Difference between revisions of "Team:uOttawa/EmilyLabNotebook"

Latest revision as of 02:53, 4 August 2019

Wednesday, May 22nd 2019

Start trying to optimize the procedure to incorporate (yeast) KanMX, prefix, and suffix into a backbone containing yeast homologies.

PCR 1: add a MfeI cut site and prefix to KanMX.

Tube	ddH2O	Buffer	dNTP	Fp (pp9)	Rp (pp10)	Phusion (homemade)	DNA (Kan prefix)
1	16.56	5	0.5	0.125	0.125	0.25	2.44
2	16.56	5	0.5	0.125	0.125	0.25	2.44
3	16.56	5	0.5	0.125	0.125	0.25	2.44
0	19	5	0.5	0.125	0.125	0.25	0
Master mix	82.8	25	2.5	0.625	0.625	1.25	0

KanMX DNA concentration = 40.9ng/µL

Approximate length = 1474 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 1474 x (15s/1000bp) + 15s

Annealing time = 37s

PCR protocol:

98°C for 3 min
98°C for 10s
70°C for 30s
72°C for 37s
Repeat 2-4 30x
72°C for 10 min
4°C for infinity

Gel PCR products:

Ladder, 0-4

The gel shows bright bands around the 1400bp mark, which confirms that the PCR was successful.

The PCR tubes were combined, purified, and the concentration was measured (209.4ng/µL).

Thursday, May 23rd 2019

PCR 2: add a suffix (and MfeI again) to the product of PCR 1.

Tube	ddH2O	Buffer	dNTP	Fp (pp19)	Rp (pp10)	Phusion (homemade)	DNA (PCR product)
1	18.5	5	0.5	0.125	0.125	0.25	0.5
2	18.5	5	0.5	0.125	0.125	0.25	0.5
3	18.5	5	0.5	0.125	0.125	0.25	0.5
0	19	5	0.5	0.125	0.125	0.25	0
Master mix	92.5	25	2.5	0.625	0.625	1.25	0

Use the same annealing temperature, time, and PCR protocol since the fragment lengths are approximately the same.

Gel PCR products:

Ladder, 0-4

The gel shows bright bands around the 1400bp mark, which confirms that the PCR was successful.

The PCR tubes were combined, purified, and the concentration was measured.

The PCR product as well as the plasmid (IAde2 PSB1K3) were digested with MfeI-HF and PstI-HF.

Tube	DNA	CutSmart	MfeI-HF	PstI-HF	ddH2O
IAde2 PSB1K3	8.9	5	1	1	34.1
KanMX, MfeI, prefix, suffix	11.5	5	1	1	31.5

Incubate at 37°C overnight.

Friday, May 24th 2019

Add phosphatase to the IAde2 PSB1K3 digest to dephosphorylate the 5’ end and prevent recircularization.

Reagent	Volume (µL)
ddH2O	1
Antarctic phosphatase	3
Buffer	6
IAde2 PSB1K3 digest	50

Incubate at 37°C for 30 minutes.

Heat shock both digest solutions at 80°C for 20 minutes.

Ligate IAde2 PSB1K3 and the KanMX insert.

The vector and insert are 2389bp and 1474bp long, respectively.

Math:

50ng vector x (60µL/1000ng) = 3µL vector

2389 bp x (650g/mol bp) = 1 552 850g/mol

50x10^-9g x (mol/1 552 850g) = 3.22x10^-14 mol vector

3.22x10^-14mol x 4 = 1.29x10^-13 mol insert

1474bp x (650g/mol bp) = 958 100g/mol

1.29x10^-13 mol x (958 100g/mol) = 1.23x10^-7 g = 123ng

123ng x (50µL/1000ng) = 6.15µL

Reagent	Volume (µL)
KanMX-EXSP digest	6.15
IAde2 PSB1K3 digest	3
T4 ligase	1
T4 ligase buffer	2
ddH2O	7.85

Incubate ligation mixture at room temperature for 2 hours, and then heat shock at 65 degrees for 10 minutes.

Transform the entire ligation mixture using the following protocol:

Thaw competent cells in ice.
Add 50µL of competent cells and the entire ligation mixture into a 1.5mL tube. For the zero control, pipette 50µL of competent cells (without DNA) into another 1.5mL tube.
Incubate on ice for 30 minutes.
Heat shock at 42°C for 30s.
Incubate on ice for 5 minutes.
Add 950µL of SOC to each tube.
Incubate at 37°C for 1 hour, shaking at 200-300rpm.
Add 100µL of the transformation and 100µL of the zero control onto LB Kan plates.
Incubate plates overnight at 37°C.

Monday, May 27th 2019

Setti took the plates out on Saturday. There was good growth on the plate (approximately 30 colonies) and no growth on the negative.

Inoculate 12 (half) colonies as well as 2 negatives in 3mL of LB Kan at 37°C for 6 hours.

Also inoculate 4 tubes of PSB1K3 IAde2 homo, IAde4 homo, IGal4 homo, and 3His3 homo in 3mL of LB Kan to replenish DNA stocks.

Plate glycerol stocks of PSB1K3 IAde2 homo and KanMX::EXSP on LB Kan to create negative and positive controls for the colony PCR tomorrow.

Miniprep the 12 inoculations.

Tuesday, May 28th 2019

The PSB1K3 IAde2 homo, IAde4 homo, IGal4 homo, and 3His3 homo inoculations grew, but it took a long time (it still looked clear after 6 hours yesterday). Miniprep and double check the contents of the stock with PCR.

Primers:

His3: pp17, pp18

Ade2: pp15, pp16

Ade4: pp11, pp12

Gal4: pp13, pp14

Tube	ddH2O	Buffer	dNTP	Fp	Rp	Phusion (homemade)	DNA (Kan prefix)
His3	17.8	5	0.5	0.125	0.125	0.25	1.2
Ade2	17.6	5	0.5	0.125	0.125	0.25	1.4
Ade4	17.9	5	0.5	0.125	0.125	0.25	1.1
Gal4	16.7	5	0.5	0.125	0.125	0.25	2.3
0	19	5	0.5	0.125	0.125	0.25	0

Annealing temp = 65°C

Approximate length = 270 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 270 x (15s/1000bp) + 15s

Annealing time = 19s

PCR protocol:

98°C for 3 min
98°C for 10s
65°C for 30s
72°C for 19s
Repeat 2-4 30
72°C for 10 min
4°C for infinity

Gel PCR products.

Ladder, His3, Ade2, Ade4, Gal4, 0

The gel has the correct bands around 270bp, which means that the stocks are fine.

Proceed with the digestion of the mini-prepped colonies, positive, and negative with PmeI. Only digest 500ng of each DNA sample, since the digested DNA will not be used for further applications.

Tube	DNA	CutSmart	PmeI	ddH2O
1	13.3	5	1	30.7
2	11.4	5	1	32.6
3	9.8	5	1	34.2
4	10.4	5	1	33.6
5	15.8	5	1	28.2
6	19.6	5	1	24.4
7	7.5	5	1	36.5
8	9.9	5	1	34.1
9	10.8	5	1	33.2
10	8.3	5	1	35.7
11	9.7	5	1	34.3
12	11.5	5	1	32.5
Negative (Ade2 homo)	6.8	5	1	37.2
Positive (Ade2::KanMX)	4.9	5	1	39.1

Incubate at 37°C for 2 hours.

Additionally, perform a colony PCR using the other half of the colonies from the plates, as well as the colonies from the Ade2 homo and Ade2::KanMX EXSP PSB1K3 plates that were made yesterday.

Tube	ddH2O	Buffer	dNTP	Fp (pp9)	Rp (pp10)	Phusion (homemade)	DNA
1-12	14	5	0.5	0.125	0.125	0.25	5
Negative	14	5	0.5	0.125	0.125	0.25	5
Positive	14	5	0.5	0.125	0.125	0.25	5
Master mix	210	75	7.5	1.875	1.875	3.75	0

PCR protocol (same as initial PCR to add MfeI and prefix)

98°C for 3 min
98°C for 10s
70°C for 30s
72°C for 37s
Repeat 2-4 30x
72°C for 10 min
4°C for infinity

Gel digest and PCR products.

Large gel:

Ladder, 1-12 digest, negative digest, positive digest

Colonies 4, 6, and 10 are very strange since the bands do not correspond to the bands in the positive lane.

Small gel:

Ladder, 1-12 PCR, negative PCR, positive PCR

Nothing showed up in the gel, including the positive. Since this PCR was recently performed at the same annealing temperature with the homemade enzyme, the actual PCR protocol cannot be the issue.

Wednesday, May 29th 2019

The problem with the gel can be traced back to the initial digestion of the Ade2 PSB1K3 backbone. Although KanMX-MfeI-EXSP should be digested with MfeI and PstI, Ade2 PSB1K3 should be digested with EcoRI and PstI. However, I digested with MfeI and EcoRI create the same sticky ends, but recognize different cutsites, allowing the restriction sites to be eliminated.

So, redigest Ade2 PSB1K3 with EcoRI-HF and PstI-HF according to the following table:

Tube	DNA	CutSmart	EcoRI-HF	PstI-HF	ddH2O
Ade2 PSB1K3	8.9	5	1	1	34.1

I kept the KamMX-MfeI-EXSP digest with MfeI and PstI, so I don’t have to redigest.

Incubate the Ade2 digest at 37°C for 2 hours.

Add phosphatase to the Ade2 PSB1K3 digest to dephosphorylate the 5’ end and prevent recircularization.

Reagent	Volume (µL)
ddH2O	1
Antarctic phosphatase	3
Buffer	6
Ade2 PSB1K3 digest	50

Incubate at 37°C for 30 minutes.

Heat shock the solution at 80°C for 20 minutes.

Ligate the digests together (math is the same as the previous ligation).

Reagent	Volume (µL)
KanMX-EXSP digest	6.15
Ade2 PSB1K3 digest	3
T4 ligase	1
T4 ligase buffer	2
ddH2O	7.85

Incubate ligation mixture at room temperature for 2 hours, and then heat shock at 65 degrees for 10 minutes.

Transform the entire ligation mixture using the following protocol:

Thaw competent cells in ice.
Add 50µL of competent cells and the entire ligation mixture into a 1.5mL tube. For the zero control, pipette 50µL of competent cells (without DNA) into another 1.5mL tube.
Incubate on ice for 30 minutes.
Heat shock at 42°C for 30s.
Incubate on ice for 5 minutes.
Add 950µL of SOC to each tube.
Incubate at 37°C for 1 hour, shaking at 200-300rpm.
Add 100µL of the transformation and 100µL of the zero control onto LB Kan plates.
Incubate plates overnight at 37°C.

Thursday, May 30th 2019

There was ok growth on the plates (approximately 20 colonies). Regardless, go ahead with the inoculation without a colony PCR, since the colonies are quite small. Inoculate 13 colonies as well as a negative in 3mL of LB Kan at 37°C for 6 hours and miniprep.

Friday, May 31st 2019

Nanodrop the inoculations and digest 500ng of each tube as well as a positive with PmeI.

Tube	DNA	CutSmart	PmeI	ddH2O
1	21.3	5	1	22.7
2	7.7	5	1	36.3
3	15.3	5	1	28.7
4	5.8	5	1	38.2
5	8.4	5	1	35.6
6	7.9	5	1	36.1
7	11.0	5	1	33.0
8	9.0	5	1	35.0
9	8.2	5	1	35.8
10	8.3	5	1	35.7
11	7.1	5	1	36.9
12	9.3	5	1	34.7
13	7.7	5	1	36.3
Positive (Ade2::KanMX)	4.5	5	1	39.5

Incubate at 37°C for 2 hours and gel:

Ladder, 1-13, positive (Ade2::KanMX)

Only one band (backbone) appeared in each lane, which means that KanMX was not successfully incorporated.

Did some research and found that ThermoFisher offers a version of MfeI (MunI) that can be heat-shocked - this would allow us to bypass the column purification step that has been causing issues. However, ThermoFisher and NEB use different buffers, so Setti has asked Matt for advice.

Monday, June 3rd 2019

While waiting for Matt to get back to us, look into the 2017 and 2019 kit plates to find a part that can be improved (preferably involving dCas9 as an inhibitor since we already have a plasmid with dCas9-MxiI).

We found the following part: http://parts.igem.org/Part:BBa_K1723000 in well 7P of kit plate 7 from 2017. We’re in the process of thinking about different ways to use dCas9 from this part to improve http://parts.igem.org/Part:BBa_K1723021 (they have the same amino acid sequence) by adding MxiI from our part, or replacing VP64 with MxiI.

The problem is, dCas9 is quite long (4kb), which means that adding MxiI (150bp) by using multiple PCRs would be pretty difficult. As a result, we might have to resort to Gibson assembly.

Tuesday, June 4th 2019

Transform the BioBrick from the 2017 kit plate and plate 100µL, 200µL, and a negative on LB CM using the following protocol:

Resuspend DNA from the kit plate in 10µL ddH2O.
Add 50µL of competent cells and the entire DNA volume into a 1.5mL tube. For the zero control, pipette 50µL of competent cells (without DNA) into another 1.5mL tube.
Incubate on ice for 30 minutes.
Heat shock at 42°C for 30s.
Incubate on ice for 5 minutes.
Add 950µL of SOC to each tube.
Incubate at 37°C for 1 hour, shaking at 200-300rpm.
Add 100µL and 200µL of the transformation as well as 100µL of the zero control onto LB CM plates.
Incubate plates overnight at 37°C.

Wednesday, June 5th 2019

There was no growth on the plates from yesterday. Given that Taylor’s transformation also didn’t work, we have to troubleshoot the transformation procedure.

We suspect that there is an issue with the plates. To test this, I streaked a glycerol stock of RFP PSB1C3 on a CM plate to incubate at 37°C overnight.

Additionally, we can’t request more parts from the iGEM registry, so we have to find a new BioBrick to improve.

Matt got back to us about the enzyme buffers and said they were ok to combine. SpeI and MunI have 100% efficiency in NEBuffer 2.1 and buffer G, respectively. We also found that both the buffers are identical, we means we can just use buffer G (which is supplied with MunI) for the digest.

Thursday, June 13th 2019

Start troubleshooting the transformation protocol. Try to digest Setti’s plasmid (Ade2::KanMX EXSP PSB1K3) and RFP PSB1A3.

Tube	DNA	CutSmart	SpeI	EcoRI-HF	ddH2O
Ade2:KanMX	12.5	5	1	1	30.5
RFP amp	11.9	5	1	1	31.1

Incubate at 37°C for 2 hours.

Add phosphatase to the backbone (Ade2:KanMX):

Reagent	Volume (µL)
ddH2O	1
Antarctic phosphatase	3
Buffer	6
Ade2:KanMX	50

Incubate at 37°C for 30 mins.

Heat shock both digests at 80°C for 20 minutes.

Ligation math:

50ng vector x (60µL/1000ng) = 3µL vector

3862 bp x (650g/mol bp) = 2 510 300g/mol

50x10^-9g x (mol/2 510 300g) = 1.99x10^-14 mol vector

1.99x10^-14mol x 4 = 7.967x10^-14 mol insert

1091bp x (650g/mol bp) = 709 150g/mol

7.967x10^-14 mol x (709 150 100g/mol) = 56.5ng

56.5ng x (50µL/1000ng) = 2.825µL

Reagent	Volume (µL)
Ade2::KanMX digest	3
RFP PSB1A3 digest	2.8
T4 ligase	2
T4 ligase buffer	1
ddH2O	11.2

Incubate ligation mixture at room temperature for 2 hours, and then heat shock at 65 degrees for 10 minutes.

Transform the entire ligation mixture using the following protocol:

Thaw competent cells in ice.
Add 50µL of competent cells and the entire ligation mixture into a 1.5mL tube. For the zero control, pipette 50µL of competent cells (without DNA) into another 1.5mL tube.
Incubate on ice for 30 minutes.
Heat shock at 42°C for 30s.
Incubate on ice for 5 minutes.
Add 950µL of SOC to each tube.
Incubate at 37°C for 1 hour, shaking at 200-300rpm.
Add 100µL of the transformation and 100µL of the zero control onto LB Kan plates.
Incubate plates overnight at 37°C.

Friday, June 14th 2019

Yesterday’s transformation worked, albeit with a little less growth than usual. However, that means that our transformation protocols and materials are fine - the failed transformations may have just been bad luck.

MunI came in, so I can try to optimize the procedure to incorporate (yeast) KanMX, prefix, and suffix into a backbone containing yeast homologies.

We looked into the reaction volumes, and it seems like sticking with the normal ratios should be okay. Buffer G was used for both tubes instead of Cutsmart to minimize any errors that could be associated with combining two different buffers during the ligation.

Tube	DNA	Buffer G	EcoRI-HF	SpeI	ddH2O
Ade2 PSB1K3	4.5, 2, 3.4	5	1	1	33.1

Tube	DNA	Buffer G	MunI	SpeI	ddH2O
MfeI-KanMX-EXSP	11.5	5	1	1	31.5

Incubate at 37°C for 2 hours.

Add phosphatase to the IAde2 PSB1K3 digest to dephosphorylate the 5’ end and prevent recircularization.

Reagent	Volume (µL)
ddH2O	1
Antarctic phosphatase	3
Buffer	6
IAde2 PSB1K3 digest	50

Incubate at 37°C for 30 minutes.

Heat shock both digest solutions at 80°C for 20 minutes.

Ligate IAde2 PSB1K3 and the KanMX insert.

Reagent	Volume (µL)
KanMX-EXSP digest	6.15
IAde2 PSB1K3 digest	3
T4 ligase	1
T4 ligase buffer	2
ddH2O	7.85

Incubate ligation mixture at room temperature for an hour, and then heat shock at 65°C for 10 minutes.

Monday, June 17th 2019

Transform the ligation mixture from Friday on LB kan.

Also streak glycerol stock of any part in PSB1K3 on the new LB kan plates to ensure that the new tryptone we’re using is okay.

Also found a Biobrick to improve: http://parts.igem.org/Part:BBa_K592009

AmilCP, blue chromoprotein is obtained from coral. There is some evidence from other sources where it has been incorporated into yeast. So, the plan is to take this Biobrick, a promoter, a terminator, and add overlapping ends via PCR. All 3 parts will be incorporated into yeast, which should combine the parts for us. Then the DNA will be extracted and incorporated into a backbone.

So today, I started off by transforming the DNA from the 2019 kit plate 1, well 19E on LB CM.

Tuesday, June 18th 2019

The plates from yesterday all grew! The glycerol stock test on the new LB kan plates grew, which means that the new tryptone is fine.

The plate with the kit plate DNA had very good growth, whereas the plate for the standard assembly optimization protocol only had approximately 6 colonies on it.

Wednesday, June 19th 2019

Inoculated 3 colonies with the kit plate DNA in 5mL LB CM, and 6 colonies from the MunI optimization test in 3mL LB Kan for 6 hours, and then miniprep.

Thursday, June 20th 2019

Digested 500ng from each Ade2::KanMX tube with PmeI and incubated at 37°C for 2 hours.

Tube	DNA	CutSmart	PmeI	ddH2O
1	8.6	5	1	35.4
2	10.3	5	1	33.7
3	9.2	5	1	34.8
4	14.1	5	1	29.9
5	8.7	5	1	35.3
6	7.2	5	1	36.8
Positive	5.1	5	1	38.9

Gel digestion products:

Ladder, 1-6, positive

The gel did not visualize very well the first time around with 5uL of DNA. I tried again using 15uL of DNA, but there were no bands at all. It’s possible that the enzymes are interfering with the visualization, so heat shock the tubes at 80°C for 20 minutes.

Friday, June 21st 2019

Continue troubleshooting the gel: try gelling the tubes again after the heat shock using 10uL of DNA in the small cassette (instead of the large cassette).

Only the ladder and positive control showed up, so I redigested using 1000ng of DNA.

Tube	DNA	CutSmart	PmeI	ddH2O
1	17.2	5	1	26.8
2	20.6	5	1	23.4
3	18.4	5	1	25.6
4	28.2	5	1	15.8
5	17.4	5	1	26.6
6	14.4	5	1	29.6
Positive	10.2	5	1	33.8

Incubate at 37°C for an hour and then heat shock at 65°C for 20 minutes.

Gel 10uL of DNA in the small cassette.

Nothing visualized on the gel.

Emma suggested replacing the TAE buffer in the gel machines, letting the gel cool longer before adding RedSafe, and trying her alternative of RedSafe.

I gelled the following:

Large gel (RedSafe)

Digest 1, undigested 1, …, digest positive, undigested positive

*10uL for all except 5uL undigested 2, no undigested 4, 5uL undigested positive

Small gel (ViewSafe)

Digest 1, undigested 1, …, digest positive, undigested positive

10uL for all except 2uL undigested 2, 5uL undigested 3, 5uL undigested 5, 5uL undigested positive

Both gels visualized fine, but only singular bands (of varying lengths) were seen, none of which were indicative of successfully incorporating the insert.

We’re out of DNA from the minipreps, so we should take a step back: instead of trying to incorporate KanMX EXSP into Ade2 PSB1K3 directly, we should try to incorporate RFP into Ade2 PSB1K3 BEFORE adding KanMX to reduce the number of false positives via colorimetric selection.

Monday, June 24th 2019

Miniprep Ade2 PSB1K3 and Ade2::KanMX PSB1K3 inoculations from Sunday.

PCR prefix and suffix on RFP PSB1A3 using a gradient between 66°C and 70°C.

Temp (°C)	Tube	ddH2O	Buffer	dNTP	Fp (oo69)	Rp (oo70)	Phusion (homemade)	DNA (PCR product)
70	1	17.4	5	0.5	0.125	0.125	0.25	1.6
68.5	2	17.4	5	0.5	0.125	0.125	0.25	1.6
67	3	17.4	5	0.5	0.125	0.125	0.25	1.6
66	4	17.4	5	0.5	0.125	0.125	0.25	1.6
66	0	19	5	0.5	0.125	0.125	0.25	0
-	Master mix	104.4	30	3	0.75	0.75	1.5	0

Approximate length = 1069 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 1069bp x (15s/1000bp) + 15s

Annealing time = 31s

PCR protocol:

98°C for 3 min
98°C for 10s
66-70°C for 30s
72°C for 31s
Repeat 2-4 30x
72°C for 10 min
12°C for infinity

Gel PCR products:

Ladder, 1-4, 0

Only primer dimers could be seen on the gel. But instead of trying to PCR again, we should directly digest RFP in PSB1A3 and ligate it into Ade2 PSB1K3. However, I will hold off on this until materials for competent cells arrive - I find that digesting, ligating, and transforming in the same day tends to give me better results.

Tuesday, June 25th 2019

Prepared primers for the amilCP improvement.

Start by amplifying URA3 to the GPD promoter (plus amilCP homo).

Temp (°C)	Tube	ddH2O	Buffer	dNTP	Fp (qq10)	Rp (qq11)	Phusion (homemade)	DNA (GFP 3B)
60.9	1	18.6	5	0.5	0.125	0.125	0.25	0.37
59.8	2	18.6	5	0.5	0.125	0.125	0.25	0.37
58.4	3	18.6	5	0.5	0.125	0.125	0.25	0.37
57.4	4	18.6	5	0.5	0.125	0.125	0.25	0.37
57.4	0	19	5	0.5	0.125	0.125	0.25	0
-	Master mix	111.6	30	3	0.75	0.75	1.5	0

Approximate length = 1793 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 1793bp x (15s/1000bp) + 15s

Annealing time = 42s

PCR protocol:

98°C for 3 min
98°C for 10s
56-62°C for 30s
72°C for 42s
Repeat 2-4 30x
72°C for 10 min
12°C for infinity

Gel PCR products:

Ladder, 1-4, 0

There were bright bands between the 1500-2000bp bands, so PCR purify and nanodrop.

Also PCR the CYC1 terminator (plus amilCP homo) - URA3.

Temp (°C)	Tube	ddH2O	Buffer	dNTP	Fp (qq17)	Rp (qq19)	Phusion (homemade)	DNA (GFP 3B)
64.7	1	18.6	5	0.5	0.125	0.125	0.25	0.37
63.1	2	18.6	5	0.5	0.125	0.125	0.25	0.37
62	3	18.6	5	0.5	0.125	0.125	0.25	0.37
60.5	4	18.6	5	0.5	0.125	0.125	0.25	0.37
60.5	0	19	5	0.5	0.125	0.125	0.25	0
-	Master mix	111.6	30	3	0.75	0.75	1.5	0

Approximate length = 762 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 762bp x (15s/1000bp) + 15s

Annealing time = 26s

PCR protocol:

98°C for 3 min
98°C for 10s
60-65°C for 30s
72°C for 26s
Repeat 2-4 30x
72°C for 10 min
12°C for infinity

Gel PCR products:

Ladder, 1-4, 0

There were bright bands around the 700bp mark, which means the PCR worked.

Wednesday, June 26th 2019

PCR purify and nanodrop the DNA.

PCR GPD homo - amilCP CYC1 - terminator homo.

Temp (°C)	Tube	ddH2O	Buffer	dNTP	Fp (qq13)	Rp (qq15)	Phusion (homemade)	DNA (amilCP)
61.3	1	17.8	5	0.5	0.125	0.125	0.25	1.2
60.5	2	17.8	5	0.5	0.125	0.125	0.25	1.2
59.6	3	17.8	5	0.5	0.125	0.125	0.25	1.2
58.4	4	17.8	5	0.5	0.125	0.125	0.25	1.2
58.4	0	19	5	0.5	0.125	0.125	0.25	0
-	Master mix	106.8	30	3	0.75	0.75	1.5	0

Approximate length = 669 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 669bp x (15s/1000bp) + 15s

Annealing time = 27s

PCR protocol:

98°C for 3 min
98°C for 10s
57-62°C for 30s
72°C for 27s
Repeat 2-4 30x
72°C for 10 min
12°C for infinity

Gel PCR products:

Ladder, 1-4, 0

Bright bands can be seen around the 600-700bp mark, so the DNA was purified and nano-dropped.

PCR Ura3 - GPD promoter - amilCP homo (longer) using yesterday’s PCR product.

Temp (°C)	Tube	ddH2O	Buffer	dNTP	Fp (qq10)	Rp (qq12)	Phusion (homemade)	DNA (Ura3 - GPD promoter - amilCP)
60.1	1	15.2	5	0.5	0.125	0.125	0.25	3.8
59	2	15.2	5	0.5	0.125	0.125	0.25	3.8
58.2	3	15.2	5	0.5	0.125	0.125	0.25	3.8
57	4	15.2	5	0.5	0.125	0.125	0.25	3.8
57	0	19	5	0.5	0.125	0.125	0.25	0
-	Master mix	91.2	30	3	0.75	0.75	1.5	0

Approximate length = 1807 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 1807bp x (15s/1000bp) + 15s

Annealing time = 42s

PCR protocol:

98°C for 3 min
98°C for 10s
57-61°C for 30s
72°C for 42s
Repeat 2-4 30x
72°C for 10 min
12°C for infinity

Gel PCR products:

Ladder, 1-4, 0

Although there are some faint bands around the 1500-2000bp mark, they are not significantly bright - throw out the tubes and try again tomorrow using a larger temperature gradient.

PCR amilCP homo (longer) - CYC1 terminator - URA3 UTR.

Temp (°C)	Tube	ddH2O	Buffer	dNTP	Fp (qq18)	Rp (qq19)	Phusion (homemade)	DNA (amil CP homo - CYC1 terminator - URA3 UTR)
63.3	1	16.7	5	0.5	0.125	0.125	0.25	2.3
62.5	2	16.7	5	0.5	0.125	0.125	0.25	2.3
61.6	3	16.7	5	0.5	0.125	0.125	0.25	2.3
60.4	4	16.7	5	0.5	0.125	0.125	0.25	2.3
60.4	0	19	5	0.5	0.125	0.125	0.25	0
-	Master mix	100.2	30	3	0.75	0.75	1.5	0

Approximate length = 780 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 780bp x (15s/1000bp) + 15s

Annealing time = 27s

PCR protocol:

98°C for 3 min
98°C for 10s
60-64°C for 30s
72°C for 42s
Repeat 2-4 30x
72°C for 10 min
12°C for infinity

The bands were faint - redo the PCR with a larger gradient.

PCR amilCP homo (longer) - CYC1 terminator - URA3 UTR.

Temp (°C)	Tube	ddH2O	Buffer	dNTP	Fp (qq18)	Rp (qq19)	Phusion (homemade)	DNA (amilCP homo - CYC1 terminator - URA3 UTR
68.7	1	16.7	5	0.5	0.125	0.125	0.25	2.3
67.4	2	16.7	5	0.5	0.125	0.125	0.25	2.3
65.8	3	16.7	5	0.5	0.125	0.125	0.25	2.3
63.7	4	16.7	5	0.5	0.125	0.125	0.25	2.3
61.6	0	19	5	0.5	0.125	0.125	0.25	0
-	Master mix	102.6	30	3	0.75	0.75	1.5	0

Thursday, June 27th 2019

Gel PCR products from yesterday’s PCR of amilCP homo (longer) - CYC1 terminator - URA3 UTR: Ladder, 1-4, 0

The gel shows bands around the 780bp mark, so purify and nanodrop.

PCR GPD homo (longer) - amilCP - CYC1 terminator homo (longer).

Temp (°C)	Tube	ddH2O	Buffer	dNTP	Fp (qq14)	Rp (qq16)	Phusion (homemade)	DNA (GPD homo - amilCP - CYC1 terminator)
66.6	1	17.1	5	0.5	0.125	0.125	0.25	1.9
64.4	2	17.1	5	0.5	0.125	0.125	0.25	1.9
62.8	3	17.1	5	0.5	0.125	0.125	0.25	1.9
61.6	4	17.1	5	0.5	0.125	0.125	0.25	1.9
61.6	0	19	5	0.5	0.125	0.125	0.25	0
-	Master mix	102.6	30	3	0.75	0.75	1.5	0

Approximate length = 705 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 705bp x (15s/1000bp) + 15s

Annealing time = 26s

PCR protocol:

98°C for 3 min
98°C for 10s
60-67°C for 30s
72°C for 26s
Repeat 2-4 30x
72°C for 10 min
12°C for infinity

Gel PCR products:

Ladder, 0-4

The bands are present but very faint, so it should be redone using a larger temperature gradient.

Redo PCR for URA3 - GPD promoter - amil CP homo (longer) using a larger temperature gradient.

Temp (°C)	Tube	ddH2O	Buffer	dNTP	Fp (qq10)	Rp (qq12)	Phusion (homemade)	DNA (URA3 - GPD promoter - amilCP homo)
69	1	15.1	5	0.5	0.125	0.125	0.25	3.9
67.5	2	15.1	5	0.5	0.125	0.125	0.25	3.9
65	3	15.1	5	0.5	0.125	0.125	0.25	3.9
61.5	4	15.1	5	0.5	0.125	0.125	0.25	3.9
56.9	5	15.1	5	0.5	0.125	0.125	0.25	3.9
53.5	6	15.1	5	0.5	0.125	0.125	0.25	3.9
50.8	7	15.1	5	0.5	0.125	0.125	0.25	3.9
49	8	15.1	5	0.5	0.125	0.125	0.25	3.9
49	0	19	5	0.5	0.125	0.125	0.25	0
-	Master mix	151	50	5	1.25	1.25	2.5	0

Approximate length = 1807 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 1807bp x (15s/1000bp) + 15s

Annealing time = 42s

PCR protocol:

98°C for 3 min
98°C for 10s
49-69°C for 30s
72°C for 42s
Repeat 2-4 30x
72°C for 10 min
12°C for infinity

Gel PCR products:

Ladder, 0-8

Very faint bands can be seen in the second-fourth wells - redo the PCR with a gradient in this range tomorrow.

Redo PCR for GPD homo (longer) - amilCP - CYC1 terminator homo (longer) with larger temperature gradient.

Temp (°C)	Tube	ddH2O	Buffer	dNTP	Fp (qq10)	Rp (qq12)	Phusion (homemade)	DNA (URA3 - GPD promoter - amilCP homo)
74	1	17.1	5	0.5	0.125	0.125	0.25	1.9
72.5	2	17.1	5	0.5	0.125	0.125	0.25	1.9
70.1	3	17.1	5	0.5	0.125	0.125	0.25	1.9
66.5	4	17.1	5	0.5	0.125	0.125	0.25	1.9
61.9	5	17.1	5	0.5	0.125	0.125	0.25	1.9
58.6	6	17.1	5	0.5	0.125	0.125	0.25	1.9
55.8	7	17.1	5	0.5	0.125	0.125	0.25	1.9
54	8	17.1	5	0.5	0.125	0.125	0.25	1.9
54	0	19	5	0.5	0.125	0.125	0.25	0
-	Master mix	171	50	5	1.25	1.25	2.5	0

Approximate length = 705 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 705bp x (15s/1000bp) + 15s

Annealing time = 26s

PCR protocol:

98°C for 3 min
98°C for 10s
54-74°C for 30s
72°C for 26s
Repeat 2-4 30x
72°C for 10 min
12°C for infinity

Gel PCR products:

Ladder, 0-8

Bright bands can be seen at the right length - PCR purify and nanodrop.

PCR URA3 - GPD promoter - amil CP homo (longer) again with a gradient between 65-69°C.

Friday, June 28th 2019

Gel yesterday’s URA3 - GPD promoter - amil CP homo PCR.

Ladder, 0-8

Bands can be seen in almost all wells, so purify and nanodrop.

Perform a yeast transformation:

Yesterday, inoculated 10mL of 2x YPD with adenine and a yeast colony. Grow overnight at 30°C at 200-300 RPM.

This morning, measure OD600. Add 1000uL of sodium citrate to a cuvette as a blank. Add 900uL of sodium citrate and 100uL of the inoculation to another cuvette and measure the absorbance.

OD600 = 0.624 = 6.24

V culture = (0.2 x 50)/OD600

V culture = (0.2 x 50)/6.24

V culture = 1.6mL - add to 50mL of YPD.

Add 1.6mL of the culture to an Erlenmeyer flask containing 50mL YPD, and grow for 3 hours at 30°C at 200-300rpm.

Measure absorbance after 3 hours:

OD600 = 0.045 = 0.45

Not close enough to 0.6 - leave to incubate for 1 more hour.

Add the culture to a Falcon tube and spin down the culture at 4000rpm for 5 minutes. Discard the supernatant and gently resuspend the pellet in 10mL of sterile ddH2O.

Spin down the culture again at 4000rpm for 5 minutes.

Discard the supernatant and gently resuspend the pellet in 1mL 100mM lithium acetate. Transfer the entire suspension to a sterile 1.5mL microcentrifuge tube. Spin down at top speed for 10s.

Discard supernatant and gently resuspend cells with 100mM lithium acetate (approx. 280uL) to a final volume of 500uL. Vortex cells for 1s before distributing 50uL aliquots into labelled 1.5mL labelled microcentrifuge tubes.

Spin down cells at top speed for 10s and remove supernatant.

Add the following ingredients in the following order (on top):

240uL PEG

36uL 1M LiAc

50uL ssDNA (boiled at 100°C for 5 mins and cooled on ice)

36uL DNA mixture

0.1-1ug of DNA mixed with ddH2O to 36uL

Vortex cells vigorously for 1 minute.

Incubate in the shaker at 30°C for 45 minutes.

Heatshock cells by incubating cells in 42°C heat bath for 20 min.

Spin down cells at 6000rpm for 2 minutes. Remove supernatant and add 150uL sterile H2O to each tube to resuspend.

Plate entire samples on -Ura plates.

Tuesday, July 2nd 2019

There was growth on the plates from Friday, but none of the colonies are blue. Check colonies via yeast PCR:

Scrape off half of a colony using a pipette tip and resuspend in 20uL 20mM NaOH. Heatshock at 100°C for 10 minutes. Centrifuge for 5 seconds, vortex for 5 seconds, and centrifuge for 5 seconds again.

PCR using 0.5uL of the DNA.

Tube	ddH2O	Buffer	dNTP	Fp (qq13)	Rp (qq15)	Phusion (homemade)	DNA
1	18.5	5	0.5	0.125	0.125	0.25	0.5
2	18.5	5	0.5	0.125	0.125	0.25	0.5
3	18.5	5	0.5	0.125	0.125	0.25	0.5
4	18.5	5	0.5	0.125	0.125	0.25	0.5
5	18.5	5	0.5	0.125	0.125	0.25	0.5
6	18.5	5	0.5	0.125	0.125	0.25	0.5
7	18.5	5	0.5	0.125	0.125	0.25	0.5
8	18.5	5	0.5	0.125	0.125	0.25	0.5
9	18.5	5	0.5	0.125	0.125	0.25	0.5
10	18.5	5	0.5	0.125	0.125	0.25	0.5
11	18.5	5	0.5	0.125	0.125	0.25	0.5
12	18.5	5	0.5	0.125	0.125	0.25	0.5
13	18.5	5	0.5	0.125	0.125	0.25	0.5
14	18.5	5	0.5	0.125	0.125	0.25	0.5
0	19	5	0.5	0.125	0.125	0.25	0
neg	18.5	5	0.5	0.125	0.125	0.25	0.5
Master mix	259	70	7	1.75	1.75	3.5	0

Annealing time = 26s

Annealing temp = 61.5°C

Gel PCR products:

Ladder, 1-12, 0, neg

There were faint bands in lanes 4, 5, 6, 8, 10.

Also make a patch plate: take the other half of the colony and scrape it on a patch of a -Ura plate. Leave to incubate at 30°C.

Resume optimizing the standard protocol, starting with inserting RFP into Ade2 PSB1K3 (and the other homologies).

Tube	DNA	CutSmart	EcoRI	SpeI	ddH2O
Ade2 PSB1K3	10.9	5	1	1	32.1
Ade4 PSB1K3	10.8	5	1	1	26.2
3His3 PSB1K3	6.8	5	1	1	32.2
IGal4 PSB1K3	23.1	5	1	1	36.2
RFP amp	16.8	5	1	1	19.9

Incubate digests for 90 minutes and add phosphatase to the backbones.

Reagent	Volume (µL)
ddH2O	1
Antarctic phosphatase	3
Buffer	6
Ade2, Ade4, His3, Gal 4 PSB1K3 digest	50

Incubate at 37°C for 30 minutes.

Heat shock both digest solutions at 80°C for 20 minutes.

Ligate the homologies and RFP.

Reagent	Volume (µL)
RFP amp	4.57
Ade2, Ade4, His3, Gal4 PSB1K3 digest	3
T4 ligase	1
T4 ligase buffer	2
ddH2O	9.43

Incubate ligation mixture at room temperature for an hour, and then heat shock at 65°C for 10 minutes.

Wednesday, July 3rd 2019

Transform yesterday’s ligation on LB kan plates. The cells have poor efficiency, so plate 500uL.

PCR colonies 4, 5, 6, 8, and 10 yesterday using the first primer (Ura3 forward) and second-last primer (CYC1 terminator reverse) since the last primer’s melting temperature is too different from the first primer.

Tube	ddH2O	Buffer	dNTP	Fp (qq10)	Rp (qq15)	Phusion (homemade)	DNA
4	18.5	5	0.5	0.125	0.125	0.25	0.5
5	18.5	5	0.5	0.125	0.125	0.25	0.5
6	18.5	5	0.5	0.125	0.125	0.25	0.5
8	18.5	5	0.5	0.125	0.125	0.25	0.5
10	18.5	5	0.5	0.125	0.125	0.25	0.5
0	19	5	0.5	0.125	0.125	0.25	0
neg	18.5	5	0.5	0.125	0.125	0.25	0.5
Master mix	148	40	4	1	1	2	0

Annealing temp = 60°C

Annealing time = 52s

Gel colony PCR:

Ladder, 0, 4, 5, 6, 8, 10, neg

The gel showed bands of the correct length between 2000-3000bp.

Thursday, July 4th 2019

There was growth on all plates from yesterday’s transformation (albeit some plates had more growth than others). Inoculate 3 red colonies from each plate in 3mL LB Kan and leave to grow for 6 hours. Miniprep and nanodrop.

Friday, July 5th 2019

Digest minipreps with EcoRI and SpeI to see if they contain the correct plasmids.

Tube	DNA	CutSmart	EcoRI	SpeI	ddH2O
Gal4 1	23.2	5	1	1	21.8
Gal4 2	14.3	5	1	1	28.7
Gal4 3	20.5	5	1	1	22.5
Ade2 1	17.7	5	1	1	25.3
Ade2 2	20.5	5	1	1	22.5
Ade2 3	18.8	5	1	1	24.2
Ade4 1	14.5	5	1	1	28.5
Ade4 2	13.1	5	1	1	29.9
Ade4 3	12.3	5	1	1	30.7
His3 1	14.5	5	1	1	28.5
His3 2	12.8	5	1	1	30.2
His3 3	11.2	5	1	1	31.8

Incubate at 37°C for 2 hours, and then heatshock at 80°C for 20 minutes.

Gel digestion products:

Ladder, Gal4, Ade2, Ade4, His3 1-3

Bands at the correct length can be seen in all lanes except for Gal4 2 and Ade4 3.

Redo second KanMX PCR to restock in preparation for Monday.

Tube	ddH2O	Buffer	dNTP	Fp (pp19)	Rp (pp10)	Phusion (homemade)	DNA (PCR product)
1	18.5	5	0.5	0.125	0.125	0.25	0.5
2	18.5	5	0.5	0.125	0.125	0.25	0.5
3	18.5	5	0.5	0.125	0.125	0.25	0.5
4	18.5	5	0.5	0.125	0.125	0.25	0.5
5	18.5	5	0.5	0.125	0.125	0.25	0.5
6	18.5	5	0.5	0.125	0.125	0.25	0.5
7	18.5	5	0.5	0.125	0.125	0.25	0.5
8	18.5	5	0.5	0.125	0.125	0.25	0.5
0	19	5	0.5	0.125	0.125	0.25	0
Master mix	185	50	5	1.25	1.25	2.5	0

Gel PCR product:

Ladder, 0-8

Bands of the correct length (just under 1500bp) can be seen, so PCR purify and nanodrop samples.

Monday, July 8th 2019

Inoculate yeasts 4, 5, 6, 8, 10 (plus negative) from the URA3-GPD promoter-amilCP-CYC1 terminator patch plate in 5mL YPD. Incubate overnight at 30°C. Also patch 4, 5, 6, 8, 10 on another -Ura plate.

Digest homologies-RFP-PSB1K3 plus MfeI-KanMX-EXSP.

Tube	DNA	Buffer G	SpeI	EcoRI-HF	ddH2O
Ade2 PSB1K3	26, 9.9	5	1	1	7.1
Ade4 PSB1K3	29.0	5	1	1	14.0
Gal4 PSB1K3	28.7	5	1	1	14.3
His3 PSB1K3	28.9	5	1	1	14.1
Tube	DNA	Buffer G	SpeI	MunI	ddH2O
MfeI-KanMX-EXSP	10.6	5	1	1	32.4

Incubate at 37°C for 2 hours, and then heat shock for 20 minutes at 80°C.

Ligate:

Reagent	Volume (µL)
KanMX-EXSP digest	6.17
Ade2/Ade4/His3/Gal4 PSB1K3 RFP digest	2.5
T4 ligase	1
T4 ligase buffer	2
ddH2O	8.33

Incubate at RT for 1 hour, and then heatshock at 65°C for 10 minutes.

Transform 500uL of each ligation onto LB Kan plates and incubate overnight at 37°C.

Tuesday, July 9th 2019

Redo the Gal4 transformation - I forgot to throw out the bad tubes from last week’s gel.

Digest Gal4:

Tube	DNA	Buffer G	SpeI	EcoRI-HF	ddH2O
Gal4 PSB1K3	24, 19.3	5	1	1	0

Incubate at 37°C for 2 hours and heat-shock at 80°C for 20 minutes.

Ligate:

Reagent	Volume (µL)
KanMX-EXSP digest	6.17
Gal4 PSB1K3 RFP digest	2.5
T4 ligase	1
T4 ligase buffer	2
ddH2O	8.33

Incubate at room temperature for 1 hour and heatshock at 65°C for 10 minutes.

Transform 500uL onto LB kan plates and let incubate overnight at 37°C. Also transform 100ng of Ade2, Ade4, Gal4,and His3 RFP to make glycerol stock.

Tube	Volume (µL)
Ade2	3.8
Ade4	2.6
Gal4	4.7
His3	2.9

Make glycerol stock of yesterday’s yeast inoculation (750uL 30% glycerol and 750uL inoculation). Store in iGEM 2019 box in -80°C freezer.

Inoculate 9 colonies and a negative overnight from the Ade2 plate from yesterday’s transformation.

Wednesday, June 10th 2019

Make glycerol stock of yesterday’s Ade2 KanMX inoculation and miniprep it.

Lots of growth (too much) on yesterday’s transformations.

Digest the minipreps with PmeI to verify:

Tube	DNA	CutSmart	PmeI	ddH2O
1	12.2	5	1	31.8
2	12.2	5	1	31.8
3	11.8	5	1	32.2
5	14.6	5	1	29.4
6	12.5	5	1	31.5
7	12.3	5	1	31.7
8	11.1	5	1	32.9
9	12.2	5	1	31.8
Positive (Ade2::KanMX)	11.0	5	1	33.0

Incubate at 37°C for 90 minutes and then heatshock at 80°C for 20 minutes.

Gel digestion products:

Ladder, 1-9, positive

The backbone and a slightly larger band can be seen in 1, 2, and 5. Unfortunately, Ade2 was not present in any of the lanes.

Monday, July 15th 2019

PCR KanMX Ade2 PSB1K3 and do 12 colony PCRs to check if KanMX is present.

Tube	ddH2O	Buffer	dNTP	Fp (pp9)	Rp (pp10)	Phusion (homemade)	DNA
1 mini	17.8	5	0.5	0.125	0.125	0.25	1.2
2 mini	17.8	5	0.5	0.125	0.125	0.25	1.2
5 mini	17.5	5	0.5	0.125	0.125	0.25	1.5
Pos mini	17.4	5	0.5	0.125	0.125	0.25	1.6
1	14	5	0.5	0.125	0.125	0.25	5
2	14	5	0.5	0.125	0.125	0.25	5
3	14	5	0.5	0.125	0.125	0.25	5
4	14	5	0.5	0.125	0.125	0.25	5
5	14	5	0.5	0.125	0.125	0.25	5
6	14	5	0.5	0.125	0.125	0.25	5
7	14	5	0.5	0.125	0.125	0.25	5
8	14	5	0.5	0.125	0.125	0.25	5
9	14	5	0.5	0.125	0.125	0.25	5
10	14	5	0.5	0.125	0.125	0.25	5
11	14	5	0.5	0.125	0.125	0.25	5
12	14	5	0.5	0.125	0.125	0.25	5
0	19	5	0.5	0.125	0.125	0.25	0
Pos	14	5	0.5	0.125	0.125	0.25	5
Master mix	266	95	9.5	2.375	2.375	4.75	0

Gel PCR products:

Colony PCR:

Ladder, 0-12, positive

Miniprep PCR:

Ladder, 0, 1, 2, 5, positive

Nothing appeared on the small gel (not even the positive), so the colony PCR didn’t work. However, various bands of different lengths an be seen on the large gel. Check if the primers are annealing non-specifically by PCRing with Ade2 primers (pp15 and pp16).

Tube	ddH2O	Buffer	dNTP	Fp (pp15)	Rp (pp16)	Phusion (homemade)	DNA
1 mini	17.8	5	0.5	0.125	0.125	0.25	1.2
2 mini	17.8	5	0.5	0.125	0.125	0.25	1.2
5 mini	17.5	5	0.5	0.125	0.125	0.25	1.5
Pos mini	17.4	5	0.5	0.125	0.125	0.25	1.6
1	14	5	0.5	0.125	0.125	0.25	5
2	14	5	0.5	0.125	0.125	0.25	5
3	14	5	0.5	0.125	0.125	0.25	5
4	14	5	0.5	0.125	0.125	0.25	5
5	14	5	0.5	0.125	0.125	0.25	5
6	14	5	0.5	0.125	0.125	0.25	5
7	14	5	0.5	0.125	0.125	0.25	5
8	14	5	0.5	0.125	0.125	0.25	5
9	14	5	0.5	0.125	0.125	0.25	5
10	14	5	0.5	0.125	0.125	0.25	5
11	14	5	0.5	0.125	0.125	0.25	5
12	14	5	0.5	0.125	0.125	0.25	5
0	19	5	0.5	0.125	0.125	0.25	0
Pos	14	5	0.5	0.125	0.125	0.25	5
Master mix	266	95	9.5	2.375	2.375	4.75	0

Annealing time = 1763bp x 15s/1000bp + 15s = 41s

Streak CEN PSB1K3 and RFP amp on plates to prepare for DEGREE.

Tuesday, July 16th 2019

Gel yesterday’s PCR product.

Colony PCR:

Ladder, 0-12, positive

Miniprep PCR:

Ladder, 0, 1, 2, 5, positive

Ade2 is present in all lanes on the large gel, but not KanMX. There are other bands in the small gel, but the lengths are wrong and there is unspecific binding.

Inoculate 3 colonies from the CEN PSB1K3 plate and 3 from RFP amp in LB kan and LB amp, respectively.

Wednesday, July 17th 2019

Take a step back - the issue may be with MfeI. Make primers to PCR MfeI-RFP-EXSP instead of KanMX to see if it works properly.

Miniprep CEN PSB1K3. The RFP inoculations didn’t turn red, so streak another RFP amp stock.

Thursday, July 18th 2019

The colonies on the RFP plate were red, so inoculate 3 colonies in LB amp overnight.

Friday, July 19th 2019

Miniprep RFP inoculations and nanodrop.

PCR MfeI-RFP-prefix to prepare for protocol optimization.

Temp (°C)	Tube	ddH2O	Buffer	dNTP	Fp (qq39)	Rp (qq40)	Phusion (homemade)	DNA (RFP amp)
73	1	16.7	5	0.5	0.125	0.125	0.25	2.3
72.5	2	16.7	5	0.5	0.125	0.125	0.25	2.3
71.5	3	16.7	5	0.5	0.125	0.125	0.25	2.3
70	4	16.7	5	0.5	0.125	0.125	0.25	2.3
68.2	5	16.7	5	0.5	0.125	0.125	0.25	2.3
66.9	6	16.7	5	0.5	0.125	0.125	0.25	2.3
65.8	7	16.7	5	0.5	0.125	0.125	0.25	2.3
65	8	16.7	5	0.5	0.125	0.125	0.25	2.3
65	0	16.7	5	0.5	0.125	0.125	0.25	2.3
-	Master mix	167	50	5	1.25	1.25	2.5	0

Approximate length = 1069 bp

Annealing time = length x (15s/1000bp) + 15s

Annealing time = 1069bp x (15s/1000bp) + 15s

Annealing time = 31s

PCR protocol:

98°C for 3 min
98°C for 10s
65-73°C for 30s
72°C for 31s
Repeat 2-4 30x
72°C for 10 min
12°C for infinity

Monday, July 22nd 2019

Start testing the DEGREE protocol!

Tube	DNA	CutSmart	SpeI	EcoRI-HF	ddH2O
CEN PSB1K3	21.5	5	1	1	21.5
RFP amp	16.4	5	1	1	26.6

Incubate at 37°C for 1 hr and heat shock at 80°C for 20 minutes.

Ligate:

Reagent	Volume (µL)
CEN PSB1K3	2.5
RFP amp	4.85
T4 ligase	1
T4 ligase buffer	2
ddH2O	9.65

Incubate at RT for 1 hour and heat-shock at 65°C for 10 minutes.

Transform on LB kan.

Gel PCR product from Friday:

Ladder, 0-8

There is a band in the zero lane, so redo the PCR at 70°C.

Tube	ddH2O	Buffer	dNTP	Fp (qq39)	Rp (qq40)	Phusion (homemade)	DNA (RFP amp)
1	17.6	5	0.5	0.125	0.125	0.25	1.4
2	17.6	5	0.5	0.125	0.125	0.25	1.4
3	17.6	5	0.5	0.125	0.125	0.25	1.4
4	17.6	5	0.5	0.125	0.125	0.25	1.4
0	19	5	0.5	0.125	0.125	0.25	0
Master mix	105.6	30	3	0.75	0.75	1.5	0

Gel PCR products:

Ladder, 0-4

There is nothing in the zero lane and the bands are the right length.

Tuesday, July 23rd 2019

PCR purify MfeI-RFP-prefix and PCR again to add the suffix.

Tube	ddH2O	Buffer	dNTP	Fp (qq39)	Rp (pp19)	Phusion (homemade)	DNA (MfeI-RFP-prefix)
1	18.3	5	0.5	0.125	0.125	0.25	0.7
2	18.3	5	0.5	0.125	0.125	0.25	0.7
3	18.3	5	0.5	0.125	0.125	0.25	0.7
4	18.3	5	0.5	0.125	0.125	0.25	0.7
0	19	5	0.5	0.125	0.125	0.25	0
Master mix	109.8	30	3	0.75	0.75	1.5	0

Inoculate 3 colonies from yesterday’s transformation and do a colony PCR. Repeat 1 colony multiple times to find the best temperature for the RFP colony PCR.

Tube	ddH2O	Buffer	dNTP	Fp (oo69)	Rp (oo70)	Phusion (homemade)	DNA
1	14	5	0.5	0.125	0.125	0.25	5
2	14	5	0.5	0.125	0.125	0.25	5
3	14	5	0.5	0.125	0.125	0.25	5
4	14	5	0.5	0.125	0.125	0.25	5
5	14	5	0.5	0.125	0.125	0.25	5
6	14	5	0.5	0.125	0.125	0.25	5
7	19	5	0.5	0.125	0.125	0.25	0
Master mix	112	40	4	1	1	2	0

Wednesday, July 24th 2019

Gel PCR products from yesterday.

Ladder, 0-4 MfeI-RFP-EXSP, 0-7 RFP colony PCR

Bands can be seen in the right lanes at the right length. PCR purify MfeI-RFP-EXSP.

For the colony PCR, 67.1°C-70°C seem like the best temperatures.

Miniprep yesterday’s inoculations. Digest with PstI and AatII for 90 minutes to test.

Tube	DNA	CutSmart	PstI-HF	AatII	ddH2O
RFP PSB1K3 1	19.6	5	1	1	23.4
RFP PSB1K3 2	7.9	5	1	1	35.1
RFP PSB1K3 3	6.8	5	1	1	36.2

Gel RFP PSB1K3 digestion products to verify the Biobrick:

Ladder, 1-3

The gel shows 2 bands of the right length in the first lane, which confirms that RFP was successfully incorporated into PSB1K3. However, the other two lanes have a third unknown band.

With this final step, the entire DEGREE training protocol has been performed and verified to work.

Digest MfeI-RFP-EXSP and CEN PSB1K3 for 75 minutes and heat shock at 80°C for 20 minutes.

Tube	DNA	CutSmart	EcoRI-HF	SpeI	ddH2O
CEN PSB1K3	21.6	5	1	1	21.4

Tube	DNA	Buffer G	MunI	SpeI	ddH2O
MfeI-RFP-EXSP	12.5	5	1	1	30.5

Ligate MfeI-RFP-EXSP with PSB1K3.

MfeI-RFP-EXSP length = 1130bp

Reagent	Volume (µL)
CEN PSB1K3	2.5
MfeI-RFP-EXSP	5.13
T4 ligase	1
T4 ligase buffer	2
ddH2O	9.37

Incubate at RT for 1 hour and heat shock at 65°C for 10 minutes.

Transform on LB kan plates.

Thursday, July 25th 2019

Count ratio of red:white colonies from yesterday’s transformation - there were approximately 350 colonies in total, with 35 of them being red.

We want to obtain more quantitative data about the efficiency of this experiment - prepare another MfeI-RFP-EXSP and PSB1K3 ligation.

Reagent	Volume (µL)
CEN PSB1K3	2.5
MfeI-RFP-EXSP	5.13
T4 ligase	1
T4 ligase buffer	2
ddH2O	9.37

Transform onto 2 LB kan plates.

Also transform RFP PSB1K3 on LB kan plates as a positive control for the colony PCR (100ng=1.96µL).

Friday, July 26th 2019

All plates from yesterday had good growth.

Count the ratio of red:white colonies on the RFP-EXSP-PSB1K3 plates.

Plate 1: ~700 colonies, 84 red

Plate 2: 363 colonies, 30 red

PCR KanMX and RFP with Type IIS primers.

KanMX (71°C):

Tube	ddH2O	Buffer	dNTP	Fp (qq45)	Rp (pp46)	Phusion	DNA
1	16.6	5	0.5	0.125	0.125	0.25	2.4
2	16.6	5	0.5	0.125	0.125	0.25	2.4
3	16.6	5	0.5	0.125	0.125	0.25	2.4
4	16.6	5	0.5	0.125	0.125	0.25	2.4
0	19	5	0.5	0.125	0.125	0.25	0
Master mix	99.4	30	3	0.75	0.75	1.5	0

RFP (70°C):

Tube	ddH2O	Buffer	dNTP	Fp (qq47)	Rp (pp48)	Phusion	DNA
1	17.4	5	0.5	0.125	0.125	0.25	1.6
2	17.4	5	0.5	0.125	0.125	0.25	1.6
3	17.4	5	0.5	0.125	0.125	0.25	1.6
4	17.4	5	0.5	0.125	0.125	0.25	1.6
0	19	5	0.5	0.125	0.125	0.25	0
Master mix	104.2	30	3	0.75	0.75	1.5	0

Monday, July 29th 2019

Start the DEGREE shadowing protocols.

Digest RFP and PSB1K3.

Tube	DNA	Buffer G	EcoRI	SpeI	ddH2O
RFP PSB1A3	23	5	1	1	20
CEN PSB1K3	19	5	1	1	24

Incubate for 1 hour at 37°C and heat-shock at 80°C for 20 minutes.

Ligate.

Reagent	Volume (µL)
RFP PSB1A3	4.85
CEN PSB1K3	2.5
T4 ligase	1
T4 ligase buffer	2
ddH2O	9.65

Ligate for 1 hour at room temperature and heat-shock at 65°C for 10 minutes.

Transform 100uL onto LB kan plates.

Perform a PCR for RFP (Type IIS):

Tube	ddH2O	Buffer	dNTP	Fp (qq47)	Rp (pp48)	Phusion (homemade)	DNA (RFP PSB1A3)
1	17.6	5	0.5	0.125	0.125	0.25	1.4
2	17.6	5	0.5	0.125	0.125	0.25	1.4
3	17.6	5	0.5	0.125	0.125	0.25	1.4
4	17.6	5	0.5	0.125	0.125	0.25	1.4
5	17.6	5	0.5	0.125	0.125	0.25	1.4
6	17.6	5	0.5	0.125	0.125	0.25	1.4
7	17.6	5	0.5	0.125	0.125	0.25	1.4
8	17.6	5	0.5	0.125	0.125	0.25	1.4
0	19	5	0.5	0.125	0.125	0.25	0
Master mix	175.6	50	5	1.25	1.25	2.5	0

Tuesday, July 30th 2019

Perform a yeast colony PCR for KanMX:

Tube	ddH2O	Buffer	dNTP	Fp (qq45)	Rp (pp46)	Phusion (homemade)	DNA (RFP PSB1A3)
1	18.5	5	0.5	0.125	0.125	0.25	1.4
2	17.6	5	0.5	0.125	0.125	0.25	1.4
3	17.6	5	0.5	0.125	0.125	0.25	1.4
4	17.6	5	0.5	0.125	0.125	0.25	1.4
5	17.6	5	0.5	0.125	0.125	0.25	1.4
6	17.6	5	0.5	0.125	0.125	0.25	1.4
7	17.6	5	0.5	0.125	0.125	0.25	1.4
8	17.6	5	0.5	0.125	0.125	0.25	1.4
0	19	5	0.5	0.125	0.125	0.25	0
Master mix	175.6	50	5	1.25	1.25	2.5	0