanodrop 1000. </p>
@@ Line 4,528: / Line 4,530: @@
 <tr> <td> pLK04RCH +lig 4/2 </td> <td> 58.1 </td> </tr>
 <tr> <td> pLK04RCH +lig 4/3 </td> <td> 431.4 </td> </tr>
-<p> pLK04RCHTag was digested with Sfi1 according to manufacturer’s instructions and incubated for 2 hours at 50°C. </p>
+<p> pLK04RCHTag was digested with Sfi1 according to manufacturer’s instructions and incubated for 2 hours at 50 °C. </p>
 <p> <table style="width:5%">
 <tr> <td> Sample </td> <td> 1/1 </td> <td> 1/2 </td> <td> 1/3 </td> <td> 2/1 </td> <td> 2/2 </td> <td> 2/3 </td> <td> 3/1 </td> <td> 3/2 </td> <td> 3/3 </td> <td> 4/1 </td> <td> 4/2 </td> <td> 4/3 </td> </tr>
@@ Line 4,535: / Line 4,537: @@
 <tr> <td> Buffer (FD-green) </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> </tr>
 <tr> <td> FD-SfiI </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> <td> 1 </td> </tr>
-<tr> <td> <b> Total volume (ul) </b> </td> <td>  20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> </tr> </table> </p>
+<tr> <td> <b> Total volume (µL) </b> </td> <td>  20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> <td> 20 </td> </tr> </table> </p>
-<p>0.5 µl Midori Direct was added to 19 µl of samples and 5 µl of ladder.</p>
+<p>0.5 µL Midori Direct was added to 19 µL of samples and 5 µL of ladder.</p>
 <p> <table style=”width:5%”>
 <tr> <td> 1st gel </td> 1/1 </td> <td> 1/2 </td> <td> 1/3 </td> <td> 2/1 </td> <td> 2/2 </td> <td> 2/3 </td> <td> ladder </td> <td> 3/1 </td> <td> 3/2 </td> <td> 3/3 </td> <td> 4/1 </td> <td> 4/2 </td> <td> 4/3 </td> </tr>
@@ Line 4,545: / Line 4,547: @@
 <p> <b> July 5th </b> </p>
 <p> Present: Ossi, Oskari </p>
-<p> pLK04RCH with Digox harmonized (digd2) was re-plated as the growth was again too dense. Cells were suspensed with 600µl of LB and 1/10000 and 1/100000 dilutions were re-plated. Cells were grown overnight at 37°C. </p>
+<p> pLK04RCH with Digox harmonized (digd2) was re-plated as the growth was again too dense. Cells were suspensed with 600 µL of LB and 1/10000 and 1/100000 dilutions were re-plated. Cells were grown overnight at 37 °C. </p>
-<p> Miniprepkit extraction and Nanodrop measurements were made for Fab0 and plk04RCHTag: 3.5 and 37.7 ng/µl respectively. </p>
+<p> Miniprepkit extraction and Nanodrop measurements were made for Fab0 and plk04RCHTag: 3.5 and 37.7 ng/µL respectively. </p>
 <p> Ligation:
 <br>
@@ Line 4,556: / Line 4,558: @@
 </td>
 <td width="41">
-<p>ng/&micro;l</p>
+<p>ng/µL</p>
 </td>
 <td width="95">
@@ Line 4,571: / Line 4,573: @@
 </td>
 <td width="103">
-<p> MQ &gt;20&micro;l</p>
+<p> MQ &gt;20µL</p>
 </td>
 </tr>
@@ Line 4,671: / Line 4,673: @@
 <p> <a href="https://nebiocalculator.neb.com/#!/ligation"> nebiocalculator </a> was used for calculations. </p>
 <p> Ligation controls didn’t include the Fab0 sequence. </p>
-<p> 35µl of XL-1 blue cells were transformed with 1µl of pLK04RCH + Fab0 ligation reaction with electroporation. </p>
+<p> 35 µL of XL-1 blue cells were transformed with 1 µL of pLK04RCH + Fab0 ligation reaction with electroporation. </p>
-<p> Electroporated cells were incubated in 5 ml LB at 37°C and in 300 rpm shaking for 1 hour. </p>
+<p> Electroporated cells were incubated in 5 mL LB at 37°C and in 300 rpm shaking for 1 hour. </p>
-<p> Cells were plated to LA plates with ampicillin and incubated overnight at 37°C. </p>
+<p> Cells were plated to LA plates with ampicillin and incubated overnight at 37 °C. </p>
 <p> <b> July 6th </b> </p>
 <p> Present: Riku </p>
-<p> Digid2 1/100000 was washed with 500 µl LB medium and cells were put into another container. 1/100 and 1/10000 dilutions were made and 100 µl of dilutions was put into LB+ampicillin dish. Dishes were put 37 C for overnight. </p>
+<p> Digid2 1/100000 was washed with 500 µL LB medium and cells were put into another container. 1/100 and 1/10000 dilutions were made and 100 µL of dilutions was put into LB+ampicillin dish. Dishes were put 37 °C for overnight. </p>
 <p> <b> July 7th </b> </p>
 <p> Present: Riku </p>
-<p> Glycerol preps were made from 500 µl overnight cultures and the rest of the medium was centrifuged in 2000g 10 minutes. </p>
+<p> Glycerol preps were made from 500 µL overnight cultures and the rest of the medium was centrifuged in 2000g 10 minutes. </p>
-<p> Supernatant was thrown away and plasmid was extracted with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin EasyPure -kit, </a> according to manufacturer’s instructions. Elution was made twice with 25 µl 70C prewarmed Elution buffer. </p>
+<p> Supernatant was thrown away and plasmid was extracted with <a href="https://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/Plasmid%20DNA%20Purification/UM_pDNA_NSEP.pdf"> NucleoSpin EasyPure -kit, </a> according to manufacturer’s instructions. Elution was made twice with 25 µL 70C prewarmed Elution buffer. </p>
 <p> Concentrations were measured with NanoDrop ND-1000. Results as follows:
   <br>
 <table style=”width:5%”>
-<tr> <td> <b> Sample </td> <td> ng/ul </b> </td> </tr>
+<tr> <td> <b> Sample </td> <td> ng/µL </b> </td> </tr>
 <tr> <td> 1/1 </td> <td> 86.0 </td> </tr>
 <tr> <td> 1/2 </td> <td> 74.4 </td> </tr>
 <tr> <td> 1/3 </td> <td>65.4 </td> </tr>
 <tr> <td> 100/1</td> <td> 86.7</td> </tr> </table> </p>
-<p> 3 LB + ampicillin cultures were started and put into 30 C 300 rpm overnight. </p>
+<p> 3 LB + ampicillin cultures were started and put into 30 °C 300 rpm overnight. </p>
 <p> <b> July 8th </b> </p>
 <p> Present: Riku </p>
@@ Line 4,697: / Line 4,699: @@
 <br>
 <table style=”width:5%”>
-<tr> <td> <b> Sample </td> <td> ng/ul </b> </td> </tr>
+<tr> <td> <b> Sample </td> <td> ng/µL </b> </td> </tr>
 <tr> <td> 1</td> <td> 15.4</td> </tr>
 <tr> <td>2 </td> <td> 16.4</td> </tr>
@@ Line 4,727: / Line 4,729: @@
 <p> <b> June 17th </b> </p>
 <p> Present: Janniina, Jutta, Pyry </p>
-<p> 50 µl 321.A with pEVOL-pAZF and/or pLK04RCHTag-harm were transformed. Digox Fab according to the following table + one control transformation with 1 µl of MQ. </p>
+<p> 50 µL 321.A with pEVOL-pAZF and/or pLK04RCHTag-harm were transformed. Digox Fab according to the following table + one control transformation with 1 µL of MQ. </p>
 <p> <table style=”width:5%”>
 <tr> <td> <b> pEVOL-pAzF </b> </td> <td> <b> pLK04RCHTag-harm. digox fab </b> </td> </tr>
-<tr> <td> x (0,5 µl) </td> <td> x (0,5 µl) </td> </tr>
+<tr> <td> x (0.5 µL) </td> <td> x (0.5 µL) </td> </tr>
-<tr> <td> x ( 0,5 µl) </td> <td> </td> </tr>
+<tr> <td> x (0.5 µL) </td> <td> </td> </tr>
-<tr> <td> </td> <td> x ( 0,5 µl) </td> </tr> </table> </p>
+<tr> <td> </td> <td> x (0.5 µL) </td> </tr> </table> </p>
-<p> Reactions were put to 37 °C at 15:17, taken out at 16:00. The cells were centrifuged to the bottom, the supernatant removed and the pellet was resuspended to 100 µl LB before dispensing it on the plates containing zeocin, chloramphenicol and ampicillin. The plates were put to 37 °C overnight. </p>
+<p> Reactions were put to 37 °C at 15:17, taken out at 16:00. The cells were centrifuged to the bottom, the supernatant was removed and the pellet was resuspended to 100 µL LB before dispensing it on the plates containing zeocin, chloramphenicol and ampicillin. The plates were put to 37 °C overnight. </p>
 <p> <b> June 18th </b> </p>
 <p> Present: Janniina </p>
@@ Line 4,739: / Line 4,741: @@
 <p> <b> June 19th </b> </p>
 <p> Present: Janniina </p>
-<p> 50 ml of LB with 25 µl of zeocin (1 x), 75 µl ampicillin (1,5 x), 50 µl chloramphenicol (1 x) and 1 % glucose was made. A colony was taken from a plate containing 321.A with pLK04RCHTag+harm digox Fab and pEVOL-pAzF and put in 4 ml of above mentioned LB to grow overnight. </p>
+<p> 50 mL of LB with 25 µL of zeocin (1 x), 75 µL ampicillin (1.5 x), 50 µL chloramphenicol (1 x) and 1 % glucose was made. A colony was taken from a plate containing 321.A with pLK04RCHTag+harm digox Fab and pEVOL-pAzF and put in 4 mL of above mentioned LB to grow overnight. </p>
 <p> <b> June 20th </b> </p>
 <p> Present: Janniina, Linnea </p>
-<p> The OD 600 from the overnight precultured 4 ml growth was measured from 1:10 dilution yielding 0,145 which is 1,45 undiluted at 9:21. The growth was diluted into OD of 0,1 by adding 280 µl of the growth into 3,7 ml of LB with glucose, zeocin, 1,5 x ampicillin and chloramphenicol and put into 37 °C 230 rpm at 9:30. </p>
+<p> The OD 600 from the overnight precultured 4 mL growth was measured from 1:10 dilution yielding 0.145 which is 1.45 undiluted at 9:21. The growth was diluted into OD of 0.1 by adding 280 µL of the growth into 3,7 mL of LB with glucose, zeocin, 1,5 x ampicillin and chloramphenicol and put into 37 °C 230 rpm at 9:30. </p>
-<p> OD was measured at 11:34, (200 µl of growth into 800 µl LB): 0.04 in dilution, 0.2 undiluted. </p>
+<p> OD was measured at 11:34, (200 µL of growth into 800 µL LB): 0.04 in dilution, 0.2 undiluted. </p>
 <p> At 13:36 OD was 0.021 diluted and 0.21 undiluted.
 <p> <b> June 24th </b> </p>
@@ Line 4,750: / Line 4,752: @@
 Pyry </p>
-<p> 4,5 ml of above prepared LB was inoculated from a glycerolprep made on 20th June and put in 37 °C. </p>
+<p> 4.5 mL of above prepared LB was inoculated from a glycerol prep made on 20th June and put in 37 °C. </p>
 <p> <b> June 25th </b> </p>
@@ Line 4,757: / Line 4,759: @@
 Pyry </p>
-<p> Calculated amount for 5 ml, OD 0,1:
+<p> Calculated amount for 5 mL, OD 0.1:
 <br>
 <table width="0">
@@ Line 4,816: / Line 4,818: @@
 </p>
-<p> Culture was diluted into 0,1 OD600 by adding 225 µl into 5 ml LB medium with 1% glucose, 25 µg/ml cloramphenicol, 100 µg/ml ampicillin, 50 µg/ml zeocin.
+<p> Culture was diluted into 0.1 OD600 by adding 225 µL into 5 mL LB medium with 1% glucose, 25 µg/mL cloramphenicol, 100 µg/mL ampicillin, 50 µg/mL zeocin.
-  <br> Culture was put into 37 C 300 rpm. </p>
+  <br> Culture was put into 37 °C 300 rpm. </p>
 <p> OD was measured as follows:
-<br> 1/10 dilution is made with 100 µl growth and 900 µl LB medium. </p>
+<br> 1/10 dilution is made with 100 µL growth and 900 µL LB medium. </p>
 <p>
 <table width="0">
@@ Line 4,883: / Line 4,885: @@
 <p> LB medium was changed to SB medium.
-Cells were centrifuged in bottom of Falcon tube and supernatant was thrown away. Cells were resuspensed in 5 ml SB medium (1% glucose, 25 µg/ml cloramphenicol, 100 µg/ml ampicillin, 50 µg/ml zeocin). </p>
+Cells were centrifuged in bottom of Falcon tube and supernatant was thrown away. Cells were resuspensed in 5 mL SB medium (1% glucose, 25 µg/mL cloramphenicol, 100 µg/mL ampicillin, 50 µg/mL zeocin). </p>
 <p>
@@ Line 4,940: / Line 4,942: @@
-<p> After OD had reached 0.72 cells were centrifuged in 2000g for 10 min. Supernatant was thrown away and cells were resuspensed in SB medium (25 µg/ml cloramphenicol, 100 µg/ml ampicillin, 50 µg/ml zeocin, 0,1 % arabinose). </p>
+<p> After OD had reached 0.72 cells were centrifuged in 2000 g for 10 min. Supernatant was thrown away and cells were resuspensed in SB medium (25 µg/mL cloramphenicol, 100 µg/mL ampicillin, 50 µg/mL zeocin, 0.1 % arabinose). </p>
 <p> Cells were incubated 26 ℃ 200 rpm for 10 min.
 <br> After incubation P-azidophenylalanine and IPTG was added in growth medium to final concentration 1mM
-<br> After 4h from induction, a 1 ml sample was taken and centrifuged to pellet cells.
+<br> After 4h from induction, a 1 mL sample was taken and centrifuged to pellet cells.
-<br> After 4h incubation 1 ml sample was taken, centrifuged (16,1 g, 1 min) and the pellet put into freezer. </p>
+<br> After 4h incubation 1 mL sample was taken, centrifuged (16.1 g, 1 min) and the pellet put into freezer. </p>
@@ Line 4,951: / Line 4,953: @@
 <p> <b> June 26th </b> </p>
 <p> Present: Linnea </p>
-<p> 5 µl of glycerol prep (321.A) was used to inoculate 5 ml of LB with 25 µg/ml chloramphenicol and 0,5 % glucose and put into Multitron (300 rpm, 37 °C) at 10:53. </p>
+<p> 5 µL of glycerol prep (321.A) was used to inoculate 5 mL of LB with 25 µg/mL chloramphenicol and 0.5 % glucose and put into Multitron (300 rpm, 37 °C) at 10:53. </p>
-<p> Two XL1 colonies with the biobrick were used to inoculate 5 ml of LB with 25 µg/ml chloramphenicol and 0,5 % glucose and put into Multitron (300 rpm, 37 °C) at 12:20. </p>
+<p> Two XL1 colonies with the biobrick were used to inoculate 5 mL of LB with 25 µg/ml chloramphenicol and 0.5 % glucose and put into Multitron (300 rpm, 37 °C) at 12:20. </p>
 <p> <b> June 27th </b> </p>
-<p> The cell pellets were sonicated with biotechnology sonicator. Cells were resuspended into 1 ml of Assay Buffer per 1 ml of culture before sonication. </p>
+<p> The cell pellets were sonicated with biotechnology sonicator. Cells were resuspended into 1 mL of Assay Buffer per 1 mL of culture before sonication. </p>
 <p> <b> Sonication procedure: </b> </p>
-<p> 1 minute, low setting, power level 40, cycle 0,5 seconds. Cells were held on ice while sonicating. </p>
+<p> 1 minute, low setting, power level 40, cycle 0.5 seconds. Cells were held on ice while sonicating. </p>
 <p> <b> Assay procedure: </b> </p>
-<p> The cell lysate was diluted 1/10 and 1/100 and 200 µl was incubated 30 minutes at slow shake on Goat Anti Human 96-well plate. </p>
+<p> The cell lysate was diluted 1/10 and 1/100 and 200 µL was incubated 30 minutes at low shake on Goat Anti Human 96-well plate. </p>
 <p> Plate was washed 4x with Wash Buffer (kaivogen). </p>
-<p> 2A11-Europium labeled anti human antibody was used to detect Fabs. Assay concentration was 0,25 ng/µl/well. Assay volume 200 µl
+<p> 2A11-Europium labeled anti human antibody was used to detect Fabs. Assay concentration was 0.25 ng/µL/well. Assay volume 200 µL
   Plate was washed 4x with WAsh Buffer (kaivogen)
-Delfia ES was used to develop signal for 10 mins
+Delfia Enhancement Solution was used to develop signal for 10 min.
   Hidex multilabel reader was used for signal detection </p>
@@ Line 4,986: / Line 4,988: @@
 <p> Electroporation reactions were mixed as follows: </p>
 <p> <table width="0"> <tbody> <tr> <td width="125"> <p>Digox Fab</p> </td>
-<td width="107"> <p>Digox Fab Plasmid (&micro;l)</p> </td>
+<td width="107"> <p>Digox Fab Plasmid (µL)</p> </td>
-<td width="116"> <p>pEVOL-pAzF (&micro;l)</p> </td>
+<td width="116"> <p>pEVOL-pAzF (µL)</p> </td>
-<td width="79"> <p>MQ H<sub>2</sub>O (&micro;l)</p> </td>
+<td width="79"> <p>MQ H<sub>2</sub>O (µL)</p> </td>
-<td width="176"> <p>Electrocompetent C321.&Delta;A.exp cells (&micro;l)</p> </td> </tr>
+<td width="176"> <p>Electrocompetent C321.&Delta;A.exp cells (µL)</p> </td> </tr>
 <tr> <td width="125"> <p>DigoxFab108</p> </td> <td width="107"> <p>0.5</p> </td>
 <td width="116"> <p>0</p> </td> <td width="79"> <p>0.5</p> </td> <td width="176"> <p>35</p> </td> </tr>
@@ Line 5,015: / Line 5,017: @@
 <p> Time constants were 5.0 ms for all samples. </p>
-<p> Electroporated cells were put into 5 ml of LB for 45 min in 37 °C and 250 rpm shaking. </p>
+<p> Electroporated cells were put into 5 mL of LB for 45 min in 37 °C and 250 rpm shaking. </p>
 <p> Plating was done as follows:
 <br>
 <table width="0"> <tbody>
-<tr> <td width="217"> <p>Dilution</p> </td> <td width="62"> <p>1/1 (&micro;l)</p> </td> <td width="72"> <p>1/100 (&micro;l)</p>
+<tr> <td width="217"> <p>Dilution</p> </td> <td width="62"> <p>1/1 (µL)</p> </td> <td width="72"> <p>1/100 (µL)</p>
 </td> </tr>
 <tr> <td width="217"> <p>DigoxFab108</p> </td> <td width="62"> <p>50</p> </td> <td width="72"> <p>&nbsp;</p> </td> </tr>
@@ Line 5,037: / Line 5,039: @@
 <p> Present: Linnea </p>
-<p> Two colonies from each 1/100 diluted plate (DigoxFab108 + pEVOL-pAzF, DigoxFab191 + pEVOL-pAzF, DigoxFab0 + pEVOL-pAzF) were used to inoculate 4 ml LB with ampicillin, chloramphenicol, zeocin and glucose. Tubes were grown o/n at 37 °C, 300 rpm. </p>
+<p> Two colonies from each 1/100 diluted plate (DigoxFab108 + pEVOL-pAzF, DigoxFab191 + pEVOL-pAzF, DigoxFab0 + pEVOL-pAzF) were used to inoculate 4 mL LB with ampicillin, chloramphenicol, zeocin and glucose. Tubes were grown o/n at 37 °C, 300 rpm. </p>
 <p> <b> July 11th </b> </p>
 <p> Present: Janniina, Oskari </p>
-<p> OD600 was measured from each of the two replicate growths. Measurements were made from a 1:10 diluted sample. The OD600 was used to calculate the growth volume needed for inoculation of 4 ml of SB. </p>
+<p> OD600 was measured from each of the two replicate growths. Measurements were made from a 1:10 diluted sample. The OD600 was used to calculate the growth volume needed for inoculation of 4 mL of SB. </p>
 <p>
 <table width="0"><tbody><tr><td width="168"><p>Growth</p></td><td width="91"> <p>OD600 (1:10)</p></td><td width="87"><p>OD600 (1:1)</p></td><td width="256">
-<p>Volume need for dilution to OD600 of 0.1 in 4ml of SB (&micro;l)</p></td></tr>
+<p>Volume need for dilution to OD600 of 0.1 in 4ml of SB (µL)</p></td></tr>
 <tr><td width="168"><p>DigoxFab191 + pEVOL-pAzF</p></td><td width="91"><p>0.219</p></td><td width="87"><p>2.19</p></td><td width="256"> <p>183</p></td></tr>
 <tr><td width="168"><p>DigoxFab108 + pEVOL-pAzF</p></td><td width="91"> <p>0.303</p></td><td width="87"><p>3.03</p></td><td width="256"><p>132</p></td></tr>
@@ Line 5,085: / Line 5,087: @@
-<p> Arabinose (final c = 5 %) and pAzF (1 mM) was added to DigoxFab191 + pEVOL-pAzF test growth and after 10 min incubation in 37 °C 0.1 mM IPTG was added to induce production of DigoxFab191. Once induced the growth was left to grow at 26 °C, while a 20 % glycerol prep was made from the control growth. </p>
+<p> Arabinose (final concentration = 5 %) and pAzF (1 mM) was added to DigoxFab191 + pEVOL-pAzF test growth and after 10 min incubation in 37 °C 0.1 mM IPTG was added to induce production of DigoxFab191. Once induced the growth was left to grow at 26 °C, while a 20 % glycerol prep was made from the control growth. </p>
 <p> The OD600 was measured again from DigoxFab108 + pEVOL-pAzF and DigoxFab0 + pEVOL-pAzF test growths from 1:10 dilutions (SB) resulting in unexpectedly low OD compared to the previous OD600 but the growths were induced anyway. Arabinose (5 %) and pAzF (1 mM) were added after 4.5 h. After 10 min incubation in RT 0.1 mM IPTG was added. After adding IPTG, growths were left to grow o/n at 26 °C, 300 rpm (multitron). </p>
@@ Line 5,122: / Line 5,124: @@
 <p> Present: Fanny, Janniina, Linnea, Oskari, Pyry </p>
-<p> An aliquot of 1 ml of overnight growths was harvested by centrifugation and resuspended to 1 ml of assay buffer. Rest of the growth was stored in -20 °C. The cells were sonicated with Braun Labsonic'u for a minute (duty cycle 0.5; output -053). </p>
+<p> An aliquot of 1 mL of overnight growths was harvested by centrifugation and resuspended to 1 mL of assay buffer. Rest of the growth was stored in -20 °C. The cells were sonicated with Braun Labsonic'u for a minute (duty cycle 0.5; output -053). </p>
-<p> After lysis the cells were pelleted by centrifugation to collect and dilute supernatant to 1:10 ratio in AB. 200 µl of the dilution was added to each well (goat anti-human plate) with three parallel samples of each. </p>
+<p> After lysis the cells were pelleted by centrifugation to collect and dilute supernatant to 1:10 ratio in AB. 200 µL of the dilution was added to each well (goat anti-human plate) with three parallel samples of each. </p>
 <p>
@@ Line 5,201: / Line 5,203: @@
-<p> The 96-well plate was incubated for 1 h on a shaker. Europium labeled 2A11 antibody was diluted with AB to 0.25 ng/µl from stock (390 µg/ml).The dilution was filtered through ⌀ 0.22 µm filter. The plate was washed using Delfia platewasher (Wallac) 4 times. 200 µl of 2A11 (0.25 ng/µl) is added into each well. The dispenser dispensed some of the liquid that was supposed to be in well C1 (AB control) into D1 (DigoxFab108 test). So DigoxFab108 test should give lower results than what might be expected. The plate is incubated on a shaker for 30 min after which the plate was washed again 4 times. 200 µl of EFI was dispensed into each well. After incubating for 10 minutes, the fluorescence was measured with Hidex Sense. </p>
+<p> The 96-well plate was incubated for 1 h on a shaker. Europium labeled 2A11 antibody was diluted with AB to 0.25 ng/µL from stock (390 µg/mL). The dilution was filtered through ⌀ 0.22 µm filter. The plate was washed using Delfia platewasher (Wallac) 4 times. 200 µL of 2A11 (0.25 ng/µL) is added into each well. The dispenser dispensed some of the liquid that was supposed to be in well C1 (AB control) into D1 (DigoxFab108 test). So DigoxFab108 test should give lower results than what might be expected. The plate is incubated on a shaker for 30 min after which the plate was washed again 4 times. 200 µL of Delfia Enhancement Solution was dispensed into each well. After incubating for 10 minutes, the fluorescence was measured with Hidex Sense. </p>
-<p>To transform the plasmids (pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1) into cells (C321.ΔA.exp) for production; 0.5 µl of each plasmid was electroporated with 30 µl of cells. Three transformation controls were made; only pEVOL-pAzF, only pLK04RCHTag+DigoxFab0 1/1 (ligation date?) and MQ water only. After electroporation the cells were moved to a round-bottomed culture tube and grown for 1 h in 3 ml of SB at 37 °C.</p>
+<p>To transform the plasmids (pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1) into cells (C321.ΔA.exp) for production; 0.5 µL of each plasmid was electroporated with 30 µL of cells. Three transformation controls were made; only pEVOL-pAzF, only pLK04RCHTag+DigoxFab0 1/1 (ligation date?) and MQ water only. After electroporation the cells were moved to a round-bottomed culture tube and grown for 1 h in 3 mL of SB at 37 °C.</p>
 <p>Transformation cultures were diluted to 1/50 and plated with tetracycline, ampicillin and zeocine.</p>
@@ Line 5,217: / Line 5,219: @@
-<p> Two colonies from the plate were used to inoculate 4 ml of SB containing zeocin, ampicillin and chloramphenicol and grown at 37 °C, 300 rpm o/n. Since the plate was dense from colonies, a couple were used to restreak a new plate, which was grown at 37 °C o/n. </p>
+<p> Two colonies from the plate were used to inoculate 4 mL of SB containing zeocin, ampicillin and chloramphenicol and grown at 37 °C, 300 rpm o/n. Since the plate was dense from colonies, a couple were used to restreak a new plate, which was grown at 37 °C o/n. </p>
 <p> <b> July 15th </b> </p>
@@ Line 5,230: / Line 5,232: @@
-<p> Colonies from the plates were used to inoculate again 100 ml of SB containing zeocin, ampicillin and chloramphenicol and grown o/n at 37 °C, 300 rpm.  </p>
+<p> Colonies from the plates were used to inoculate again 100 mL of SB containing zeocin, ampicillin and chloramphenicol and grown o/n at 37 °C, 300 rpm.  </p>
@@ Line 5,237: / Line 5,239: @@
 <p> Present: Janniina, Jutta, Linnea, Oskari, Pyry, Robin </p>
-<p> The growths were diluted to 1:10 in 1 ml of SB for measurement of OD600.  </p>
+<p> The growths were diluted to 1:10 in 1 mL of SB for measurement of OD600.  </p>
 <p>
 <table width="0">
@@ Line 5,288: / Line 5,290: @@
 </table> </p>
-<p> Growths were diluted to OD600 of 0.1 in 400 ml of SB and antibiotics in working concentrations. These growths were left to grown at 37 °C, 230 rpm. RPM was increased to 300 rpm after 2h. OD600 was measured again after 4h; giving 0.104 for DigoxFab0, 0.086 for DigoxFab191 and 0.088 forDigoxFab108. After 5h 0.05 % glucose was added to each culture to accelerate growth. OD600 measured again after 8h were: DigoxFab108: 1.01; DigoxFab191: 0.69; DigoxFab0: 0.65. </p>
+<p> Growths were diluted to OD600 of 0.1 in 400 mL of SB and antibiotics in working concentrations. These growths were left to grown at 37 °C, 230 rpm. RPM was increased to 300 rpm after 2h. OD600 was measured again after 4h; giving 0.104 for DigoxFab0, 0.086 for DigoxFab191 and 0.088 forDigoxFab108. After 5h 0.05 % glucose was added to each culture to accelerate growth. OD600 measured again after 8h were: DigoxFab108: 1.01; DigoxFab191: 0.69; DigoxFab0: 0.65. </p>
 <p> 0.2 % arabinose was added to all growths. After 10 min of incubation in RT growths were induced with 0.4 mM IPTG and 1 mM pAzF. Growths were covered in foil and left to grown o/n at 26 °C and 300 rpm shaking. </p>
@@ Line 5,294: / Line 5,296: @@
-<p> Again no growth observed in the preculture. Transformed pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1 into C321.ΔA.exp. After electroporation the cells were moved to a round-bottomed culture tube and grown for 1 h in 3 ml of SB at 37 °C. After incubation the cultures were diluted to 1/100 and plated with tetracycline, ampicillin and zeocine and gronw o/n at 37 °C, 300 rpm. </p>
+<p> Again no growth observed in the preculture. Transformed pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1 into C321.ΔA.exp. After electroporation the cells were moved to a round-bottomed culture tube and grown for 1 h in 3 mL of SB at 37 °C. After incubation the cultures were diluted to 1/100 and plated with tetracycline, ampicillin and zeocine and grown o/n at 37 °C, 300 rpm. </p>
 <p> <b> July 21st </b> </p>
@@ Line 5,300: / Line 5,302: @@
-<p> Two cultures of 20 ml of SB containing zeocin, ampicillin, chloraphenicol and 0,5 % glucose were made, with cells inoculated from the plate containing C321.ΔA.exp transformed with pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1. Cultures grown o/n at 37 °C, 300 rpm. </p>
+<p> Two cultures of 20 mL of SB containing zeocin, ampicillin, chloraphenicol and 0.5 % glucose were made, with cells inoculated from the plate containing C321.ΔA.exp transformed with pEVOL-pAzF and pLK04RCHTag + DigoxFab0 1/1. Cultures grown o/n at 37 °C, 300 rpm. </p>
 <p> <b> July 22nd </b> </p>
@@ Line 5,309: / Line 5,311: @@
 <p> Glycerol prep was made of the cultures. </p>
-<p> Cultures were diluted to maximum OD600 of 0.1 in 400 ml of SB (antibiotics: ZCA, 0.05 % glucose). When OD600 was around 0.7 added 1 % arabinose and 1 mM p-azido-L-phenylalanine (pAzF). Cultures were covered with aluminium foil and after 10 min 1 mM of IPTG was added to induce production of DigoxFab0. Cultures were grown o/m at 26 °C. </p>
+<p> Cultures were diluted to maximum OD600 of 0.1 in 400 mL of SB (antibiotics: ZCA, 0.05 % glucose). When OD600 was around 0.7 added 1 % arabinose and 1 mM p-azido-L-phenylalanine (pAzF). Cultures were covered with aluminium foil and after 10 min 1 mM of IPTG was added to induce production of DigoxFab0. Cultures were grown o/m at 26 °C. </p>
 <p> <b> July 23rd </b> </p>
@@ Line 5,315: / Line 5,317: @@
-<p> Prepared histag column prior to purifications. A sample (500 µl) was taken from the growth.
+<p> Prepared histag column prior to purifications. A sample (500 µL) was taken from the growth.
-Cells were washed and lysed according to protocol. Weights of the two lysate pellets were 6,46 g and 6,28 g. Second pellet was resuspended into 15 ml NPI-lysis buffer. Another 15 ml was added since the pellet did not completely dissolve. Once resuspended the second tube was poured to the first one.
+Cells were washed and lysed according to protocol. Weights of the two lysate pellets were 6,46 g and 6,28 g. Second pellet was resuspended into 15 mL NPI-lysis buffer. Another 15 mL was added since the pellet did not completely dissolve. Once resuspended the second tube was poured to the first one.
-Lysate (25 ml) were purified according to protocol and collected in 5 fractions of 1 ml. </p>
+Lysate (25 mL) were purified according to protocol and collected in 5 fractions of 1 mL. </p>
 <p> <h3> <b> ANTIBODY PRODUCTION AND PURIFICATION </b> </h3> </p>
@@ Line 5,389: / Line 5,391: @@
 <p>Present:  Janniina</p>
-<p>Precultures for production were started by inoculating pLK04RCHTag (DigoxFab108Tag, DigoxFab191Tag and DigoxFab0HistagTag) and pEVOL-pAzF containing C321.ΔA.exp-cells from a glycerol prep into 20 ml of SB. Inoculated cells were incubated o/n at 37 °C, 250 rpm. </p>
+<p>Precultures for production were started by inoculating pLK04RCHTag (DigoxFab108Tag, DigoxFab191Tag and DigoxFab0HistagTag) and pEVOL-pAzF containing C321.ΔA.exp-cells from a glycerol prep into 20 mL of SB. Inoculated cells were incubated o/n at 37 °C, 250 rpm. </p>
 <p> <b> August 23rd </b> </p>
 <p>Present:  Ossi, Pyry</p>
-<p>Yesterdays growths were inoculated inoculated into the main culture (400 ml) so that the  main culture’s OD600 was 0.1 with chloramphenicol, ampicillin and 0.05 % glucose. Inoculated main growth was left to grow at 37 °C, 250 rpm until OD600 was 0.6 - 0.8. Once ready, cells were induced with 1 mM pAzF, 0.5 mM IPTG and 0.5 % arabinose and incubated o/n 26 °C, 250 rpm. </p>
+<p>Yesterdays growths were inoculated inoculated into the main culture (400 mL) so that the main culture’s OD600 was 0.1 with chloramphenicol, ampicillin and 0.05 % glucose. Inoculated main growth was left to grow at 37 °C, 250 rpm until OD600 was 0.6 - 0.8. Once ready, cells were induced with 1 mM pAzF, 0.5 mM IPTG and 0.5 % arabinose and incubated o/n 26 °C, 250 rpm. </p>
 <p> <b> August 24th </b> </p>
@@ Line 5,409: / Line 5,411: @@
 Lysate was added to the column and the column was then capped. The resin lysate mixture was then on rotation for 1 h at 4 °C. </p>
-<p>All the supernatant was combined  to a 50ml bottle and the mixing with resin continued for 30 min.</p>
+<p>All the supernatant was combined to a 50ml bottle and the mixing with resin continued for 30 min.</p>
 <p>The fractions were eluted in 1ml batches. 5 each. Preliminary results showed good yields of DigoxFab0Histag and control DigoxFab0 (Histag fraction 2 2.9mg/ml and Fab0 fraction 1 0.90 mg/ml). Other fractions had lower yields and were interfered by imidazole from elution buffer. Buffer was changed into PBS, except for the negative control.
@@ Line 5,457: / Line 5,459: @@
 <p> <b> July 18th </b> </p>
 <p> Present: Oskari, Robin, Janniina </p>
-<p>The Dengue-Fab sequences from Integrated DNA Technologies were suspended in 20 µl MQ-H2O to a final concentration of 50 ng/µl. We have four different Dengue-Fab fragments to work with, three of which have had an amino acid replaced with an amber stop codon (TAG): <br> DengueFab Ser221 > TAG (DengueFab221), <br>DengueFab Thr196 > TAG (DengueFab196) <br>and DengueFab Asp152 > TAG (DengueFab152). <br>The last one is an unmodified Dengue-Fab fragment (DengueFab0). The concentration of the previously SfiI-digested vector backbones were: </p>
+<p>The Dengue-Fab sequences from Integrated DNA Technologies were suspended in 20 µL MQ-H2O to a final concentration of 50 ng/µL. We have four different Dengue-Fab fragments to work with, three of which have had an amino acid replaced with an amber stop codon (TAG): <br> DengueFab Ser221 > TAG (DengueFab221), <br>DengueFab Thr196 > TAG (DengueFab196) <br>and DengueFab Asp152 > TAG (DengueFab152). <br>The last one is an unmodified Dengue-Fab fragment (DengueFab0). The concentration of the previously SfiI-digested vector backbones were: </p>
-<p> pLK04RCH: 44.8 ng/µl
+<p> pLK04RCH: 44.8 ng/µL
-<br> pLK04RCH-Tag: 37.7 ng/µl </p>
+<br> pLK04RCH-Tag: 37.7 ng/µL </p>
 <p> The Dengue-Fabs were digested with SfiI: </p>
 <p>
@@ Line 5,469: / Line 5,471: @@
 </td>
 <td width="231">
-<p>volume (&micro;l)</p>
+<p>volume (µL)</p>
 </td>
 </tr>
@@ Line 5,514: / Line 5,516: @@
 </tbody>
 </table> </p>
-<p> The reactions were incubated for 15 minutes at 50 oC. SfiI was inactivated by addition of 20 mM EDTA. </p>
+<p> The reactions were incubated for 15 minutes at 50°C. SfiI was inactivated by addition of 20 mM EDTA. </p>
 <p> We then attempted ligation of the Dengue-Fab inserts into the vector backbones. DengueFab0 was cloned into the pLK04RCH and the pLK04RCH-Tag vector. The DengueFab152, DengueFab196 and DengueFab221 were cloned into the pLK04RCH vector: </p>
 <p>
@@ Line 5,623: / Line 5,625: @@
 <tr>
 <td width="77">
-<p>total volume (&micro;l)</p>
+<p>total volume (µL)</p>
 </td>
 <td width="97">
@@ Line 5,640: / Line 5,642: @@
 </tbody>
 </table> </p>
-<p> The reactions were incubated for 1 hour at room temperature. Then 1 µl of the ligated product was electroporated into 40 µl of XL-1 electrocompetent cells, according to our electroporation protocol. Electroporation was followed by a 45 minute incubation at 37 oC and 300rpm. 200 µl of the mixture was plated onto selective LA plates containing tetracyclin and ampicillin and incubated o/n at 37 oC. </p>
+<p> The reactions were incubated for 1 hour at room temperature. Then 1 µL of the ligated product was electroporated into 40 µL of XL-1 electrocompetent cells, according to our electroporation protocol. Electroporation was followed by a 45-minute incubation at 37°C and 300rpm. 200 µL of the mixture was plated onto selective LA plates containing tetracyclin and ampicillin and incubated o/n at 37°C. </p>
 <p> <b> July 19th </b> </p>
 <p> Present: Oskari </p>
@@ Line 5,723: / Line 5,725: @@
 <p> <b> July 21st </b> </p>
 <p> Present: Janniina, Jutta </p>
-<p> 40 ml of SB medium, containing ampicillin and 0.5 % glucose, was prepared. Two colonies were picked from the pLK04RCH-Tag+DengueFab0, pLK04RCH+DengueFab152, -196 and -221, and inoculated to 2x4 ml of SB medium. The growths were incubated o/n at 37 oC.</p>
+<p> 40 mL of SB medium, containing ampicillin and 0.5 % glucose, was prepared. Two colonies were picked from the pLK04RCH-Tag+DengueFab0, pLK04RCH+DengueFab152, -196 and -221, and inoculated to 2x4 mL of SB medium. The growths were incubated o/n at 37°C.</p>
 <p> <b> July 22nd </b> </p>
 <p> Present: Jutta</p>
@@ Line 5,747: / Line 5,749: @@
 </td>
 <td width="151">
-<p>136.4 ng/&micro;l</p>
+<p>136.4 ng/µL</p>
 </td>
 <td width="151">
-<p>124.5 ng/&micro;l</p>
+<p>124.5 ng/µL</p>
 </td>
 </tr>
@@ Line 5,758: / Line 5,760: @@
 </td>
 <td width="151">
-<p>120.7 ng/&micro;l</p>
+<p>120.7 ng/µL</p>
 </td>
 <td width="151">
-<p>130.1 ng/&micro;l</p>
+<p>130.1 ng/µL</p>
 </td>
 </tr>
@@ Line 5,769: / Line 5,771: @@
 </td>
 <td width="151">
-<p>129 ng/&micro;l</p>
+<p>129 ng/µL</p>
 </td>
 <td width="151">
-<p>431.3 ng/&micro;l</p>
+<p>431.3 ng/µL</p>
 </td>
 </tr>
@@ Line 5,780: / Line 5,782: @@
 </td>
 <td width="151">
-<p>448.1 ng/&micro;l</p>
+<p>448.1 ng/µL</p>
 </td>
 <td width="151">
-<p>137.9 ng/&micro;l</p>
+<p>137.9 ng/µL</p>
 </td>
 </tr>
@@ Line 5,797: / Line 5,799: @@
 </td>
 <td width="232">
-<p>volume (&micro;l)</p>
+<p>volume (µL)</p>
 </td>
 </tr>
@@ Line 5,842: / Line 5,844: @@
 </tbody>
 </table> </p>
-<p> The reaction was incubated for 30 minutes at 50 oC. 0.5 µl Midori Green Direct (Nippon Genetics) was added to each digestion reaction and 1 µl to 1 kB ladder.  </p>
+<p> The reaction was incubated for 30 minutes at 50 °C. 0.5 µL Midori Green Direct (Nippon Genetics) was added to each digestion reaction and 1 µL to 1 kB ladder.  </p>
 <p> The digested vectors and inserts were separated on a 1 % agarose gel:</p>
 <p> <img src="https://2019.igem.org/wiki/images/0/01/T--ABOA--Fanny_notes_img2.png" style="width:779px;height:300px;">
 <br> From left to right: ladder, pLK04RCH-DengueFab152 (miniprep 1), pLK04RCH-DengueFab152 (miniprep 2), pLK04RCH-DengueFab196 (miniprep 1), pLK04RCH-DengueFab196 (miniprep 2), pLK04RCH-DengueFab221 (miniprep 1), pLK04RCH-DengueFab221 (miniprep2), pLK04RCHTag-DengueFab0 (miniprep 1), pLK04RCHTag-DengueFab0 (miniprep 2).</p>
-<p>We repeat the ligation of DengueFab0 and the pLK04RCH vector (vector concentration: 18.8 ng/µl):</p>
+<p>We repeat the ligation of DengueFab0 and the pLK04RCH vector (vector concentration: 18.8 ng/µL):</p>
 <p>
 <table>
@@ Line 5,919: / Line 5,921: @@
 <tr>
 <td width="151">
-<p>total volume (&micro;l)</p>
+<p>total volume (µL)</p>
 </td>
 <td width="160">
@@ Line 5,930: / Line 5,932: @@
 </tbody>
 </table> </p>
-<p>Reaction was incubated for 1 hour at room temperature. XL-1 electrocompetent cells were transformed with the ligation mixture according to our electroporation protocol, and then incubated for 45 minutes at 37 oC and 300 rpm, plated onto selective plates and incubated o/n at 37 oC.</p>
+<p>Reaction was incubated for 1 hour at room temperature. XL-1 electrocompetent cells were transformed with the ligation mixture according to our electroporation protocol, and then incubated for 45 minutes at 37 °C and 300 rpm, plated onto selective plates and incubated o/n at 37 °C.</p>
 <p> <b> July 23rd </b> </p>
 <p> Present: Ossi </p>
-<p> Colonies from the plates were inoculated into 4 ml SB cultures containing ampicillin and 0.5 % glucose. The growths were incubated o/n at 37 oC and 300 rpm. </p>
+<p> Colonies from the plates were inoculated into 4 mL SB cultures containing ampicillin and 0.5 % glucose. The growths were incubated o/n at 37 °C and 300 rpm. </p>
 <p> The pLK04RCH-DengueFab196, pLK04RCH-DengueFab221, pLK04RCH-DengueFab152 and pLK04RCH-Tag-DengueFab0 were sent for sequencing. </p>
 <p> <b> July 24th </b> </p>
 <p> Present: Ossi </p>
 <p> Glycerol preps and minipreps were made from the o/n cultures, according to our protocols. The DNA concentration as measured with Nanodrop:</p>
-<p> pLK04RCH-DengueFab0 (prep 1): 518.8 ng/µl
+<p> pLK04RCH-DengueFab0 (prep 1): 518.8 ng/µL
-<br> pLK04RCH-DengueFab0 (prep 2): 471.9 ng/µl </p>
+<br> pLK04RCH-DengueFab0 (prep 2): 471.9 ng/µL </p>
 <p> To verify if the cloning worked we re-digested the vectors with SfiI: </p>
 <p>
@@ Line 5,979: / Line 5,981: @@
 <tr>
 <td width="242">
-<p>total volume (&micro;l)</p>
+<p>total volume (µL)</p>
 </td>
 <td width="229">
@@ Line 5,987: / Line 5,989: @@
 </tbody>
 </table> </p>
-<p>The reaction was incubated for 10 minutes at 50 oC. The digestion products were separated on a 1 % agarose gel:</p>
+<p>The reaction was incubated for 10 minutes at 50 °C. The digestion products were separated on a 1 % agarose gel:</p>
 <p> <img src="https://2019.igem.org/wiki/images/0/05/T--ABOA--Fanny_notes_img3.png" style="width:554px;height:269px;"> </p>
@@ Line 5,996: / Line 5,998: @@
 <br> we attempted test production on a small scale of the DengueFab0 in the 321.A strain, but we soon realized that it has a mutation in a stop codon (TAA > TAG), leading to a nonsense peptide that is not able to bind anything. </p>
 <p> <b> July 30th </b> </p>
-<p> New 4 ml SB growths containing ampicillin and 0.5 % glucose were inoculated with pLK04RCH-Tag-DengueFab0 and incubated o/n at 37 oC. </p>
+<p> New 4 mL SB growths containing ampicillin and 0.5 % glucose were inoculated with pLK04RCH-Tag-DengueFab0 and incubated o/n at 37°C. </p>
 <p> <b> July 31st </b> </p>
 <p> Glycerol preps and minipreps were made from the o/n cultures according to our protocols. The DNA concentration in the minipreps:</p>
-<p> pLK04RCH-Tag-DengueFab0 (prep 1): 27.9 ng/µl
+<p> pLK04RCH-Tag-DengueFab0 (prep 1): 27.9 ng/µL
-<br> pLK04RCH-Tag-DengueFab0 (prep 2): 7.9 ng/µl
+<br> pLK04RCH-Tag-DengueFab0 (prep 2): 7.9 ng/µL
-<br> pLK04RCH-Tag-DengueFab0 (prep 3): 40.8 ng/µl </p>
+<br> pLK04RCH-Tag-DengueFab0 (prep 3): 40.8 ng/µL </p>
 <p> The vector (prep 1 and 3) was re-digested with SfiI: </p>
 <p>
@@ Line 6,041: / Line 6,043: @@
 <tr>
 <td width="301">
-<p>total volume (&micro;l)</p>
+<p>total volume (µL)</p>
 </td>
 <td width="301">
@@ Line 6,049: / Line 6,051: @@
 </tbody>
 </table> </p>
-<p>The reaction was incubated for 10 minutes at 50 oC, and the digestion fragments separated on a 1 % agarose gel.</p>
+<p>The reaction was incubated for 10 minutes at 50°C, and the digestion fragments separated on a 1 % agarose gel.</p>
 <p>There was a 8 kB band visible in the gel, which is too large to be the vector.</p>
 <p> <b> August 5th </b> </p>
 <p> Present: Janniina </p>
-<p> 2 new colonies from the plate containing XL-1 with pLK04RCHTag-Dengue Fab0 were picked and inoculated to 4 ml of SB with ampicillin and 0.5 % glucose and incubated o/n at 37 °C, 300 rpm.</p>
+<p> 2 new colonies from the plate containing XL-1 with pLK04RCHTag-Dengue Fab0 were picked and inoculated to 4 mL of SB with ampicillin and 0.5 % glucose and incubated o/n at 37 °C, 300 rpm.</p>
 <p> <b> August 6th </b> </p>
 <p> Present: Janniina </p>
 <p> Glycerol preps and minipreps were made from the o/n cultures according to our protocols. The DNA concentration in the minipreps: </p>
-<p> pLK04RCH-Tag-DengueFab0 (prep 1): 413.6 ng/µl
+<p> pLK04RCH-Tag-DengueFab0 (prep 1): 413.6 ng/µL
-<br> pLK04RCH-Tag-DengueFab0 (prep 2): 512.7 ng/µl </p>
+<br> pLK04RCH-Tag-DengueFab0 (prep 2): 512.7 ng/µL </p>
 <p> SfiI digestion: </p>
 <p>
@@ Line 6,098: / Line 6,100: @@
 <tr>
 <td width="301">
-<p>total volume (&micro;l)</p>
+<p>total volume (µL)</p>
 </td>
 <td width="301">
@@ Line 6,106: / Line 6,108: @@
 </tbody>
 </table> </p>
-<p> The reaction was incubated for 10 minutes at 50 oC, and the digestion fragments separated on a 1 % agarose gel. </p>
+<p> The reaction was incubated for 10 minutes at 50 °C, and the digestion fragments separated on a 1 % agarose gel. </p>
-<p> Comment: We now had bands of the correct size in the gel. So the pLK04RCH-Tag-DengueFab0 vector was sent for sequencing. </p>
+<p> Comment: We now had bands of the correct size in the gel.Therefore, pLK04RCH-Tag-DengueFab0 vector was sent for sequencing. </p>
@@ Line 6,133: / Line 6,135: @@
 <p> E.coli 321.A was transformed with pEVOL-pAzF and pLK04RCH-AntiDengueFab plasmids according to electroporation protocol. </p>
-<p> After transformation, cells were plated on LA+ampicillin+cloramphenicol plates. Plates were incubated o/n in 37 C. </p>
+<p> After transformation, cells were plated on LA+ampicillin+cloramphenicol plates. Plates were incubated o/n in 37 °C. </p>
 <p> <b> Friday July 26th</b> </p>
-<p> After o/n incubation, plates were put on +4 C. </p>
+<p> After o/n incubation, plates were put on +4 °C. </p>
 <p> <b> Sunday July 28th </b> </p>
-<p> 4 ml precultures or test production was started. Precultures were done on each AntiDengueFab. Preculture was done in SB-medium containing 0,5 % glucose ampicillin, cloramphenicol and zeocin. </p>
+<p> 4 mL precultures or test production was started. Precultures were done on each AntiDengueFab. Preculture was done in SB-medium containing 0.5 % glucose ampicillin, cloramphenicol and zeocin. </p>
-<p> All precultures were put on 250 rpm shaking 37 C for o/n. </p>
+<p> All precultures were put on 250 rpm shaking 37 °C for o/n. </p>
 <p> <b>  July 28th </b> </p>
-<p> Each preculture was diluted to OD 0.1 with similar SB medium as before, so that the final volume of cultures were 4 ml. Cultures were put to grow in 37 C 250 rpm shaking. </p>
+<p> Each preculture was diluted to OD 0.1 with similar SB medium as before, so that the final volume of cultures were 4 mL. Cultures were put to grow in 37 °C 250 rpm shaking. </p>
-<p> When OD reached 0.6-0.8, 20 µl of 20 % arabinose and 80 µl of 50 mM pAzF was added to growth medium. Cultures were protected from the light from this point on due to light sensitivity of p-Azidophenylalanine. </p>
+<p> When OD reached 0.6-0.8, 20 µL of 20 % arabinose and 80 µL of 50 mM pAzF was added to growth medium. Cultures were protected from the light from this point on due to light sensitivity of p-Azidophenylalanine. </p>
-<p> After 10 min of incubation 4 µl 0.1 M IPTG was added to growth medium and temperature was lowered to 26 C and cultures were incubated o/n. </p>
+<p> After 10 min of incubation 4 µL 0.1 M IPTG was added to growth medium and temperature was lowered to 26 °C and cultures were incubated o/n. </p>
 <p> <b> July 29th </b> </p>
@@ Line 6,183: / Line 6,185: @@
 <p> Riku, Linnea, Pyry, Janniina </p>
-<p> We came to suspect, that at some point we have mixed at least the plasmids pUC19 and the original pLK04RCH we had been multiplying and purifying ourselves. Therefore we decided to SfiI digest and separate on a 1% agarose gel everything we thought might have become compromised in addition to the constructs we had just cloned and purified. </p>
+<p> We came to suspect, that at some point we have mixed at least the plasmids pUC19 and the original pLK04RCH we had been multiplying and purifying ourselves. Therefore, we decided to SfiI digest and separate on a 1 % agarose gel everything we thought might have become compromised in addition to the constructs we had just cloned and purified. </p>
-<p> SfiI digestions were done using the enzyme from thermofisher according to their protocol. The digested constructs were:
+<p> SfiI digestions were done using the enzyme from Thermo Fisher according to the manufacturer’s protocol. The digested constructs were:
 <br>pLK04RTagCH+harm. (5 colonies: ⅕-5/5)
-<br>pLK04RCH (1 colony from a plate (1/1) and 3 from another plate (3/1, 3/2 & 3/3)). <br>Also, two copies of the plasmid we had been multiplying in 321.A, that was thought to be the pLK04RCHTag from professor Huovinen was also SfiI digested. In the last well 1 µl of the vector gotten from Huovinen was loaded undigested by adding 10 µl of MQ. </p>
+<br>pLK04RCH (1 colony from a plate (1/1) and 3 from another plate (3/1, 3/2 & 3/3)). <br>Also, two copies of the plasmid we had been multiplying in 321.A, that was thought to be the pLK04RCHTag from professor Huovinen was also SfiI digested. In the last well 1 µL of the vector gotten from Huovinen was loaded undigested by adding 10 µL of MQ. </p>
-<p> The samples and 1 kb ladder (Nippon Genetics) were loaded with MidoriDirect (BioRad) according to the manufacturer (0,5 µl in a sample and 1 µl to a DNA ladder) to visualize the bands under blue light and orange plastic filter.
+<p> The samples and 1 kb ladder (Nippon Genetics) were loaded with MidoriDirect (BioRad) according to the manufacturer (0.5 µL in a sample and 1 µL to a DNA ladder) to visualize the bands under blue light and orange plastic filter.
-After the gel run we discovered, that the pLK04RTagCH+harm. ⅕ - ⅘ transformants as well as the pLK04RCH 3/3 did not show the supposed 3.7 and 1.5 kb bands (figure X). The 5/5, 1/1, 3/1-3/3 were chosen to be sequenced. </p>
+After the gel electrophoresis we discovered, that the pLK04RTagCH+harm. ⅕ - ⅘ transformants as well as the pLK04RCH 3/3 did not show the supposed 3.7 and 1.5 kb bands (figure X). The 5/5, 1/1, 3/1-3/3 were chosen to be sequenced. </p>
 <p> <img src="https://2019.igem.org/wiki/images/a/af/T--ABOA--Janniina_notes_img1.png" style="width:800px;height:370px;">
@@ Line 6,198: / Line 6,200: @@
 <p>From the gel, it can also be seen that the original vector was possibly present as a double plasmid. More disturbingly, the gel run also revealed that the original pLK04RCHTag plasmid that we have thought we have been multiplying did not create the correct sized bands indicating that it was in fact some other similar sized plasmid we have been working with. We had been multiplying pUC19 at the same time to be able to clone the biobrick into it. We have also plated the GFP-TAG-RFP gene from the biobrick gotten from the iGEM plate that was cloned into pUC19. </p>
-<p> Since both pUC19 and the pLK04 vectors we had, also had XbaI site, we decided to digest the suspicious plasmids with it (FastDigest XbaI, ThermoFisher) and run them on a gel. However, we later figured out, that all our strains has the native methylation enzymes, which create methylations sites blocking XbaI (see ____). </p>
+<p> Since both pUC19 and the pLK04 vectors we had, also had XbaI site, we decided to digest the suspicious plasmids with it (FastDigest XbaI, ThermoFisher) and run them on a gel. However, we later figured out, that all our strains have the native methylation enzymes, which create methylations sites blocking XbaI (see ____). </p>
 <p> Since either the plasmid or MidoriDirect was forgotten from the digestion sample, there was nothing to see in the gel. Regardless, it was quite clear that the plasmids had been mixed up at some point, so we got rid of all the plasmids we thought were possibly mixed. Since we still needed pUC19 and pLK04RCH backbones, we transformed the original pLK04RCH plasmid achieved from Tuomas and the pUC19 (2nd try) we got from professor Käpylä. </p>
@@ Line 6,223: / Line 6,225: @@
 <p> Janniina & Pyry </p>
-<p> We were encountering some difficulties with extracting pLK04-based vectors with our Fabs containing PelB signal sequence; the colonies with Fab0 we were able to pick from the plate and grow in liquid medium seemed to have disturbingly often a deletion in the PelB signal sequence according to our sequencing services purchased from Eurofins. However the sequences containing amber stop codons showed at least one good sequence per cloning with XL-1 as the host. This could be due to the toxicity of Fabs to E. coli, since XL-1 is not able to translate the complete Fab sequence due to the amber stop codon and the absence of corresponding tRNA synthetase. </p>
+<p> We were encountering some difficulties with extracting pLK04-based vectors with our Fabs containing PelB signal sequence; the colonies with Fab0 we were able to pick from the plate and grow in liquid medium seemed to have disturbingly often a deletion in the PelB signal sequence according to our sequencing services purchased from Eurofins. However, the sequences containing amber stop codons showed at least one good sequence per cloning with XL-1 as the host. This could be due to the toxicity of Fabs to E. coli, since XL-1 is not able to translate the complete Fab sequence due to the amber stop codon and the absence of corresponding tRNA synthetase. </p>
 <p> So we decided to use a sledgehammer to crack a nut and change the signal sequence with restriction enzymes XbaI and XhoI. To maximize the probability to succeed, we tried a pLK04 vector with harmonized Fab and a signal sequence obtained from utu (not shown) as the “donor”.</p>
-<p> Today, we transformed the donor from utu into XL-1 and digested the recipient vector using the enzymes from Thermofisher. The sizes of the bands were not checked with benchling’s virtual digestion, but a band close to the size of the vector was cut from the 1 % agarose gel after electrophoresis. A colony from the transformation today was picked to inoculate 4 ml of SB containing ampicillin and grown over night for plasmid extraction.</p>
+<p> Today, we transformed the donor from utu into XL-1 and digested the recipient vector using the enzymes from Thermo Scientific. The sizes of the bands were not checked with Benchling’s virtual digestion, but a band close to the size of the vector was cut from the 1 % agarose gel after electrophoresis. A colony from the transformation today was picked to inoculate 4 mL of SB containing ampicillin and grown over night for plasmid extraction.</p>
-<p> The following day, the signal sequence donor plasmid was extracted and 1394 ng of it was digested with FastDigest XhoI and XbaI. While the digestion was incubating, using the virtual digestion attribute of benchling I checked the sizes of the insert and the vector to calculate the ligation reactions. Based on the virtual digestion we learned that instead of one XhoI and two XbaI sites there were actually two of both of them in the pLK04RCHTag+Fab0 with deletion making it too difficult to change the signal sequence by digesting and ligating. Also this meant that the fragment cut from the gel yesterday was missing all of the Fab sequence and little extra making our customary SfiI digestion just as useful. Therefore we did not continue to change the signal sequence, but to try to pick new colonies and to try to clone the correct Fab0 sequence from ____ vector to the pLK04RCHTag backbone by SfiI digestion. </p>
+<p> The following day, the signal sequence donor plasmid was extracted and 1394 ng of it was digested with FastDigest XhoI and XbaI. While the digestion was incubating, using the virtual digestion attribute of benchling I checked the sizes of the insert and the vector to calculate the ligation reactions. Based on the virtual digestion we learned that instead of one XhoI and two XbaI sites there were actually two of both of them in the pLK04RCHTag+Fab0 with deletion making it too difficult to change the signal sequence by digesting and ligating. Also this meant that the fragment cut from the gel yesterday was missing all of the Fab sequence and little extra making our customary SfiI digestion just as useful. Therefore we did not continue to change the signal sequence, but to try to pick new colonies and to try to clone the correct Fab0 sequence from vector to the pLK04RCHTag backbone by SfiI digestion. </p>
                                  </div>
@@ Line 6,271: / Line 6,273: @@
                                          </p>
                                          <p>
-                                             Goat-anti-human plates were used in determination of Fab amount in samples. Fab standard was made so that there was 10, 50, 100, 150, 300 ng (rhFab in TSA, 3,62 mg/mL, DIBT-kurssi) of fab in the wells.
+                                             Goat-anti-human plates were used in determination of Fab amount in samples. Fab standard was made so that there was 10, 50, 100, 150, 300 ng (rhFab in TSA, 3,62 mg/mL) of fab in the wells.
-<br>Fabs and lysates were diluted 1/10, 1/100 and 1/1000 before adding to the wells in the volume or 200 µL. Each sample was added to the plateas triplicates. The samples were incubated for 30 minutes in shaking before washing of the unbounds.
+<br>Fabs and lysates were diluted 1/10, 1/100 and 1/1000 before adding to the wells in the volume or 200 µL. Each sample was added to the plate as triplicates. The samples were incubated for 30 minutes in shaking before washing of the unbounds.
 					</p>
                                          <p>
-µL of 250 ng/µL Eu-labeled antibody solution was put into each well and plate incubated in shaking for 30 minutes. Plate was washed and 200 µl of EFI per well was added with delfia plate dispenser. After 10 min incubation, time-resolved fluorescence was measured with Hidex. As a result it was noticed that there were no dengue fabs in the reaction.
+µL of 250 ng/µL Eu-labeled antibody solution was put into each well and plate incubated in shaking for 30 minutes. Plate was washed and 200 µL of Delfia Enhancement Solution per well was added with delfia plate dispenser. After 10 min incubation, time-resolved fluorescence was measured with Hidex. As a result, it was noticed that there were no dengue fabs in the reaction.
                                          </p>
                                          <p>
@@ Line 6,293: / Line 6,295: @@
 </td>
 <td>
-<p>ng/&micro;l</p>
+<p>ng/µL</p>
 </td>
 <td>
-<p>&micro;l of 2400 ng/&micro;l stock</p>
+<p>µL of 2400 ng/µL stock</p>
 </td>
 <td>
@@ Line 6,302: / Line 6,304: @@
 </td>
 <td>
-<p>&micro;l AB</p>
+<p>µL AB</p>
 </td>
 <td>
-<p>V_tot (&micro;l)</p>
+<p>V_tot (µL)</p>
 </td>
 <td>
-<p>Add &micro;l of standard Fab</p>
+<p>Add µL of standard Fab</p>
 </td>
 <td>
-<p>&micro;l AB</p>
+<p>µL AB</p>
 </td>
 </tr>
@@ Line 6,504: / Line 6,506: @@
 					</p>
 					<p>
-ml of Eu-2A11 0.25 ng/mL dilution was made from 20 µg/ml stock to Assay Buffer and the solution was filtered. Add 200 µl of the 2A11 dilution to each well, The tracer was incubated for 30 minutes. Unbounds were removed by washing the wells four times. 200 µL of Delfia Enhancement solution was added to each well and incubated 10 min in shaking. Time-resolved fluorescence was measured with Hidex.
+mL of Eu-2A11 0.25 ng/mL dilution was made from 20 µg/ml stock to Assay Buffer and the solution was filtered. Add 200 µL of the 2A11 dilution to each well, The tracer was incubated for 30 minutes. Unbounds were removed by washing the wells four times. 200 µL of Delfia Enhancement solution was added to each well and incubated 10 min in shaking. Time-resolved fluorescence was measured with Hidex.
 					</p>
 					<p>
@@ Line 6,513: / Line 6,515: @@
 					</p>
 					<p>
-ml of biotinylated digoxigenin (0,1 ng/µl concentration) was made. 200 µl of the 0,1 ng/µl bio-dig dilution was added to each well and incubated for 30. 15 ml of 0.125 ng/µL dilution of Eu-streptavidin (stock concentration 0.1 mg/mL) was made by adding 18.8 µL of Eu-Streptavidin to 15 mL of Assay Buffer.
+mL of biotinylated digoxigenin (0.1 ng/µL concentration) was made. 200 µL of the 0.1 ng/µL bio-dig dilution was added to each well and incubated for 30. 15 mL of 0.125 ng/µL dilution of Eu-streptavidin (stock concentration 0.1 mg/mL) was made by adding 18.8 µL of Eu-Streptavidin to 15 mL of Assay Buffer.
 					</p>
 					<p>
@@ Line 6,556: / Line 6,558: @@
                                          </p>
                                          <p>
-                                             Since the Fabs had started to decay, a new set of MBswas coated with digoxigenin Fabs 191, 108 and Histag as well as Fab NHS-AZ and Fab. 17.1 µg of MBs was used in each reaction, but the MBs were pipetted by accident in the protein stocks instead of the reaction tubes. After 5 minutes the mistake was noticed and the Fab stocks were pipetted into new tubes while holding the MBs with magnets. Assuming that there was 40 nmol of DBCO on 1 mg of MBs, 1.5 molar excess of digoxigenin Fabs was used to coat the 17.1 µg of MBs. PBS was added to the total volume of 250 µl, after which the SPAAC was incubated for 16 hours in +4 °C covered with aluminium foil. However the reaction with Fab 191 was forgotten in RT and disposed due to that.
+                                             Since the Fabs had started to decay, a new set of MBswas coated with digoxigenin Fabs 191, 108 and Histag as well as Fab NHS-AZ and Fab. 17.1 µg of MBs was used in each reaction, but the MBs were pipetted by accident in the protein stocks instead of the reaction tubes. After 5 minutes the mistake was noticed and the Fab stocks were pipetted into new tubes while holding the MBs with magnets. Assuming that there was 40 nmol of DBCO on 1 mg of MBs, 1.5 molar excess of digoxigenin Fabs was used to coat the 17.1 µg of MBs. PBS was added to the total volume of 250 µL, after which the SPAAC was incubated for 16 hours in +4 °C covered with aluminium foil. However the reaction with Fab 191 was forgotten in RT and disposed due to that.
                                          </p>
                                          <p>
@@ Line 6,563: / Line 6,565: @@
                                          </p>
                                          <p>
-                                             The MBs coated with digoxigenin Fab 108, Histag and NHS-AZ in addition to the MBs with Fab0 were washed first with 1000 µL of PBS. Since it took a long time for the MBs to aggregate in such a volume, the MBs were next washed with only 350 µL of PBS (500 µL last time). The MBs were washed twice with 250 µL of washing buffer (Kaivogen) before resuspending the coated MBs into 0.5 µg/µL of MBs in PBS with 0,05% 2-methyl-4-isothiazolin-3-one to preserve the Fabs. However it was visually observed that the size of the MB precipitates varied between the coated MBs. The coated MBs were stored in +4 °C for later use.
+                                             The MBs coated with digoxigenin Fab 108, Histag and NHS-AZ in addition to the MBs with Fab0 were washed first with 1000 µL of PBS. Since it took a long time for the MBs to aggregate in such a volume, the MBs were next washed with only 350 µL of PBS (500 µL last time). The MBs were washed twice with 250 µL of washing buffer (Kaivogen) before resuspending the coated MBs into 0.5 µg/µL of MBs in PBS with 0.05% 2-methyl-4-isothiazolin-3-one to preserve the Fabs. However, it was visually observed that the size of the MB precipitates varied between the coated MBs. The coated MBs were stored in +4 °C for later use.
                                          </p>
                                      </div>
@@ Line 6,570: / Line 6,572: @@
                              <div class="card">
+                                <!-- Card header -->
+                                <div class="card-header" role="tab" id="heading1094">
+                                    <a data-toggle="collapse" data-parent="#accordionEx" href="#collapse1094" aria-expanded="true"
+                                    aria-controls="collapse1094">
+                                    <h4>
+                                    Relative Fab activity determination on DBCO beads
+                                    </h4>
+                                    </a>
+                                </div>
+                                <!-- Card body -->
+                                <div id="collapse1094" class="collapse" role="tabpanel" aria-labelledby="heading1094"
+                                data-parent="#accordionEx2">
+                                    <div class="card-body">
+                                            <b>21th of August</b>
+<br>Karinsalo J. & Lindfors J.
+                                        </p>
+                                        <p>
+                                            <b>This is continuation to “Coating DBCO beads with NHS-containing Fab molecules”.</b>
+                                        </p>
+                     			<p>
+.5 ng/µL working solution of Eu-2A11 was made into Assay Buffer. The solution was filtered before use. 100 µL of 2A11 mAb working solution was added into wells. Therefore, there was 50 ng of Eu-2A11 mAb in each well.
+                                        </p>
+					<p>
+, 30, 60, and 100 ng of DBCO magnetic beads was added to each well in three replicates. In addition, non-coated DBCO bead control was used. 100 µL of Eu-2A11 mAb was added to wells and reaction was incubated for 1 hour in shaking.
+					</p>
+					<p>
+					    The beads were washed three times and 200 µL of DELFIA Enhancement Solution. Enhancement solution was incubated for 10 minutes in shaking and time-resolved fluorescence was measured with Hidex.
+					</p>
+                                    </div>
+                                </div>
+                            </div>
+                            <div class="card">
+                                <!-- Card header -->
+                                <div class="card-header" role="tab" id="heading1095">
+                                    <a data-toggle="collapse" data-parent="#accordionEx" href="#collapse1095" aria-expanded="true"
+                                    aria-controls="collapse1095">
+                                    <h4>
+                                    Optimization of click immunomagetic bead (CIMB) concentration in assay
+                                    </h4>
+                                    </a>
+                                </div>
+                                <!-- Card body -->
+                                <div id="collapse1095" class="collapse" role="tabpanel" aria-labelledby="heading1095"
+                                data-parent="#accordionEx2">
+                                    <div class="card-body">
+                                            <b>21th of August</b>
+<br>Karinsalo J. & Lindfors J.
+                                        </p>
+                                        <p>
+µg of beads was added to each well/tube in 100 µL. 10 µg/mL stock from empty MB was made by adding 180 µL of the 100 µg/ml stock to 1620 µL of Assay Buffer.
+                                        </p>
+                     			<p>
+                                            Because there wasn’t enough of 10 µg/mL stock 15 µg/mL stock was diluted by adding 600 µL of beads to 300 µL of Assay Buffer. 0.01, 0.1, 1, 10, 50 ng of Eu-2A11 was added to each well in the volume of 100 µL in Assay Buffer. The Eu-226A2 solution was filtered before use to reduce %CV.
+The tracer was incubated at shaking for 1h.
+                                        </p>
+					<p>
+					    The wells were washed three times with Kaivogen wash buffer on a magnetic plate holder. (Invitrogen). 200 µL of Delfia Enhancement solution was added to wells and incubated for 10 minutes in shaking. Time-resolved fluorescence was measured with Hidex
+					</p>
+					<p>
+					    Table 1. Results
+					</p>
+                                    </div>
+                                </div>
+                            </div>
+                            <div class="card">
                                  <!-- Card header -->
                                  <div class="card-header" role="tab" id="heading1097">
@@ Line 6,591: / Line 6,665: @@
 					</p>
                                          <p>
-µl of azido derivatized Dengue Fab0 (stock concentration 0.09 mg/mL) and Digox fab 0 (0.58 mg/mL) were biotinylated. Additionally, 500 µL of HistagTAG Digox Fab (stock concentration 0.80 mg/mL) was biotinylated with three-fold molar excess of DBCO-PEG4-Biotin.
+µL of azido derivatized Dengue Fab0 (stock concentration 0.09 mg/mL) and Digox fab 0 (0.58 mg/mL) were biotinylated. Additionally, 500 µL of HistagTAG Digox Fab (stock concentration 0.80 mg/mL) was biotinylated with three-fold molar excess of DBCO-PEG4-Biotin.
                                          </p>
                                          <p>
@@ Line 6,601: / Line 6,675: @@
                                          </p>
 					<p>
-µl of Fab0 was incubated with NHS-PEG4-azide (stock concentration 0.23 mg/mL).
+µL of Fab0 was incubated with NHS-PEG4-azide (stock concentration 0.23 mg/mL).
 					</p>
 					<p>
@@ Line 6,621: / Line 6,695: @@
 </td>
 <td style="width: 148.65px;">
-<p>DBCO-PEG4-biotin (&micro;L)</p>
+<p>DBCO-PEG4-biotin (µL)</p>
 </td>
 </tr>
@@ Line 7,769: / Line 7,843: @@
                              <div class="card">
-<!-- Päivä alkaa tästä-->
-                            <!-- Card header -->
-                            <div class="card-header" role="tab" id="heading302">
-                                <a data-toggle="collapse" data-parent="#accordionEx" href="#collapse302" aria-expanded="true"
-                                aria-controls="collapse302">
-                                <h4>
-nd of April
-                                </h4>
-                                </a>
-                            </div>
-                            <!-- Card body -->
-                            <div id="collapse302" class="collapse" role="tabpanel" aria-labelledby="heading302"
-                            data-parent="#accordionEx302">
-                                <div class="card-body">
-                                    <p>
-                                        Alotettiin kasvattaa
-                                    </p>
-                                    <p>
-                                        Puhastettiin ja testattiin antibodyjä
-                                    </p>
-                                </div>
-                            </div>
-                        </div>
-                        <!--Päivä loppuu tähän!-->
                      </div>
                      <!-- Alaotsikko loppuu tähän!-->
@@ Line 7,800: / Line 7,849: @@
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 </html>
+{{Aboa/Footer}}

Difference between revisions of "Team:Aboa/Notebook"

Latest revision as of 18:28, 21 October 2019

Team Aboa Notebook

Table of contents

Work with Cell lines

Week 1

Week 2

Testing the amber suppression of E.coli 321A and pEVOL- pAzF with BBa_K567011 (GFP-tag-RFP)

Week 3

Week 4

Week 5

Week 6

Week 7

Week 8

Week 9

Week 10

Week 11

Producing Digoxigenin Fabs

Cloning Digoxigenin Fabs into pLK04RCHTag

Digoxigenin-Fab-TAG cloning of ordered variants

One-Pot Triple fragment cloning of pEB04CH SfiI/SfiI and SfiI/HindIII fragments to replace existing pLK04 Histag

Cloning Digoxigenin fabs into pLK04RCHTag

Cloning Synthesised Digox Fabs into pLK04RCH

Production of Digoxigenin Fabs

Production of pLK04RCHTag-harmonized digoxigenin Fab

Production of DigoxFab0, DigoxFab108 and DigoxFab191 in pLK04RCH

ANTIBODY PRODUCTION AND PURIFICATION

Last Production of Azido-Modified Fabs

Producing Dengue Fabs

Cloning DengueFabs to pLK04RCH/Tag

Test production of dengue Fabs

Troubleshooting

Testing all previously restricted plasmids

Changing signal sequence to pLK04RCHTag+Fab0 digox

Immunoassays

Fab activity determination on Goat Anti-Human plates

Coating DBCO beads with NHS-containing Fab molecules

Relative Fab activity determination on DBCO beads

Optimization of click immunomagetic bead (CIMB) concentration in assay

Biotinylation of Fabs with DBCO Biotin

Determination of Streptavidin plate binding capacity with Fabs biotinylated with different biotinylation strategies