Team:FAU Erlangen/Notebook

Notebook

May

Aim / Task Generation of pACYC184 plasmid
Protocol
Participants Eva Neugebauer
  • bacteria from -80°C: HVS11 pEcoE, HVS11 pEcoF
  • inoculation of both samples in 5 mL LB medium with Chloramphenicol
  • overnight culture at 37°C with 200 rpm

Aim / Task Generation of pACYC184 plasmid
Protocol
Participants Eva Neugebauer
  • no turbidity in the ON culture visible
  • new bacteria from -80°C: HVS11 pEcoE, HVS11 pEcoF, HVS11 pEcoD
  • inoculation of the three samples in 5 mL LB medium with Tetracyclin (2 µg/mL)
  • ON culture at 37°C with 200 rpm

Aim / Task Generation of pACYC184 plasmid
Protocol Zymo research Miniprep - Classic (Cat.No. D4015, Lot.No. 202653)
Participants Eva Neugebauer

Turbidity in the ON culture visible

Miniprep:

  1. Centrifugate the ON culture at 4000 rpm at room temperature
  2. Discard the supernatant
  3. Add 200 µL P1 buffer
  4. Add 200 µL P2 buffer and mix by inverting two times
  5. Add 400 µL P3 buffer and mix by inverting three times
  6. Centrifugate the samples for 2 min in a microcentrifuge
  7. Place the column in a collection tube and transfer the supernatant
  8. Centrifugate for 30s
  9. Discard the flow-through and return of the column to the collection tube
  10. Add 200 µL Endo-Wash buffer and centrifugate for 30 s
  11. Add 400 µL Plasmid Wash buffer and centrifugate for 1 min
  12. Place the column in a 1.5 mL tube, add 30 µL DNA Elution Buffer and centrifugate for 30 s

Storage of the purified plasmid DNA at 4°C

Aim / Task Generation of pACYC184 plasmid
Protocol -
Participants Eva Neugebauer

inoculation of 6 clones in 5 mL LB medium with Chloramphenicol for the ON culture

Aim / Task Generation of pACYC184 plasmid
Protocol -
Participants Eva Neugebauer

Nanodrop measurement of the samples from 2019-05-23 * pEcoD: 46.1 ng/µL * pEcoE: 70.9 ng/µL * pEcoF: 27.6 ng/µL

restriction enzyme digest:

setpoint EcoD EcoE EcoF
DNA 500 ng 10.85 µL 7.05 µL 17.5 µL
H20 20 µL 6.65 µL 10.45 µL 0 µL
CutSmart Buffer 2 µL 2 µl 2 µL 2 µL
EcoRI 0.5 µL 0.5 µ 0.5 µL 0.5 µL

Digestion 1h at 37°C and 350 rpm, afterwards adding of 4 µL BPB

agarose gel 1%: 1x TBE |60 mL agarose |0.6 g ethidiumbromid |2 µL

  • load the gel with 20 µL of the samples and 3 µL Gene Ruler Mix
  • run the gel at 130 V for 1 h

Aim / Task Generation of pACYC184 plasmid
Protocol -
Participants Eva Neugebauer

Miniprep:

  1. Centrifugate the ON culture at 4000 rpm at room temperature
  2. Discard the supernatant
  3. Add 200 µL P1 buffer
  4. Add 200 µL P2 buffer and mix by inverting two times
  5. Add 400 µL P3 buffer and mix by inverting three times
  6. Centrifugate the samples for 2 min in a microcentrifuge
  7. Place the column in a collection tube and transfer the supernatant
  8. Centrifugate for 30s
  9. Discard the flow-through and return of the column to the collection tube
  10. Add 200 µL Endo-Wash buffer and centrifugate for 30 s
  11. Add 400 µL Plasmid Wash buffer and centrifugate for 1 min
  12. Place the column in a 1.5 mL tube, add 30 µL DNA Elution Buffer and centrifugate for 30 s

store the purified DNA at 4°C

DNA concentration: measurement at the NanoDrop

Clone Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6
concentration [ng/µL] 30.6 21.3 26.1 23.6 28.1 25.4

Digestion:

digestion for 1.5 h at 37°C

Clone 1 2 3 4 5 6
Buffer 2 µL 2 µL 2 µL 2 µL 2 µL 2 µL
Enzyme (EcoRV) 0.5 µL 0.5 µL 0.5 µL 0.5 µL 0.5 µL 0.5 µL
DNA 16.3 µL 17.5 µL 17.5 µL 17.5 µL 17.5 µL 17.5 µL
H20 1.2 - - - - -
  • load 20 µL of each sample and 3.5 µL of the marker on a 1 % agarose gel
  • run 1.5 h at 130 V

DNA extraction from agarose gel:

  1. add 200 µL NTI per 100 mg gel piece
  2. incubate for 5 min at 50°C
  3. place the column in a collection tube and transfer the sample
  4. centrifugate at 11000 rpm for 30 s
  5. add 700 µL NT3 twice adn centrifugate each time at 11000 rpm for 30 s
  6. dry the silica membrane for 1 min at 11000 rpm
  7. place the column on a sterile 1.5 mL reaction tube
  8. add 20 µL NE buffer and centrifugate for 1 min at 11000 rpm

DNA concentration: measurement at the NanoDrop * clone 1: 11.2 ng/µL * clone 2: 22.3 ng/µl

Aim / Task Generation of pACYC184 plasmid
Protocol DNA extraction from agarose gel
Participants Eva Neugebauer
  • run the gel from 2019-05-27 for 45 min at 130 V
  • cut out the vector DNA from the gel
  • place the gel pieces into reaction tubes

DNA extraction from agarose gel:

  1. add 200 µL NTI per 100 mg gel piece
  2. incubate for 5 min at 50°C
  3. place the column in a collection tube and transfer the sample
  4. centrifugate at 11000 rpm for 30 s
  5. add 700 µL NT3 twice adn centrifugate each time at 11000 rpm for 30 s
  6. dry the silica membrane for 1 min at 11000 rpm
  7. place the column on a sterile 1.5 mL reaction tube
  8. add 20 µL NE buffer and centrifugate for 1 min at 11000 rpm

Ligation: DNA |9 µL Quick Ligation buffer |10 µL Quick Ligase |1 µL

Incubate for 10 min at room temperature

Transformation into competent DH10B e.coli via heat shock:

  1. thaw competent bacteria on ice
  2. add 10 µL of the previously ligated DNA, storage of the rest at -20°C
  3. incubate 30 min on ice
  4. incubate 1 min at 42°C
  5. incubate 2 min on ice
  6. add 500 µL Soc medium
  7. 30 min on 37°C

plate the bacteria on Cam plates, incubate at 37°C over night

Control gel for the ligation:

  • loading of 2 µL of each sample with 2 µL running buffer
  • run the gel for 1 h at 130 V
  • for each sample only one band visible

Aim / Task Generation of pACYC184 plasmid
Protocol -
Participants Eva Neugebauer
  • some colonies visible on the plates
  • store at 4°C

June

Aim / Task Generation of more open pACYC184 with EcoRV
Protocol Miniprep
Participants Eva Neugebauer

Protocol

Substance concentration
Cut Smart buffer 2 µL 2 µg (= 1 µl) 1 µg (= µl)
DNA 7,98 µL
Enzyme 1,5 µL
Water 44,52 µL

Incoulation of 200 µL sample 2 from the 27.05.2019

Miniprep

  • Adding 10 mL Equilibration buffer at the collum
  • Centrifugation of overnightculture for 10 min at 4000 x g and removement of all the medium
  • Resuspension of the pellet in 4 mL resuspension buffer (R3)
  • Adding of 4 mL Lysis buffer and mixing by capping the tube for 5 times
  • Incubation for 5 min at RT
  • Adding 4 mL precipitation buffer (N3) and inverting till it is homogenous
  • Centrifugation for 10 min at 5000 x g at RT
  • Loadng supernatant on the collum, drain by gravity
  • Adding of 10 mL washing buffer to the collum twice
  • Placing collum on a 15 mL tube and adding of 5 mL elution buffer (E4) and letting it drain by gravity
  • Adding 3,5 mL isopropanol and mixing it well
  • Centrifugation for 30 min at 5000 x g at RT
  • Discarding of the supernatant
  • Adding 3 mL 70% ethanol
  • Centrifugation for 5 min, 5000 x g at RT
  • Remove supernatant
  • Airdry pellet for 10 min
  • Resuspension of pellet in 200 µL
  • Storage at -20°C (still in virology lab)

Nanodrop Measurement

188,8 ng/ µL

Digest

500 ng for 0,5 µL enzyme

Digest of following components at 37°C foe 1 h:

Digest

500 ng for 0,5 µL enzyme

Digest of following components at 37°C foe 1 h:

*see table above

Gel

Mixing of 60 µL digest with 12 µL loading buffer

Loading a agarose gel

130 V for 1 h

Gel extraction

Cutting the digest out of the gel.

  1. Adding 200 µL /per 100 mg gel piece of NTI.

  2. 5 min 50°C

  3. Placing of the collum in a collection tube and loading of the sample.

  4. Centrifugation at 11000 rpm for 30 s.

  5. Adding twice 700 µL NT3 and centrifugation each time at 11000 rpm for 30 s.

  6. Drying of the silica membrane for 1 min at 11000 rpm.

  7. Placing collumn on a 1,5 mL sterile Eppi.

  8. Adding 20 µL NE buffer and centrifugation for 1 min at 11000 rpm.

  9. Storage at -20°C.

Nanodrop

Different results with a wide range

Aim / Task Concentration of EcoRV opend pACYC184
Protocol Nanodrop
Participants Eva Neugebauer

Protocol

Nanodrop measurement: concentration still varies

Concentration determination with agarose gel

Loading of the sample (2µL and 2µL marker on a agarose gel)

sample: 0,5 µL loading buffer on 2,5 µL digest

Running gel for 1 h at 130 V

Marker 4000 bp ~ 50 ng/µL

<agarose gel of EcoRV opend pACYC184>

Aim / Task Material and Devices
Protocol Material check
Participants Daniel Schreyer

Protocol

Material and Devices

  • E.coli are in the -80°C Refrigerator in our lab - Box 12, reserved for iGEM
    • BL21 = Blank/no name, Star = S, Tuner = T
  • 4°C Refrigerator is in our lab - top shelf
  • PCR machine is on the other site of the kitchen
  • Material for different buffers is on the left side next of the Winkler lab
  • Winkler lab is on the opposite site of our lab
  • Elektroporator in Jäck AG - Contact Felix Pfister, iGEM for more information
  • Shaker Incubator in our Lab - Connect to water flow - Temperature adjustable

Aim / Task Production of LB medium
Protocol LB-Medium Production Protocol
Participants Daniel Schreyer, Felix Fister

Protocol

Notes

  • Autoclave machine in room 02.052
  • Big incubation shaker device in 02.053
  • Create medium and label it — “Content, Name-iGEM, room of our lab, date” e.g LB-medium, DS-iGEM, 02.081, 24.06.19
  • Created 4L LB-Medium

Calculations and Results

20g of LB-Broth dissolved in 1L milli-Q H2O

Materials used

  • 80g LB-Broth

Problems faced / possible error causes

  • Medium-bottle do not close 100%
  • not 100% contamination proof -use fast
  • Our water shaker has the wrong size. Winkler is ordering new sizes for our shaker
    • Look for right size. Send product link to prof. winkler. He is going to order them for us
  • Get right flask holder for our shaker?


Pending tasks, next steps, comments for next people in the Lab

  • Cleaning ladies place the autoclaved medium into our lab. Store at 8°C. Should be finished around 2pm.
  • Adding of antibiotics for selection after transformation.
  • Use for cultivation and transformation
  • Tape and pens are necessary

Aim / Task E.coli Cultivation
Protocol Cultivation with LB-medium
Participants Daniel Schreyer, Lisa Vogg

Protocol

Notes

  • Stored LB-medium in fridge
  • Used 3 E.coli strains: Tuner, BL21 Star, BL21
  • Pipetting under bunsen burner in Winkler lab
  • For each strain: 10 mL LB-medium combined with 1 sterile pipet tip E.coli in 12 ml not fully closed reaction tubes
  • Cultivation in top incubation shaker (left side, 3 12 mL reaction tubes) in room 02.053 at 37°C with 130 rpm
  • Autoclave 100 mL flasks for tomorrow 26.06.19
  • If we are using stuff from the Winkler lab, bring it back
  • If we are using workspace in the Winkler lab - clean the bench with 70% Ethanol

Protocol:

  1. Unfreeze E.coli
  2. Centrifuge tube with Mini-Centrifuge –Solution at the bottom of the reaction tube

Next Steps are performed next to the bunsen burner

  1. Fill 10 ml LB-medium into 12 ml reaction tube under

  2. Dip 1 sterile pipet tip into E.coli reaction tube

  3. Throw pipet tip into 12 ml reaction tube and close it

  4. Cultivation in incubation shaker for 20 hours at 37°C with 130 rpm

  • Store remaining E.coli at -80°C

Problems faced / possible error causes

  • Incubation flasks are needed for further cultivation. Autoclave 100 mL flasks for tomorrow 26.06.19
  • Need lab briefing from some of winklers lab

Pending tasks, next steps, comments for next people in the Lab

  • OD measurement –further cultivation
  • Cultivation in 100mL flasks with the incubation shaker in our lab
  • Freeze 1mL from each E.coli strain at -80°C
  • Wait for autoclaved flasks. Use them to cultivate our bacteria in our shaker …

Aim / Task E. coli cultivation
Protocol Cultivation with LB-medium
Participants Lisa Vogg, Martin Borgmann

Protocol

Notes

  • Stored LB-medium in fridge
  • Used 3 E.coli strains: Tuner, BL21 Star, BL21
  • Pipetting under bunsen burner in Winkler lab
  • For each strain: 155 mL LB-medium combined the 10 ml of the previous bacteria culture (25.06.19)
  • Cultivation in top incubation shaker in room 02.053 at 37°C with 130 rpm
  • stuff flask holder with paper towels to keep the flasks in place
  • autoclaved flasks from the previous day not yet usable

Protocol:

(Performed next to a bunsen burner)

  1. Fill flask with desired volume of LB medium
  2. sterilize tip of the pipet, the lid of the bottle with medium and the edge of the flask in the flame to prevent contamination
  3. add content of the previous culture tubes to the new flasks
  4. Cultivation in incubation shaker for 20 hours at 37°C with 130 rpm

Problems faced / possible error causes

  • briefing for the lab desperately needed!
  • Which kuvettes for OD measurement?
  • flasks from the previous day not yet usable

Pending tasks, next steps, comments for next people in the Lab

  • measure OD
  • make cells competent

  • freeze a batch

    • 3x 25 ml pipette

  • gloves, paper towels, 70% ethanol

  • 490 ml LB medium

Aim / Task Overnight Cultivation of E.coli strains
Protocol E.coli Cultivation
Participants Daniel Schreyer, Leon Altmann, Lisa Vogg, Jannik Wagner

Protocol

Notes

  • Measure OD(600nm) (1:10 dilution) after 2 days incubation in incubation shaker of the E.coli strains BL21, BL21 Star, BL21 Tuner
  • OD of 0.71 =7.1

Protocol:

Inoculate overnight culture:

  1. inoculate 100 ml LB-medium with 100µl of the E.coli cultures
  2. Fill water shaker with water
  3. Place into water shaker at 37°C and 120 RPM
  4. Incubate overnight: Start 5pm –19h incubation time

Materials used

  • OD cuvette
  • 25 ml plastic tip
  • 3 yellow tips

Problems faced / possible error causes

  • NEB Kit was stored at -20°C: competent E.colis should be stored at -80°C, other lab material should be stored at 4°C (DNA Ladder, buffer, …) –Damage is possible

Pending tasks, next steps, comments for next people in the Lab

  • Pour agar plates for heat shock test: Test transformation by heat shock with NEB control vector PUC19
  • Pour agar plates for first transformations: Agar plates with chloramphenicol
  • freeze E.coli from overnight culture
  • Heat shock test

Aim / Task Pour agar plates
Protocol
Participants Daniel Schreyer, Leon Altmann, Lisa Vogg

Protocol

Notes

  • Pour agar plates:
    • 3 plates with ampicillin resistance
    • 21 plates with chloramphenicol resistance
    • 3 OD measurements of overnight culture. Each around 45 minutes apart Materials used

agar for 700 mL agar plates

July

Aim / Task Preparation Sequences
Protocol -
Participants Lena Schorr, Ruben Caspary, Eva Neugebauer

Protocol

Resuspension of the IDT sequences in 20 µL TE buffer.

Resulting concentration 50 ng/ µL.

Vortexing and incubation for 15 min at 50°C.

Vortexing and storage at -20°C.

Biotwist sequences and primer still not resuspended! 10 µL Filtertip-Spitzen

Aim / Task Growth kinetics
Protocol OD
Participants Martin Borgmann

Protocol

Freezing of E.coli

Goal: storing the 3 E.coli strains BL21, BL21 Tuner and BL21 Star to have them ready for cultivation for transformation

-Spinned for 10 minutes at 2,500 rpm

-Discarded supernatant without disturbing the pellet, while leaving ~1ml for every sample

-Resuspend the pellet in the remaining supernatant.

-Added 900μl cell culture and 300μl 99% glycerol, to a final glycerol concentration of ~25%. 6.

-Store in -80 °C freezer

Growth kinetics

Goal: get Growth kinetics of the 3 E.coli strains BL21, BL21 Tuner and BL21 Star to estimate the optimal time point for the transformation!

-Measuring OD600 of over night culture (RT)

OD600 = 1 equals 8*108 cells/mL

Stem OD Cell count
B21: 1 µg (=0,5 µl) –5,5*108 cells / mL
B21 Tuner: OD600 (1) = 0,1966 and OD600 (2) = 0,1402 (double determination!) -average: 0,1684 –1,35*109 cells / mL
B21 Star: OD600 = 0,0779 –6,29*108 cells / mL

-Seeding with same cell count -106 cells / mL in 25 mL LB medium –2,5*107 cells

Stem Calculation
B21: 5,5*108 cells / mL -2,5*107 cells / 5,5*108 cells / mL = 0,045 mL
B21 Tuner: 1,35*109 cells / mL - 2,5*107 cells / 1,35*109 cells / mL = 0,019 mL
B21 Star: 6,29*108 cells / mL - 2,5*107 cells / 6,29*108 cells / mL = 0,040 mL
Time in h OD dilution factor real OD µ(t) in 1/h OD dilution factor real OD µ(t) in 1/h OD dilution factor real OD µ(t) in 1/h
Stem: BL12 BL12 Tuner BL12 Star
0 0,0192 1 0,0192 0,0262 1 0,0262 0,0199 1 0,0199 .
1 0,0324 1 0,0324 0,523 0,0237 1 0,0237 -0,100 0,0283 1 0,0283 0,352
2 0,0674 1 0,0674 0,732 0,027 1 0,027 0,130 0,0557 1 0,0557 0,677
2,5 0,1265 1 0,1265 1,259 0,0405 1 0,0405 0,811 0,092 1 0,092 1,004
3 0,2873 1 0,2873 1,641 0,0724 1 0,0724 1,162 0,217 1 0,217 1,716
3,5 0,2609 2 0,5218 1,194 0,069 2 0,138 1,290 0,1933 2 0,3866 1,155
4 0,1663 5 0,8315 0,932 0,067 5 0,335 1,774 0,1384 5 0,692 1,164
4,5 0,2182 5 1,091 0,543 0,142 5 0,71 1,502 0,1759 5 0,8795 0,480
5 0,1992 6 1,1952 0,182 0,1605 6 0,963 0,610 0,1843 6 1,1058 0,458

-all strains in log phase at roughly 3-3,5h –Transformation should be done after 3-3,5h of incubation time after staring a culture!

<Beschreibung>

Aim / Task Preparation Sequences
Protocol
Participants Lena Schorr, Ruben Caspary, Eva Neugebauer

Protocol

Resuspension of the IDT sequences in 20 µL TE buffer.

Resulting concentration 50 ng/ µL.

Vortexing and incubation for 15 min at 50°C.

Vortexing and storage at -20°C.

Biotwist sequences and primer still not resuspended!

10 µL Filtertip-Spitzen

Aim / Task Amplify construct DNA
Protocol -
Participants Lena Schorr, Lisa Vogg
Name Attempt 1 Attempt 2 Attempt 3
DNA 1 µg (=0,5 µl) 2 µg (= 1 µl) 1 µg (= µl)
NEB buffer 3.1 (x10 ) 5 5 -

Protocol

Notes

  • dissolved primers for amplification in TE buffer to a concentration of 100mM
  • froze each primer diluted in TE with a concentration of 1mM (labeled according to construct)
  • dissolved all remaining DNA of the constructs in 20 µl TE buffer
  • prepared 2 1:100 pre-dilutions of the primer for the PCR, first in TE buffer, second in RNase-free water
  • performed PCR according to NEB protocol
  • stored PCR products in 4 °C fridge
  • prepared agarose gel for electrophresis (needs to be repeated)

Protocol

Dissolving freeze-dried DNA

  • spin down aliquots
  • add required volume of TE buffer (20 µl)
  • vortex thoroughly
  • incubate on the heating block at 50°C for 20 min

PCR (according to the NEB protocol)

template DNA 5 µl (250 ng)
forward primer (10 µM) 1.25 µl (end concentration 0.5 µM)
reverse primer (10 µM) 1.25 µl (end concentration 0.5 µM)
Master Mix 12.5 µl
RNase-free water 5 µlpr
25 µl
  • programme is saved under Thomas Winkler >Nina >iGEM
  • can otherwise be found in the NEB protocol
    • 30 cycles at 60°C

Agarose gel

  • 0.8 g Agarose
  • 100 ml TAE buffer
  • 10 µl SYBR Safe

  • medium chamber

  • slot chamber onto its sled with the open ends aligning with the edge of the sled

  • cautiously pour the liquid agarose into the chamber, avoid air bubbles

  • insert the comb

  • DO NOT MOVE AFTER AGAROSE HAS BEEN ADDED! Otherwise liquid will slosh out and gel will be too thin

  • gels can be stored in a plastic dish with a little bit of TAE buffer at 4°C or wrapped in saran film

Calculations and Results - calculated volume for dissolving the primers: V=m/(M*c) with m and M found on the aliquots and c set as 100 mM

Primer m [mg] M [g/mol] V [µl]
Tac Promotor Primer fw 2.0 0.97 6.38 1520
K1 HisStop Primer re 2.0 0.61 6.467 943
K2A HisStop re 2.0 0.21 7.084 296
K3a HisStop Primer re 2.0 0.54 6.194 886
K4a StrepStop re 2.0 0.22 6.362 346
K4b StrepStop re 2.0 0.2 6.924 314

Pending tasks, next steps, comments for next people in the Lab - run electrophoresis with PCR products

Aim / Task Primer Dilution
Protocol -
Participants Lena Schorr
Name Attempt 1 Attempt 2 Attempt 3
DNA 1 µg (=0,5 µl) 2 µg (= 1 µl) 1 µg (= µl)
NEB buffer 3.1 (x10 ) 5 5 -

Protocol

Notes

Prepare Primer Solution:

  • centrifuge primers
  • add TE-buffer to dilute primers to a concentration of 100nM
  • Vortex
  • heat and shake at 50°C in thermomixer
  • freeze and store at -20°C

Protocol

  • Ran gelelectrophoresis with PCR products 02.07.2019 with 100x
DNA 2µl
Loading Dye(6x) 2µl
H20 16µl
  • Put 20µl in each pocket
  • put 6µl DNA Ladder in one pocket

Gibson - PCR:

template DNA 5 µl (250 ng)
forward primer (10 µM) 1.25 µl (end concentration 0.5 µM)
reverse primer (10 µM) 1.25 µl (end concentration 0.5 µM)
Master Mix 12.5 µl
Autoclaved H20 5 µlpr
25 µl
  • NEW PCR PROGRAM: igem gibson anhang
  • PCR is running over night. Finishes around 9pm and is then stored at 4°C in the PCR cycler

Calculations and Results

Problems faced / possible error causes

  • No Protocol! People should make clear which protocol they want to use
  • Tested PCR Products on quantity with biophotometer: No clear results. Results are in our lab notebook

Materials used

  • 16 PCR Tubes and caps

Files used / generated

Pending tasks, next steps, comments for next people in the Lab

  • Run Gel electrophoresis. See day before
  • Gibson Assembly of Fab Fragments
  • Gibson Assembly of different constructs into vector
  • Loading Dye 6x is stored in the fridge in Winkler’s Lab

Aim / Task Create E.coli Starter Glycerol Stock
Protocol see link
Participants Daniel Schreyer, Jannik Wagner

Protocol

Notes

Problems faced / possible error causes

  • Agar dissolves in autoclave
  • work very sterile!!! No antibiotica

Materials used

  • 35g LB-Broth with agar


Pending tasks, next steps, comments for next people in the Lab

  • Cultivate 2 colonies each in 50mL flasks. See protocol day 2 morning!

Aim / Task Gibson PCR Control Gel-Electrophoresis
Protocol see link
Participants Felix Pfister

Protocol

Notes

  • 0,8% Agarose gel
  • DNA-Mix of Gibson PCR
    • 2 µl DNA
    • 2 µl Loading Dye (6x)
    • 16 µl dH2O
  • Gelelectrophoresis @130V for 60 min
  • Used only 4µl of 1kb DNA Ladder

Protocol

https://www.addgene.org/protocols/gel-electrophoresis/

Calculations and Results

Problems faced / possible error causes

  • Gel seems to run a bit crooked
  • Amplification of unknown DNA
  • Only little desired DNA amplified

Materials used

  • Gelectrophoresis
    • 0.8g Agarose
    • 100ml TAE-Buffer (1x)
  • DNA-Mix
    • 2µl DNA
    • 2µl Loading Dye (6x)
    • 16µl dH2O
  • 4µl DNA Ladder (1kb)
  • 8 PCR tubes

Files used / generated

Pending tasks, next steps, comments for next people in the Lab

  • Gibson Assembly if correct bases are seen in the gelphoto

<Beschreibung>

Aim / Task Cultivation of E.coli in 15ml flasks with 10ml LB-medium
Protocol see links
Participants Daniel Schreyer, Jannik Wagner

Protocol

Notes

  • Too many colonies on plates
  • used 2 µl of each strain and put the whole tip into 10ml LB-medium
  • shake at 37°C and 200rpm. Incubate until the evening
  • autoclave 1L erlenmeyer flask until the evening

Protocol

http://www.molbi.de/protocols/competent_cells_starter_cultures_v1_0.htm Day 2: Morning

Problems faced / possible error causes

  • Too many colonies. Use less amount for cultivation on agar plates


Materials used

  • 4 Falcons 15mL
  • 1 10ml plastic tip



Pending tasks, next steps, comments for next people in the Lab

  • Cultivate in 1L erlenmeyer flask with 250ml LB until the morning
  • OD measurement of cultivated E.coli

Aim / Task Gibson PCR (Overhang) - New 57°C
Protocol PCR (according to NEB protocol)
Participants Felix Pfister, Daniel Schreyer

Protocol

Notes

  • Defrost Primer on Ice

Protocol

Gibson - PCR:

Template DNA 5 µl (250 ng)
Primer fw (10µM) 1.25 µl (end concentration 0.5 µM)
Primer rev (10µM) 1.25 µl (end concentration 0.5 µM)
Master Mix 12.5 µl
  • NEW PCR PROGRAM: igem gibson anhang —-now with 57°C instead of 65°C
    • Also 35 cycles instead of 30 cycles
  • PCR is running over night

Calculations and Results

-

Problems faced / possible error causes

  • Gib pAC Promotor fw almost empty

Materials used

  • 8 PCR tubes

Files used / generated

Pending tasks, next steps, comments for next people in the Lab

  • Run Gel electrophoresis. See day before
  • Gibson Assembly of Fab Fragments
  • Gibson Assembly of different constructs into vector
  • Get new pAC fw primer!!!

Aim / Task Gibson Assembly - cancelled!
Protocol see link
Participants Felix Pfister, Antonio Maccataio, Daniel Schreyer

Notes

  • Checked Gel electrophoresis from Gibson PCR
    • Didnt work/not enough DNA!!!!

Protocol

https://international.neb.com/protocols/2012/09/25/gibson-assembly-master-mix-assembly

Calculations and Results

Problems faced / possible error causes

Materials used

Files used / generated

Pending tasks, next steps, comments for next people in the Lab

  • Repeat the Gibson Overhang PCR

Aim / Task Aliquot E. coli
Protocol http://www.molbi.de/protocols/competent_cells_starter_cultures_v1_0.htm Day 3: Morning
Participants Daniel Schreyer
  • PCR products from 2019-07-04 are stored at 4 °C
  • aliquots are stored at -80 °C, cells are not competent

Protocol:

  • transfer 200 mL E.coli suspension in four 50 mL flasks –Centrifuge at 4000 rpm for 10 mins with ultra centrifuge
  • dilute centrifuged E. coli in 2.5 ml glycerol and 2.5 mL LB-medium
  • use 4 mL of each flask to make 1 mL aliquots
  • making around 16x 1 mL aliquots of each strain: DH5alpha, Star, Tuner, BL21

Aim / Task Control electrophoresis Gibson Overhnag PCR from 07-04
Protocol
Participants Felix Pfister
  • checked Gibson Overhang
  • ran agarose gel at 130 V

Aim / Task Purify Konstruct DNA from Agarose Gel
Protocol -
Participants Eva Neugebauer, Leon Altmann

Protocol

Notes

  • Before the purification the according DNA bands were cut out of the gel
  • The gel pieces were transferred into Eppendorf Reaction Tubes, which were weighed beforehand;
  • The mass of the gel pieces was determined by repeated weighing, to determine the necessary amounts of reagents


Protocol

Protocol of the NucleoSpin Extract Kit

Calculations and Results

  • Reaction Tube weight (empty): K2b 1,013g K3b 1,014g K4a 1,013g K4b 1,013g

  • Weight of Gelfragments: K2b 0,287g K3b 0,356g K4a 0,288g K4b 0,287g

  • For each 100mg gel 300ul of NT1 buffer should be used

  • Amount of NT1 buffer: K2b 861ul K3b 1068ul K4a 863ul K4b 861ul

Problems faced / possible error causes

The gel fragments were quite large; This might have a negative impact on the efficiency of the purification;

As the lab with the nanodrop was already closed the Concentration of the processed DNA could not be assesed at this day;

Materials used

  • NucleoSpin Extract Kit

Files used / generated

Pending tasks, next steps, comments for next people in the Lab

  • Measure DNA concentrations

Aim / Task Production of electrocompetent Bacteria (BL21, BL21 Star, Tuner)
Protocol http://www.molbi.de/protocols/competent_cells_electro_v1_0.htm
Participants Felix Pfister, Jannik Wagner
  • used only 100 mL of LB medium for 3 flasks
  • Problems faced:
    • no ON culture, directly from -80 °C into 100 mL LB medium
    • lost Tuner completely –no competent Tuner bacteria on -80 °C
    • BL21 Star had a smeary pellet in the last washing step –could be problematic

Aim / Task PCR with K1, K2a, K3a 3% DMSO and gel-electrophoresis
Protocol -
Participants Antonio Maccataio, Lisa Vogg, Marie Wiedemann

Protocol

Notes

  • 5 yL DNA
  • 1,25 ym primer fw
  • 1,25 yL primer re
  • 5 yL DMSO 15% Stock ->3% diluted
  • Mastermix 12,5 yL

2x 1:100 Dilution

1mM TE (DNA storage at -20°)

Gibson

gel-electrophoresis

<Beschreibung>

Aim / Task Repeat failed PCR for Gibson overhangs with added DMSO
Protocol NEB kit instructions, NEB troubleshooting
Participants Lisa Vogg, Antonio Maccataio
  • aliquoted 15% DMSO
  • used original amplified DNA of K1, K2a, K3a
  • added DMSO to a final concentration of 3%
  • lowered running temperature of the PCR from 57 °C to 55 °C due to DMSO
  • prepared 1:100 dilution of the respective primers (–1mM) in TE buffer and stored them at -20 °C
  • prepared a further 1:100 dilution (–10 µM) in dH2O for the PCR
DNA 5 µL
Primer fw 1.25 µL
Primer re 1.25 µL
Master Mix 12.5 µL
DMSO (15%) 5 µL
25 µL

Aim / Task Electroporation Control
Protocol NEB electroporation protocol
Participants Felix Pfister
  • electroporation of pUC19 into BL21, BL21 Star
  • one H2O control in BL21 Star
  • generated own electroporation protocol in the GeneUser (>Users>igem>igem1)
  • used 25 µL bacteria with 1 µL 1:5 diluted pUC19
    • should be 50 µL next time instead of 25 µL

Protocol: 1. incubate labelled agar plates in the incubator at 37 °C 2. incubate medium at 37 °C 3. thaw bacteria from -80 °C on ice 4. when bacteria are thawed, add DNA 5. keep 1 mm cuvettes on ice 6. electroporation of the bacteria with 1 µL DNA –immediately add warm medium to cuvette and mix by pipetting up and down 7. put bacteria on shaker at 37 °C for 30-60 mi at 300 rpm 8. plate them on the 37 °C warm agar plates 9. incubate the bacteria on plates ON –not too long or they will grow too much

Aim / Task repeat failed PCR with correct amount of primer at 57 °C with and without DMSO
Protocol NEB PCR kit
Participants Daniel Schreyer, Lena Schorr, Lisa Vogg
  • set up two PCR reactions:
DNA 5 µL
Primer fw (10 µM) 1.25 µL
Primer re (10 µM) 1.25 µL
Master Mix 12.5 µL
dH2O or DMSO (15 %) 5 µL
25 µL
  • run PCR at 57 °C
    • last PCR might have failed due to too low temperatures
    • last PCRs for overhang generation might have failed due to much too high primer concentrations, would explain very bright bands in the previous gel
  • amplified constructs form the very first PCR were not sufficient fpr the reaction
  • set up another PCR to amplify the constructs K1, K2a and K3a in a 25 µL reaction at 60 °C
  • run 0.8 % agarose gel (small) for the amplified constructs
    • 2 µL DNA, 18 µL total volume in each pocket
    • run at 130 V
    • 3 µL 1 kb DNA ladder
  • run 0.8 % gel (medium) for the overhang PCR
    • used 2 µL DNA and 6 µL of test vector from the day before
  • autoclaved 1 L LB medium

Aim / Task Check electroporated E. coli on plates
Protocol
Participants Daniel SChreyer
  • no growth on plates –incubate longer
  • E. coli were possibily not transformed
  • use bigger amount of eletrocompetent bacteria

Aim / Task make E. coli strains Tuner and DH5alpha electrocompetent
Protocol http://www.molbi.de/protocols/competent_cells_electro_v1_0.htm
Participants Martin Borgmann
  • thawed stock bacteria on ice
  • inocculated 2 25 µL of each strain (Tuner and DH5alpha) in 100 mL LB medium and incubated for 1:45 h until OD600 was 0.4-0.6
  • not much 10 % glycerol available so the two cultures of each strain have been pooled and washed together

  • frozen in 50 µL aliquots

  • modification of used protocol: 8.7 % glycerol used instead of 10 %

Aim / Task amplification of original constructs
Protocol NEB kit instructions
Participants Lisa Vogg
  • used 1:10 dilution of 100 µM stock of the primers
  • amplified all constructs
  • PCR at 60 °C, 25 µL reaction with 5 µL DNA

Aim / Task produce SOB medium
Protocol -
Participants Antonio Maccataio

Protocol:

Per liter: To 950 mL of deionized H2O, add:

Tryptone 20 g
Yeast extract 5 g
NaCl 0.5 g

SOC medium is identical to SOB medium, except that it contains 20 mM glucose. To prepare SOB medium, combine the above ingredients and shake until the solutes have dissolved. Add 10 mL of a 250 mM solution of KCl. (This solution is made by dissolving 1.86 g of KCl in 100 mL of deionized H2O.) Adjust the pH of the medium to 7.0 with 5 N NaOH (∼0.2 mL). Adjust the volume of the solution to 1 L with deionized H2O. Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle.

Just before use, add 5 mL of a sterile solution of 2 M MgCl2 and 5ml of MGSO4. (This solution is made by dissolving 40,66 g of MgCl2 (Hydrate) and 49,3g MgSO4 (Hydrate) respectively in 90 mL of deionized H2O. Adjust the volume of the solution to 100 mL with deionized H2O and sterilize by autoclaving for 20 min at 15 psi [1.05 kg/cm2] on liquid cycle.) After the SOB medium has been autoclaved, allow it to cool to 60°C or less. Add 20 mL of a sterile 1 M solution of glucose. (This solution is made by dissolving 18 g of glucose in 90 mL of deionized H2O. After the sugar has dissolved, adjust the volume of the solution to 100 mL with deionized H2O and sterilize by passing it through a 0.22-µm filter.)

  • only produced 100 mL

Aim / Task electroporation control
Protocol https://international.neb.com/Protocols/0001/01/01/electroporation-protocol-c2986
Participants Felix Pfister
  • control if BL21 blank/star/tuner and DH5alpha are electrocompetent
  • added 2 µL of pUC19 1:5 –electroporation with iGEM protocol
  • bacteria plated on 37 °C LB-Agar plates with ampicillin

Aim / Task Heat shock of DH5alpha and BL21
Protocol
Participants Eva Neugebauer, Daniel Schreyer
  1. Thaw chemical competent bacteria on ice (200 µL each)
  2. Add 5 µL of pUC vector
  3. 30 min incubation on ice
  4. 1 min 42°C (450 rmp)
  5. 2 min on ice
  6. Add 500 µL SOC medium
  7. 30 min 37°C (450 rpm)
  8. Plate 200 µL bacs on Amp plates
  9. Incubation over night at 37°C

Aim / Task Preparation of chemically competent E. coli
Protocol https://www.helmholtz-muenchen.de/fileadmin/PEPF/Protocols/Chemically-competent-cells.pdf
Participants Eva Neugebauer, Daniel Schreyer, Ruben Caspary
  • prepared chemically competent E. coli: DH5alpha, BL21 Star, Tuner, BL21
  • used E. coli with the following OD:
Strain BL21 BL21 Star Tuner DH5alpha
OD 0.55 0.499 0.43 0.42
  • 20x 200 µL aliquots stored at -80 °C of each strain
  • CC buffer is stored at 4 °C and is sterilized through 0.22 µm filtration
  • used 1⁄5 of each medium –cultivated in 100 mL LB instead of 500 mL
  • used stock E. coli, not single colony –lower transformation rate possible

Aim / Task Check plates of transformation of 2019-07-18
Protocol
Participants Daniel Schreyer
  • checked LB agar plates with ampicillin oand our transformed bacteria of 2019-07-18

  • growth on both plates –heat shock success

  • pick on DH5alpha colony from the agar plate

  • cultivated in 250 mL LB-medium with 250 µL (50 mg/ml) Ampicillin

  • cultivate at 30 °C and 130 rpm for around 18 h

Aim / Task Cultivation of transformed DH5alpha and BL21, PCR with new settings
Protocol
Participants Eva Neugebauer, Daniel Schreyer

Cultivation for miniprep of pUC19:

  • no bacteria grew in the cultivation of 2019-07-20
  • incubator did not heat up
  • new clones picked: 5x DH5alpha, 3x BL21
  • in 5 mL LB medium with Amp each
  • incubation at 37 °C

PCR overhang K1, K2a, K3a with DMSO:

  • primers used:
    • K1: fw Gib pAC Promotor fw 2.0, re Gib pACK1HisStop prim
    • K2a: fw Gib pAC Promotor fw 2.0, re Gib pAC K2a HisStop
    • K3a: fw Gib pAC Promotor fw 2.0, re Gib pAc K3a fusion re
  • PCR program:
    • 98 °C 3 min
    • 98 °C 10 s
    • 53.7 °C 30 s
    • 72 °C 1 min
    • 72 °C 10 min
    • 35 cycles
  • PCR products stored at 4 °C

Aim / Task Overnight culture of transformed E. coli for control
Protocol
Participants Daniel Schreyer, Lisa Vogg
  • Picked 3 DH5alpha and 1 BL21 Heat shock puC19 transformed E.coli and incubated them over night in 2 ml LB+Amp at 37°C with 220 rpm
  • OD measurements in the morning to validate transformation success

Aim / Task Run test gel for new overhang PCR, mini prep of test transformation
Protocol Extract me! Mini kit instructions
Participants Felix Pfister, Lisa Vogg
  • 0.8 % gel with 2 µL DNA, 3 µL loading dye, 13 µL dH2o
  • out of 8 cultivated colonies, only 2 showed bacterial growth
  • performed mini prep for those two cultures (BL21 colonies 2 and 3)
  • ran test gel for mini prep

Protocol:

  1. Pellet cell from bacterial culture by centrifugation for 5 min at 3500g
  2. discard supernatant and resuspend cell pellet thoroughly in 250 µl resuspension buffer. Transfer resuspended cell pellet to sterile 1.5 ml tube.
  3. add 250 µl lysis buffer and invert tube 4-6 times. DO NOT VORTEX. If sample is not clear immediately, incubate for 1-2 min.
  4. add 350 µl neutralization buffer and invert tube several times. DO NOT VORTEX.
  5. centrifuge for 5 min at 11000 g. Repeat if supernatant is not clear.
  6. Carefully transfer supernatant into a purification microcolumn placed in a collection tube. Do not contaminate sample with pellet! Centrifuge for 60 s at 11000 g.
  7. Optional: add 500 µl NW buffer and centrifuge 60 s at 11000g.
  8. add 750 µl wash buffer and centrifuge for 60 s at 11000g. Discard filtrate and reuse collection tube.
  9. centrifuge for 2 min at 11000g. Discard collection tube and carefully transfer column to a sterile 1.5 ml tube.
  10. add 50-100 µl elution buffer directly onto the column. Incubate at room temperature for 60 s.
  11. centrifuge for 60 s at 11000g. Remove microcolumn. Isolated DNA should be stored at 4 or -20°C.

Aim / Task OD of over night culture DH5alpa with pUC19 and BL21 with pUC19
Protocol -
Participants Marie, Jannik

Protocol

Notes

  • OD: DH5alpha with pUC19 = 2,79
  • OD: BL21 with pUC19= 4,4
  • Argarose gel for vector extraction for BL21
  • 5 yL Eluate
  • 3 yL dye
  • 12 yL H2O
  • 4 yL Ladder
  • Cut lower band out
  • Gel volume 230 mg
  • Used Qiagen QiAquick gel-extraction kit50 (see Protocol)
  • Used 80 yL EB-buffer to eluate
  • PCR amplification of K1 with
    • 5 yL DNA
    • 1,25 yL primer re & fw
    • 12 yL Master-mix
    • 5 yL H2O

BL21 pUC19 concentration: 6,53 ng/µL

Protocol

<Beschreibung>

Aim / Task Gibson assembly and transformation of K2a, K2b, K3a and K4a in pACYC in BL21
Protocol Gibson assembly instructions, heat shock protocol
Participants Jannik Wagner, Lena Schorr
  • measured DNA concentrations of all constructs

  • calculated the amount of DNA and vector for the Gibson assembly

  • Gibson assembly of 2a, 2b, 3a+b, 4a+b

  • transformation of the constructs into BL21 via heat shock

    • 200 µL bacteria with 2 µL Gibson reagent

  • preparation of chloramphenicol plates

  • plated transformed bacteria –incubator at 37 °C

Concentrations of DNA of the constructs:

  • K1: 808.9 ng/µL
  • K2a: 868.7 ng/µL
  • K2b: 847.2 ng/µL
  • K3a: 791 ng/µL
  • K3b: 819.1 ng/µL
  • K4a: 776.1 ng/µL
  • K4b: 805.9 ng/µL

Lengths of the DNA constructs:

  • K1: 1629 bp
  • K2a: 1278 bp
  • K2b: 1218 bp
  • K3a: 801 bp
  • K3b: 753 bp
  • K4a: 801 bp
  • K4b: 759 bp

Calculation of the amount of contruct and vector needed for the Assembly:

  • our constructs and vector in pmol/µl (with the formula: amount of DNA * 1000 / bp of the constructs + 650 Da)
  • K1: 1.04 pmol/µL
  • K2a: 1.097 pmol/µL
  • K2b: 1.46 pmol/µL
  • K3a: 1.36 pmol/µL
  • K3b: 1.49 pmol/µL
  • K4a: 1.34 pmol/µL
  • K4b: 1.46 pmol/µL –1:3 dilution with H2O –1 µL was taken for the Gibson Assembly (=0.37 pmol/µl for construct 2a) –>Vector (pACYC): same amount: ~0.4 pmol/µL

Gibson Protocol:

  • 0.02-0.5 pmol/µL DNA
  • 10 µL Gibson assembly master mix
  • 10 µL dH2O

Aim / Task Transformation of E.coli BL21 with Test Vector pUC
Protocol Transformation mit Ca2+ - kompetente E.coli - Zellen
Participants Martin Borgmann, Marie Wiedemann
Name Attempt 1 Attempt 2 Attempt 3
DNA 1 µg (=0,5 µl) 2 µg (= 1 µl) 1 µg (= µl)
NEB buffer 3.1 (x10 ) 5 5 -

Protocol

Transformation of BL21 with Test vector pUC (Amp res.):

  • Protocol used is in folder page 1: Transformation mit Ca2+ - kompetente E.coli - Zellen

    • Nanodrop: 6,53ng/µL
    • 50ng DNA ( = 7,7µL) and 200 µL chemocompetent cells
    • heatshock 45s, 42 °C

  • plate on Amp plates –test vector with Amp resitance

  • incubate at 37°C overnight

Result: succesfull transfortmation since the bacteria grew on the Amp plate

Agarose gel electropheres of PCR products from the morning,

Amplification PCR of Construct 1

  • Protocol from the PCR kit
  • Q5 polymerase
  • 4µL template
  • Procedure:
    1. letting the cycler heat up and wait untilll actuall programm starts
    2. then put in the reaction tube

Result: negative

  • either bad primers
  • or because no DSMO was used

Aim / Task Preparation of Media LB and SOC
Protocol
Participants Martin Borgmann

Protocol

Notes

  • Repeated PCR from this morning (26.07.19., entry 56) –Attempt to amplify K1 –not successful
    • 2µL Template
    • 25 µL x2 Q5 MasterMix
    • 2,5 µL FW Primer
    • 2,5 µL RV Primer
    • 2µL H2O
    • 16 µL 15% DSSO –~5% final conc.

Prepartion of Media SOC and LB

1L medium -25 g/L LB broth powder dissolved in dH2O

0,2 L SOB Medium for SOC Medium to make E.coli electrocompetent

  • 1,25 g Trypton
  • 5 g yeast extract
  • 0,117 g NaCl

–autoclave 30 min 121 °C

Added the rest of the ingredients for SOC by Eva & Lisa the next day after the Medium cooled off (entry 63)

Aim / Task Transformation of BL21 with Construct 2a, 2b, 3a + 3b, 4a + 4b in pACYC184 vector, Amplification of Construct 1 with original DNA
Protocol Transformation mit Ca2+ - kompetente E.coli Zellen (Ordner Seite 1)
Participants Daniel Schreyer, Ruben Caspary

Notes

  • amplified 2µl original K1 DNA
  • H20 Control

Protocol

DNA 2µl
2x MasterMix 10µl
Primer fw 10µM 1µl
Primer re 10µM 1µl
DMSO 3% in dH20 6µl

Program used Construct Amplification (30 cycles):

Denaturation 98°C 10s
Annealing 51°C 30s
Extension 72°C 60s
  • Control with 0.8% Agarose-Gelelectrophoresis
  • 17 µl K1 Amp remaining

Transformation of BL21 with Construct 2a, 2b, 3a + 3b, 4a + 4b in pACYC184 vector:

  • Protocol used is in folder page 1: Transformation mit Ca2+ - kompetente E.coli - Zellen
  • Contructs used: 2a, 2b, 3a+b, 4a+b 2µl each

  • positive Control with NEB pUC Control vector: used 10µl -500pg + purified pUC Vector from colony 3 –4µl: around 25ng DNA

  • plate on Amp plates: positive Controls

  • plate on Chloramphenicol plates from 29.06.2019 Construc 2a, 2b, 3a + 3b, 4a + 4b

  • Incubate in the afternoon or until tomorrow morning at 37°C

Problems faced / possible error causes

  • Used old plates 29.06.2019
  • used SOC-Medium, which was opened on the 11.07.2019 –might be contaminated, even though medium was clear
  • used maybe too much DNA Ladder

Materials used

  • 3mL SOC-Medium
  • NEB pUC Vector: 10µl
  • 0,4 Agarose
  • 10µl Quick 1kb DNA Ladder

Pending tasks, next steps, comments for next people in the Lab

  • Control of Gelelectrophoresis under UV-Light
  • autoclave SOC-Medium
  • Pour new plates: Chloramphenicol and Ampicillin plates
  • Check transformed bacteria on plates in the next morning 27.07.2019

Aim / Task Preparation of Media
Protocol
Participants Lisa Vogg, Eva Neugebauer

Protocol

Notes

  1. 1% Gel

Sample from 26.07.2019 for the gel (2µL DNA, 3µL Stain, 13µL H2O)

No bands visable!

  1. Each 2 colonies of K2a, K2b, K4ab and one colonie of K3ab

Incoluation overnight at 37°C in 5 mL LB with Camp

  1. Finishing the SOC medium from th 26.06.2019 by adding 1 M MgSo4 and 0,72 g Glucose

Pending tasks, next steps, comments for next people in the Lab

Miniprep of clones

Aim / Task Store transformed E.coli, K1 Gel Extraction
Protocol
Participants Eva Neugebauer, Daniel Schreyer

Protocol

Notes

  • No mini-prep columns left!! –Wait for new order.
  • Transformed E.coli are stored at 4°C and are waiting for their mini-prep (4.5 ml left)**
  • Aliquoted 50% Glycerol in LB-medium in 15 ml Falcon

Storage of 500µl transformed E.coli at -80°C

Protocol:

  1. Mix 500µl vortexed transformed E.coli with 500µl 100% Glycerol solution
  2. Different colonies are labeled 1 and 2. Some Construct transformed E.colis have 2 aliquots, because of 2 colonies picked
  3. Store at -80°C

K1 Gel Extraction:

  • Preparation for Blunt-End Cloning
  • Extract K1 amplificat (11.07.2019) out of a 0.8% agarose gel
  • Use 6µl of K1 and 15µl of H20 control –2µl of K1 are left for further PCRs
  • Run at 110V for 90 minutes
  • Plot: 4µl Ladder - empty - K1 - empty - H20

Plated 200µl K3a+b on Agar-Chloramphenicol plates

Problems faced / possible error causes

  • Amplification PCR are not perfectly labeled. Unsure if it was K1 or Water control –Used both in our gel electrophoresis
  • NO K1 Amplificat left in tube! Gel Extraction was not possible

Materials used

  • 4µl DNA Ladder
  • Gel extraction Kit (1 prep)
  • 10ml Glycerol

Pending tasks, next steps, comments for next people in the Lab

  • Blunt-End cloning of purified K1 in pACYC184
  • Mini-prep of all our transformed E.coli
    • PCR to validate Gibson Assembly and transformation success.
  • Pick colony of K3a+b and Incubate in Lb - Chloramphenicol medium for mini-prep

Aim / Task Cultivation of K3ab for Mini-Prep
Protocol
Participants Daniel Schreyer

Protocol

Notes

  • Picked 2 colonies of K3ab plate (overnight plate from the 29.07.19)
  • Cultivate in Lb-Chloramphenicol medium

Pending tasks, next steps, comments for next people in the Lab

  • Check growth on the 30.07.19
  • Perform mini-prep
  • Validate Insertion success of our Gibson Assembly

Aim / Task Mini-Prep of the reagenz K2a (2x), K2b (2x), K3ab (3x), K4ab (2x) = 9 samples
Protocol Protocol of the Kit "ExtractMe"
Participants Martin Borgmann, Lena Schorr

Protocol

Notes

  • first we down-centrifuged the over-night culture from K3ab
  • We then put 500µl of K3ab together with 500µl of Glycerol in the freezer at -80°C (box at the bottom of rang 12)
  • Then the Mini-Prep with the Extract gel was performed
  • The DNA was then stored at 4°C

Protocol

Protocol of the Extract me Kit

Calculations and Results

Problems faced / possible error causes

We used 14ml overnight-culture, which was a little bit too much, because after the lysis there was a lot protein waste so we had to repeat this step several times

Materials used

  • Glycerol
  • Extract Me Kit

Files used / generated

Pending tasks, next steps, comments for next people in the Lab

  • Measure DNA concentrations
  • Performing a gel-electrophoresis with the isolated DNA (first cleave the DNA with restriction-enzymes)
  • Sent a part of the isolated DNA to sequencing-company

Aim / Task Concentration of Minipreps (K2a (2x), K2b (2x), K3ab (3x), K4ab (2x) = 9 samples)
Protocol Eva’s Digest Protokoll
Participants Marie Wiedemann, Ruben Caspary

Protocol

Notes

  • Nanodrop of all Mini-Preps
  • Digest at 37° 350 rpm heat-block
    • 12 yL H2O
    • 5 yL Probe
    • 2 yL CutSmart Buffer
    • 0,5 yL Hind_III
    • 0,5 yL BamHI-HF
  • Concentrations similar to Eva’s vector preperation
  • Gel, 14 lanes for vector-insert digest of:
    • K2a (2x colonie 1 & 2),
    • K2b (2x colonie 1 & 2),
    • K3ab (3x colonie 1,2,3),
    • K4ab (2x colonie 1 & 2 no Kol 3) = 9 samples

Protocol

Calculations and Results

Construct Colonie 1 [c] ng/yL Colonie 2 Colonie 3
2a 40,22 46,00
2b 46,08 41,48
3ab 62,29 36,44 32,29
4ab 33,01 3,01

Problems faced / possible error causes

Materials used

CutSmart Buffer

Hind_III

BamHI_HF

TAE-Buffer

Agarose

Files used / generated

eluate gel, send Insert

Pending tasks, next steps, comments for next people in the Lab

August

Aim / Task Transformation colony screening with PCR
Protocol Standard PCR protocol
Participants Daniel Schreyer
  • used PCR primers for amplification to screen for our inserted sequence
  • forward primer the same for all the samples, used the respective reverse primers of each sample. For the ligated samples, the “b” primers were used.
Template DNA 5 µL
Primer fw (10 µM) 1.25 µL
Primer rev (10 µM) 1.25 µL
Master Mix 12.5 µL
autoclaved H2O 5 µL
25 µL
  • checked success with a 1% agarose gel, run at 150 V for 1 h
  • PCR was negative
    • too little amplification primers
    • wrong primers used
    • or no insert in our vector

Aim / Task Transformation Colony Screening with PCR
Protocol Standard PCR Protocol
Participants Daniel Schreyer
Name Attempt 1 Attempt 2 Attempt 3
DNA 1 µg (=0,5 µl) 2 µg (= 1 µl) 1 µg (= µl)
NEB buffer 3.1 (x10 ) 5 5 -

Protocol

Notes

  • Use PCR Primers for Amplification to screen for our inserted sequence
  • Only if Primers can bind to our vector and PCR produces the right Insert size our sequence is in our vector and the colony can be used for dowstream experiments
  • Use undiluted amplification primers, because we don’t trust the 100µM note.
    • We have enough Primers anyway
  • used always the same forward Primer, but the reverse primer differed from construct to construct. Used always b re primers, if we assembled multiple constructs together
  • Used 2 x 2a, 2 x 2b, 3 x 3ab, 2 x 4ab –labeled them respectively

Gel Electrophoresis:

  • 1% Agarose gel. Run with 150 V for 1 hour

Protocol

PCR:

Primers:

fw: Tac Promoter fw 2.0 Primer

re: 2a: 2a re Primer, 2b: 2b re Primer, 3ab: 3b re Primer, 4ab: 4b re Primer

Name Volume
Template DNA 5 µl
Primer fw (10µM) 1.25 µl
Primer rev (10µM) 1.25 µl
Master Mix 12.5 µl
Autoclaved H2O 5 µl
Total Volume 25 µl

Problems faced / possible error causes

  • Too little amplification primers
  • Wrong amplification primers used
  • No Sequence in our vector

Materials used

  • 100 µl 2x MasterMix
  • 12 PCR Tubes and lid

Aim / Task Control of the Gibson assembly
Protocol
Participants Martin Borgmann, Jannik Wagner
  • restriction digest with Ase1 and Pvu1: Ase1 cuts once in the vector and once in the promoter region of the insert, Pvu1 was used to cut the insert, however it only cuts in construct 2a
    • 12 µL H2O
    • 5 µL DNA
    • 2 µL CutSmart Buffer 3.1
    • 0.5 µL Ase1
    • 0.5 µL Pvu1
  • incubation for 1 h at 37 °C
  • gel electrophoresis in 1% agarose in TAE with SYBR Safe Gel Stain

Aim / Task Control if the Assemblys worked
Protocol -
Participants Martin Borgmann, Jannik Wagner

Protocol

Notes

  • Restriction digest with Ase1 and Pcu1.

    • 12 µL H2O
    • 5 µL DNA
    • 2 µL CutSmart Buffer 3.1
    • 0,5 µL Ase1
    • 0,5 µL Pvu1

    -incubation 1h 37 °C

Gel Electrophoresis in 1% Agarose in TAE + SYBR green
Calculations and Results

Ase1 part of the vector and also part of each construct

-Ase1 cuts sticky ends -occurence of multimers

-Smallest piece is 329 kb, additinal bands due to multimers

-These bands can be found for every construct

Pvu1 cuts only 2a in two site

-many fragments from construct 2a

Result: The Construct had been inserted into the vector successfully!

Problems faced / possible error causes

Pvu1 was mistakingly used in an attempt to cut the insert. However, cutting site is only in 2a. Ase1 sufficient for the intepretation of the results.

Pending tasks, next steps, comments for next people in the Lab

confirm results with PCR of the insert

<Control if the Assemblys worked>

Aim / Task Check RE digestion of the miniprep, dissolved new DNA samples
Protocol -
Participants Lisa Vogg, Leon Altmann

Protocol

Notes

  • ran gel (2 µl plasmid DNA, 3 µl loading dye, 13 µl dH20)
  • dissolved new DNA (CHO expression and measured part) in 20 µl TE buffer and stored at -20 °C
  • DNA should be thoroughly resuspended before usage just in the case it is not already

Aim / Task Check RE digestion of the miniprep
Protocol
Participants Leon Altmann, Lisa Vogg
  • ran 1% agarose gel: 2 µL plasmid DNA, 3 µL loading dye, 13 µL dH20 at 130 V for 1 h
  • dissolved newly arrived DNA for CHO expression and measurement part in 20 µL TE buffer, stored at -20 °C

Aim / Task Gibson overhang PCR, pACYC184 EcoRV digest
Protocol
Participants Lena Schorr, Daniel Schreyer

Digest:

  • EcoRV digest with Eva’s protocol
  • digest mini-prep from 2019-08-02
  • concentration of pACYC184: 154 ng/µL
  • digest 1.5 µg plasmid: 9.74 µL at 37 °C for 60 min
CutSmart buffer 5 µL
DNA 9.74 µL
EcoRV-HF enzyme 1.5 µL
Water 33.76 µL
50 µL
  • inactivate for 20 min at 65 °C
  • run 0.8% agarose gel

PCR:

  • PCR of constructs from 07-16 K1, K2a, K3a + DMSO control
  • PCR of constructs from 07-04 K1, K2a, K2b, K3a, K3b, K4a, K4b + DMSO control
  • used residual DNA of 2b, 3b, 4a, 4b from 07-16
  • gel electrophoresis with 0.8% agarose gel
DMSO (15%) 5 µL
DNA 5 µL
fw Primer 1.25 µL
re Primer 1.25 µL
Master Mix (2x) 12.5 µL
25 µL

Second PCR with 3% DMSO:

  • used overhang PCR products from 07-16 of K1, K2a, K3a

Problems faced:

  • used wrong plasmid concentration! after mini-prep concentration of 50 ng/µL
  • used 100% DMSO instead of 15% as H2O replacement in the first PCR

Aim / Task Control of overhang PCR, purification of the cut pACYC184
Protocol Monarch DNA Gel Extraction Kit
Participants Martin Borgmann, Lena Schorr
  • Control gel:
    • used 1% agarose gel for samples of the overhang PCR and the cut vector
    • run gel at 120 V for 1 h
  • purification of the cut vector with Monarch DNA Gel Extraction Kit:
  1. Excise the DNA fragment from the agarose gel, taking care to trim excess agarose.Transfer to a 1.5 ml microfuge tube, and weigh the gel slice. Minimize exposure to UV light.
  2. Add 4 volumes of Gel Dissolving Buffer to the gel slice (e.g., 400 μl buffer per 100 μl or 100 mg agarose).
  3. Incubate the sample between 37-55°C (typically 50°C), vortexing periodically until the gel slice is completely dissolved (generally 5-10 minutes). For DNA fragments 8 kb, an additional 1.5 volumes of water should be added after the slice is dissolved to mitigate the tighter binding of larger pieces of DNA (e.g., 100 μl gel slice: 400 μl Gel Dissolving Buffer: 150 μl water).
  4. �Insert column into collection tube and load sample onto the column. Spin for 1 minute, then discard flow-through.
  5. Re-insert column into collection tube. Add 200 μl DNA Wash Buffer and spin for 1 minute. Discarding flow-through is optional.
  6. Repeat step 5.
  7. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not come into contact with the flow-through. If in doubt, re-spin for 1 minute.
  8. Add ≥ 6 μl of DNA Elution Buffer to the center of the matrix. Wait for 1 minute, and spin for 1 minute to elute DNA. Typical elution volumes are 6-20 μl. Nuclease-free water (pH 7-8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield.
  • measurement of purified vector: 4.9 ng/µL
  • storage of purified cut vector at -20 °C

  • digestion of purified cut vector with EcoRV-HF enzyme

  • run gel with samples of the overhang PCR of K1, K2a, K3a from the previous day, the digested vector and uncut vector as control

  • extraction of the digested vector of the gel, store at 4 °C

Problems faced:

  • Control gel was negative, only K3a was visible in the overhang PCR
  • Gel for purification was empty (broken slide), had to be repeated
  • Second gel also negative except for K3a

Aim / Task PCR of CFP part for measurement
Protocol
Participants Felix Pfister, Leon Altmann
  • eluted the Measurement Primers to a concentration of 100 µM
  • used for amplification via PCR: annealing temperature 57 °C, elongation time 45 s
Name Volume
Primer fw (10 µM) 1.25 µL
Primer rev (10 µM) 1.25 µL
template DNA 1 µL
Q5 2x Master Mix 12.5 µL
Nuclease free H2O 9 µL

Aim / Task Create good Gibson overhangs and perform Gibson Assembly
Protocol Q5 HiFi DNA-Pol NEB Protocol// Taq DNA-Pol NEB Protocol // NEBuilder HiFi DNA Assembly Kit Protocol
Participants Antonio Maccataio, Jannik Wagner

PCR:

  • annealing temperature: 53.7 °C
  • polymerase: Taq and Q5
  • DNA: 2 µL
  • nuclease-free water

Gel:

  • 1% agarose gel in TAE buffer
  • SYBR Safe Gel Stain: 7:100,000
  • run at 120 V

Analysis:

  • Taq PCR: total failure
  • Q5: good amplification of 3a, 3b and 4b, rest: failure
  • 3a and 3b measured by photometer
    • 500 ng 3a = 1,66µl (500ng/µl)
    • 500 ng 3b = 1,00µl (300ng/µl)
    • 370 ng pACYC = 2µl

Aim / Task gel extraction of pSec Hygro Tag, CHO constructs K1, K2a, K2b
Protocol Qiaquick Gel Extraction Kit
Participants Felix Pfister, Leon Altmann
  • gel extraction of pSec Hygro Tag, and the CHO constructs K1, K2a, K2b
  • Qiaquick Gel Extraction Kit:
  1. Add 5 volumes of Buffer PB to 1 volume of the PCR sample, and then mix. It is not necessary to remove mineral oil or kerosene. For example, add 500 μl of Buffer PB to 100 μl PCR sample (not including oil).
  2. If pH Indicator I has been added to Buffer PB, check that the mixture’s color is yellow. If the color of the mixture is orange or violet, add 10 μl of 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.
  3. Place a QIAquick spin column in a provided 2 ml collection tube.
  4. To bind DNA, apply the sample to the QIAquick column and centrifuge for 30–60 s.
  5. Discard flow-through. Place the QIAquick column back into the same tube. Collection tubes are reused to reduce plastic waste.
  6. To wash, add 0.75 ml Buffer PE to the QIAquick column and centrifuge for 30–60 s.
  7. Discard flow-through and place the QIAquick column back into the same tube. Centrifuge the column for an additional 1 min. IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.
  8. Place QIAquick column in a clean 1.5 ml microcentrifuge tube.
  9. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge. IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volumes are 48 μl from 50 μl elution buffer volume and 28 μl from 30 μl elution buffer. Elution efficiency is dependent on pH. Maximum elution efficiency is achieved between pH 7.0 and 8.5. When using water, make sure that the pH value is within this range, and store DNA at –20°C because DNA may degrade in the absence of a buffering agent. The purified DNA can also be eluted in TE buffer (10 mM Tris·Cl, 1 mM EDTA, pH 8.0), but the EDTA may inhibit subsequent enzymatic reactions.
  10. If the purified DNA is to be analyzed on a gel, add 1 volume Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting it up and down before loading the gel. Loading Dye contains 3 marker dyes – bromophenol blue, xylene cyanol and orange G – that facilitate estimation of DNA-migration distance and optimization of the agarose gel run time. Refer to Table 2 (page 17) to identify the dyes according to migration distance and agarose gel percentage and type.

Aim / Task PCR of CHO constructs
Protocol
Participants Felix Pfister, Leon Altmann
  • eluted the CHO Primers to a concentration of 100 µM
  • used for amplification via PCR: annealing temperature 57 °C, elongation time 45 s
Name Volume
Primer fw (10 µM) 1.25 µL
Primer rev (10 µM) 1.25 µL
template DNA 1 µL
Q5 2x Master Mix 12.5 µL
Nuclease free H2O 9 µL
  • many unspecific bands, especially K1
  • new agarose gel with 10 µL PCR product and gel extraction

Aim / Task Digestion of pSec Hygro Tag
Protocol Digest protocol of NEB
Participants Felix Pfister, Leon Altmann
  • digested pSec Hygro Tag with NheI
  • 15 min at 37 °C, then 20 min at 80 °C

Aim / Task Heatshock of Gibson CHO-S (K1, K2a, K2b)
Protocol NEB Heatshock
Participants Felix Pfister, Jannik Wagner
  • made 15 LB-Agar plates with Ampicilin
  • heatshock of the constructs of CHO constructs K1, K2a, K2b in BL21, BL21 Star, Tuner and DH5alpha, plus a positive and negative control in BL21 Star (=14 different transformations)
    • used 2 µL of the Gibson assembly
    • used SOC medium for the bacteria after the heatshock
  • plated bacteria afterwards on the LB-Agar plates
  • incubation at 37 °C

Aim / Task Production of single colonies (Star; K1, K2a,K2b of 11.08); Preparation of new amp-plates
Protocol
Participants Felix Pfister, Lena Schorr

Aim / Task Miniprep of ON Culture & digestion; PCR clean-up
Protocol
Participants Eva Neugebauer, Jannik Wagner

  • Miniprep of the samples:

    • K1-1 Tuner, K1-2 Tuner, K1-3 Tuner
    • K2a-1 Tuner, K2a-2 Tuner, K2a-3 Tuner
    • K2b-1 Tuner, K2b-2 Tuner, K2b-3 Tuner
    • K1 Star
    • negative control

  • digestion of isolated DNA from Miniprep:

Name Volume
EcoRV 1 µL
NheI 1 µL
CutSmart Buffer 5 µL
DNA 1 µL
dH2O ad 50 µL

  • incubation for 15 min at 37 °C

  • PCR clean up of the samples:

    • K2a, K2b, K3a from 2nd amplification from 15.7 !! no clear description on tubes of 2nd amplification and no picture of gel ==possible error source !!
    • K3b, K4a, K4b from 1st amplification
    • end volume 50 µL

Aim / Task PCR - Amplification, Overhang / Puc19 digest with Sma1 / Digested vector gel extraction / Mini-Prep CHO-S Parts
Protocol
Participants Eva Neugebauer, Daniel Schreyer

PCR Amplification and overhang:

  • used every construct besides K1 from clean up

pUC19 digest:

  • digest for 1 h with Sma1 at 25 °C in CutSmart Buffer
  • 5 µL pUC19 (Bl21 K3 from gel 07-23)
  • 2 µL restriction enzyme
  • pUC19 gel extraction: 0.8% agarose gel electrophoresis with undigested pUC19 as control
  • no pUC19 was digested! BL21 K3 is empty!

Miniprep from the ON cultures 2019-08-13:

  • extract-me miniprep kit used
  • nanodrop measurements are on the respective tubes
  • control digestion with Pst1
    • used 500 ng of each miniprep
    • used 1 µL Pst1-HF and 5 µL CutSmart Buffer
    • stopped reaction with 10 µL 6x loading dye and used 20 µL for gel electrophoresis

Aim / Task Control Digestion of pSec/Tag2/Hygro
Protocol -
Participants Jannik Pfister, Felix Wagner
  • Did single enzyme digestion of pSec
  • EcoRI –2 sites
  • EcoRV –1 site
  • NheI –1 site *See picture attachment for estimation and results

|DNA |pSec + EcorI |Laddder|pSec + EcorV|pSec + NheI| |—|—|—| |

<Hygro digestion with EcoRV,EcoRI,NheI>

Aim / Task Gel extraction of Gibson Overhang PCR (K2a -K4b) (from 14.08)
Protocol
Participants Felix Pfister, Lena Schorr
  • no bands in K2b and K3a
  • many different bands in K2a
  • K3b - K4b look good –gel extraction only of K2a, K3b, K4a, K4b

Aim / Task pSec+CHO (K1, K2a K2b) / Gel extraction
Protocol
Participants Felix Pfister, Lena Schorr
  • used 2x 20 µL of the miniprep of the vector+construct
  • did not start the gel extraction
  • ran agarose gel
  • gel extraction only of K1E1-1, K1E1-2, K2aE1-3
  • gel weight for the extraction:
    • K1E1-1: 0.17 g
    • K1E1-2: 0.16 g
    • K2aE1-3: 0.12 g

Aim / Task pUC19 / MiniPrep / Nanodrop / Gel-Extraction / Dephosphorylation Test / Heat-Shock
Protocol
Participants Felix Pfister
  • Miniprep: used 30 µL MiliQ for eluation
  • Nanodrop:
    • colony 1: 286 ng/µL
    • colony 2: 283 ng/µL
    • colony 3: 306 ng/µL
  • gel weight for the extraction:
    • pUC19 1: 0.31 g
    • pUC19 2: 0.23 g
    • pUC19 3: 0.25 g

Aim / Task Produce new Amp+ Agar Plates/ Measure Miniprep of pUC19/ Digest,De-phosphorylate pUC19 with SmaI/ Seed new single colonies from Gibson CHO-S K1, K2a, K2b
Protocol
Participants Marie Wiedemann, Leon Altmann

Protocol

Notes

Prepare new Amp plates:

Prepare 500ml LB-Broth with Agar (17,5 g Agar) for 19 new colony plates;

Autoclave the Mixture to make it sterile;

After a cooling phase Amp got added (500 µl –concentration: 50 µg/ml);

Pour the plates under sterile conditions and put them in the refrigerator @4 °C;

For the digestion of pUC19 with SmaI the miniprep samples were measured in the nanodrop;

Sample Name 260 280 260⁄280 ng/µl
pUC19 c1 1,143 0,606 1,89 57,15
pUC19 c2 1,426 0,873 1,63 71,30
pUC19 c3 1,073 0,533 2,01 53,65

As the measurement diverged greatly from the measurement the day before that was done right after the miniprep, but with out further purification two approaches were prepared. One with the old nanodrop results from the day before and one with the new results. It is thought that the approach from the day before won’t have the right amount of Vector inside but it is prepared either way to be on the safe side.

Approach one:

Name Amount [µl]
DNA 3,3
CutSmart x10 NEB 5
Arctic Buffer x10 NEB 5
Arctic Phosphatase NEB 1
SmaI NEB 0,5
H2O to 50µl 35,2

Approach two:

Name Amount [µl]
DNA 14
CutSmart x10 NEB 5
Arctic Buffer x10 NEB 5
Arctic Phosphatase NEB 1
SmaI NEB 0,5
H2O to 50µl 24,5

Incubate at 37°C for 1 h;

Inactivate at 80 °C for 20 min (80 °C necessary to inactivate the phosphatase, SmaI itself 65 °C for 20 min would be sufficient);

Preparation of 0,8 % Agarose Gel:

Weigh in 0,8g Agarose and solve in 100ml TAE-buffer –solve Agarose by microwaving;

After cooling adding of SybrGreen 7 µl;

Preparation of Single Colonies from CHO-S K1, K2a, K2b

For each of the three constructs three colonies were picked and carefully plated under sterile conditions onto the Agar (Amp+) Plates that were earlier prepared;

Problems faced / possible error causes

The Gel did not show a clear enough band to extract the digested vector. This is due incubation temperature (37 °C);
SmaI does only require 25 °C to cut properly and shows only 50 % performance at 37 °C;

Pending tasks, next steps, comments for next people in the Lab

  • Measurement part!: Repeat digestion (SmaI) and dephosphorylation of puc19 at 25 °C (Stored at 4 °C);
  • Control: Gel electrophoresis after digest;
  • Perform Gibson Assembly puc19 + CFP;
  • Check Plates seeded today (Incubator at 37 °C);
  • New Gibson Assembly with K1, K2a, K2b (important)

<pUC19>

Aim / Task Miniprep of Gibson CHO Constructs (ON Cultures 18.08) / Preperation for MidiPrep of pUC19 /Control digestion of Gibson CHO Constructs
Protocol -
Participants Felix Pfister

Protocol

Notes

  • Did a MidiPrep of ON Cultures from 18.08
    • Concentration:

Agarose-Gel electrophoresis for validation

Name K1-1 K1-2 K1-3 K2a-1 K2a-2 K2a-3 K2b-1 K2b-2 K2b-3
Conc [ng/µl] 33,9 31,9 27,4 38,7 31,7 27,4 28,5 44,4 26,1
Component Amount
DNA 3 µl
6X Loading Dye 2 µl
dH2O 1 µl

-Same Loading order as NanoDrop concentration measurment

  • Did an ON Culture of a single conlony of pUC19 (18.08)

    • Picked one colony and incubated it in 5 ml LB+Amp in the shaker @37°C
    • In the late afternoon –1:500 in 85ml LB+Amp –shake for 12-16h @37°C

  • Control digestion of CHO Constructs

    • Restriction enzymes: ApaI and HindIII-HF
Component Amount
DNA 300 ng
ApaI 1 µl
HindIII-HF 1 µl
CutSmart Buffer 5
dH2O ad 50 µl
  • - Protocol - Incubate ApaI at 25°C for 5-15 minutes - Add 1.0 µl (or 10 units) of HindIII-HF and Incubate at 37°C for 5-15 minutes - Inactivate both at 80°C for 20 minutes

Problems faced / possible error causes

  • Low concentration of construc –more LB+amp Medium next time with longer incubation time in the shaker?

Pending tasks, next steps, comments for next people in the Lab

  • MidiPrep of ON Culture of pUC19
  • Empty constructs
  • repeat gibson

<Beschreibung> <Beschreibung> <Beschreibung>

Aim / Task New Attempts for pUC19 Digest/ Heatshock pSEC and ON culture/ ON cultures from K1,K2a,K2b
Protocol -
Participants Marie Wiedemann, Leon Altmann

Protocol

Notes

Check the Plates from 17.8.19 CHO-S Constructs K1, K2a, K2b:

On all of the plates the seeding worked fine and clones can be picked later on for ON cultures;

New Attempt to digest pUC19:

Name Attempt 1 [µl] Attempt 2 [µl]
DNA 17,5 (pUC19 c1) 18,6 (pUC19 c3)
CutSmart x10 5 5
AntarcticBuffer x10 5 5
Antarctic Phosphatase 1 1
SmaI 0,5 1
H2O 21 19,4

The first attempt was @25 °C for 1 hour;

Inactivation @ 80 °C for 20 min;

Second attempt incubation @37 °C for 1 hour;

Inactivation @ 80 °C for 20 min;

Both of the samples were to find better conditions as the digest from 17.8.19 were not optimal;

25 °C is the optimal temperature for SmaI, whereas 37 °C is the optimum for the phosphatase;

With 1 hour incubation time also the 25 °C should be suitable for the phosphatase and likewise the 37 °C are acceptable for SmaI, given that the efficacy of the digest is lowered by approx 50% (information on the NEB site);

None of the Digests showed optimal results;

Both were looked at in an 0,8% agarose gel, each for their own.

The order looked like the following:

|1kb DNA Ladder (5µl)|Half Sample (30µl):5 µl Staining|25 µl Sample|Half Sample (30 µl):5 µl Staining25 µl Sample|

Attempt 1 of todays pUC19 digest did show a similar result to yesterdays digestion

Expected was one band of 2,7 kb length (see picture from virtual digest 17.8.19);

The suspected bands were cut out and extracted from the gel (QiaQuick Gel Extraction Kit);

Sample 1: 0,36 g; Sample2: 0,3 g;

–1060 µl QG; –900 µl QG

Protocoll of the Kit was used, the DNA was solved in 50 µl H2O;

The resulting DNA was measured in the Nanodrop;

Sample Name 260 280 260⁄280 ng/µl
Attempt 1 first half 0,026 0,030 0,87 1,29
Attempt 1 second half 0,033 0,041 0,81 1,66

With these low DNA concentrations Gibson Assembly is not feasible without increasing the reaction volume substantially;
Therefor one should ask one of the TAs for advise to increase the DNA concentrationin a reliable way;

ON culture of Single Colonies from CHO-S K1, K2a, K2b

For each of the three constructs three colonies were picked and carefully seeded in 5 ml LB-Medium (Amp+) under sterile conditions;

Placed in the incubator @ 37 °C 130 rpm (time approx. 18:30);

Heatshock pSec:

Stored pSec has a concentration of 2 µg/µl –a 1:10 dilution was performed to pipette 100 ng of vector DNA for the heatshock procedure;

The 100 ng of pSec vector DNA were used for 200 µl DH5a;

Evas’ protocoll was used;

A plate of LB-Agar (Amp+) was seeded and put in the incubator @ 37 °C (time approx. 18:30);

Problems faced / possible error causes

Gel bands did indicate a non optimal digest –Not suspected bands and maybe also religation??;

The other digestion Attempt worked as bad as the first or the one from 17.8.19;

Not sure which of the bands is the actual cut vector as one bamd is visible at approx. 3kb, one band looks more like 2 kb and maybe there is a very thin band at 2,7 kb which would be the expected position;

Ask TA with the gel photo for advise and maybe also advise for a proper digestion –Might be that it was not to few but rather to much enzyme??

Also DNA purification from the Gel yielded only an extremly low amount of DNA –Gibson not performable;

Pending tasks, next steps, comments for next people in the Lab

Take the overnight cultures of Gibson CHO K1, K2a, K2b out of the incubator

Take the plate from pSec out of the incubator

Mini-Prep of pSec & freeze

Speak to the TA or PhD: Discuss the gel bands of the digests & how to increase the concentration of pUC19 digest 2 to continue with gibson assembly

Gibson assembly of pUC19 and measurement part (has conc of 1000 ng in 20 µl according to TAs advice

Heatshock of pUC19 and measurement part in competent E.coli

Generate more pUC19 vector

<pUC19 Digest> <pUC19 Digest>

Aim / Task Midi-prep of overnight-culture from DH5a with puc19; Digestion of puc19 with SmaI at 37°C
Protocol Midi prep protocol
Participants Lena Schorr

Protocol

Notes

  • performance of a midi prep of the overnight culture of DH5a with puC19

    • 2 x 25ml from overnight culture
    • See procotol below
    • 467,5 ng/µl (total: 400µl)
    • Storage in -20°C in 15ml falcon

  • Digestion of the puC19 vector with SmaI under new conditions

    • all materials in the fridge of TW (-20°C)
    • digestion at 37°C for 1 h
    • Inactivation at 80°C for 20 minutes
    • Storage at -20°C (annotated with attempt 1; attempt 2)
Name Attempt 1 [µl] Attempt 2 [µl]
DNA 10µl (=6µg) 20µl (=12µg)
CutSmart x10 5 5
AntarcticBuffer x10 5 5
Antarctic Phosphatase 1 1
SmaI 0,5 0,5
H2O 28,5 18,5

Protocol Midi:

  1. Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 min at 4°C. (at the fuge in the Behrensen Lab!!!)

  2. Resuspend the bacterial pellet in 4 ml Buffer P1.

  3. Add 4 ml Buffer P2, mix thoroughly by vigorously inverting 4–6 times and incubate at room temperature (15–25°C) for 5 min. If using LyseBlue reagent, the solution will turn blue.

  4. Add 4 ml Buffer P3, mix thoroughly by vigorously inverting 4–6 times. Incubate on ice for 15 min. If using LyseBlue reagent, mix the solution until it is colorless.

  5. Centrifuge at ≥20,000 x g for 30 min at 4°C. Re-centrifuge the supernatant at ≥20,000 x g for 15 min at 4°C.

  6. Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow column to empty by gravity flow.

  7. Apply the supernatant from step 5 to the QIAGEN-tip and allow it to enter the resin by gravity flow.

  8. Wash the QIAGEN-tip with 2 x 10 ml Buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow.

  9. Elute DNA with 5 ml Buffer QF into a clean 15 ml vessel. For constructs larger than 45 kb, prewarming the elution buffer to 65°C may help to increase the yield.

  10. Precipitate DNA by adding 3.5 ml roomtemperature isopropanol to the eluted DNA and mix. Centrifuge at ≥15,000 x g for 30 min at 4°C. Carefully decant the supernatant.

  11. Wash the DNA pellet with 2 ml room-temperature 70% ethanol and centrifuge at ≥15,000 x g for 10 min. Carefully decant supernatant.

  12. Air-dry pellet for 5–10 min and redissolve DNA in a suitable volume of appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).

Pending tasks, next steps, comments for next people in the Lab

  • 0,8% agarose gel of the digested puc vector (attempt 1 and 2)
  • Performing gibson assembly with construct 1, and 2a+b in psec for CHOs

Aim / Task Digestion of pSEC with EcoR5 and NHeI for Gibson Assembly, agarose gel (0,8%) for control of digestion of pSec and pUC19, Extraction of digested pSEC vector from gel
Protocol Extraction protocol from Qiagen
Participants Lena Schorr

Protocol

Notes

  • Digestion of pSEC with EcoR5 and NHE1
Name Attempt 1 [µl] Attempt 2 [µl] Attempt 3 [µl]
DNA 1 µg (=0,5µl) 2 µg (=1µl) 1 µg (=0,5µl)
NEB buffer 3.1 (x10) 5 5 -
Cut-Smart (10x) - - 3
EcoR5 1µl 1µl 1µl
NHE1 1µl 1µl 1µl
H2O 42,5µl 42µl 42µl
  • attempt 1 and 2:
    • 15 minutes on 37°C and stop reaction by adding 10 µl 6 x loading dye
  • attempt 3:
    • 60 minutes on 37°C and inactivation at 80°C

Preparation of a 0,8% agarose gel

  • scheme see below
  • 25 µl of the samples and 5µl ladder were used
  • gel run for 45 minutes at 120 V

Extraction of line 7 (digested pSEC vector with EcoRV and NheI)

  • Extraction kit was used
  • sample stored at -20°C

Pending tasks, next steps, comments for next people in the Lab

  • new digestion of puC19 and maybe pSEC

  • Gibson Assembly of CHO constructs in pSEC and iGEM Part in Puc18

  • Heat Shock

<Beschreibung> <Beschreibung>

Aim / Task Digestion of pSEC with EcoR5 and NHeI for Gibson Assembly, agarose gel (0,8%) for control of digestion of pSec and pUC19, Extraction of digested pSEC vector from gel
Protocol Extraction protocol from Qiagen
Participants Leon Altmann

Protocol

Notes

Digestion of pSEC with EcoR5 and NHE1

Name Amount [µl]
DNA 1 µl (=2 µg)
Cut-Smart (10x) 5
EcoR5 0,5
NHE1 0,5
H2O 43
  • 60 minutes @ 37°C
  • 20 minutes inactivation @ 80°C

Digestion of pUC19 with SmaI + Antarctic Phosphatase

Name Amount [µl]
DNA 10,6 µl (of Midiprep)
Cut-Smart (10x) 5
Antarctic Buffer x10 5
Antarctic Phosphatase 1
EcoR5 0,5
H2O 27,9
  • 60 minutes @ 37°C
  • 20 minutes inactivation @ 80°C

Preparation of a 0,8% agarose gel

  • scheme see below
  • 25 µl of the samples + 5 µl of 6x loading dye
  • 5µl ladder

  • 1h @130V

  • Extraction of line 5, 6 (digested pSEC vector with EcoRV and NheI)

  • QiaGen Gelextraction kit was used

  • sample stored at -20°C

Pending tasks, next steps, comments for next people in the Lab

  • new digestion of pUC19 better would be to ask wether they have pUC19 stored somewhere as ours is out of bounds

  • Gibson Assembly of CHO constructs in pSEC and potentially pUC19

  • Heat Shock

Aim / Task Gibson Assembly of CHO K1, K2a, K2b and measurement part in pSec; Heatshock in DH5a bacs; Gel electrophoresis of Gibson Assembly Samples
Protocol Gibson Assembly; Heatshock; Gel electrophoresis
Participants Marie Wiedemann

Protocol

Notes

Gibson Assembly

According to 2.8.19: CHO constructs and measurement part were taken up in 20 µl TE buffer. –concentration: 50ng/µl

concentration pSec: 26,8 ng/µl

using 50 ng pSec 1,86 µl

using insert three-fold: 150 ng ~3 µl ; used inserts: CHO K1, CHO K2a, CHO K2b, measurement part

also performed a positive control with the provided kit.

Amount in µl Name
1,86 Vector pSec
3 Insert
10 Gibson Assembly Master Mix
5,14 dH2O

Incubated at 50 °C in thermocycler for 30mins.

Heatshock

performed a heatshock with the Gibson Assembly with DH5a bacteria, following Evas protocoll. Used 18 µl of the assembly samples.

Plated the bacteria on Amp plates and put in incubator to be continued with tomorrow.

Gel electrophoresis

prepared a 0.8% agarose gel with 0.8 g agarose in 100 ml TE buffer. microwaved and added SybrGreen after short cooling period.

Mixed 2 µl of DNA sample with 2 µl of DNA Purple Dye and 16 µl of water.

Added 7 µl DNA Ladder

Order: wayward DNA Ladder - empty - DNA Ladder - CHO K1 - CHO K2a - CHO K2b - Measurement Part - Positive control

Problems faced / possible error causes

Nothing just DNA ladder visible on gel. Might have used not enough DNA sample?

Pending tasks, next steps, comments for next people in the Lab

  • take plates out of incubator, pick colonies
  • find a new pUC19 vector to try

Aim / Task Isolation of DNA (CHO constructs) from overnight culture
Protocol Extract Me - for mini prep
Participants Lena Schorr

Protocol

Notes

  • BACS stock of
    • CHO K1, clone 2
    • CHO K1, clone 1
    • CHO K2a, clone 4
    • DH5a positive control
      • storage at -80°C (in box of electrocompetent tuner and DH5a)
  • Mini-prep of overnight culture
    • eluation with 50µl MiliQ water

Pending tasks, next steps, comments for next people in the Lab

  • Measerment of the DNA concentrations
  • gel of Mini-Prep (did Gibson work?)
  • Digestion of puC19 with ecoRV
  • Gibson Assembly
  • sent CHOs to Kiel

Aim / Task Mini Prep from ON cultures (K1,K2a, K2b), Gel electrophoresis, PCR
Protocol Extract Me - Plasmid Preparation Kit
Participants Leon Altmann

Protocol

Notes

  • Mini-prep of overnight cultures (K1, K2a, K2b) of each 5 clones
    • eluation with 50µl MiliQ water
  • Ran a 0,8% Gel
  • Measured Plasmid DNA at Nanodrop
Sample Name 260 280 260⁄280 ng/µl
K1 c1 0,525 0,293 1,79 26,25
K1 c2 1,408 0,748 1,88 70,42
K1 c3 1,088 0,619 1,76 54,39
K1 c4 1,275 0,694 1,84 63,77
K1 c5 1,404 0,760 1,85 70,21
K2a c1 1,809 0,983 1,84 90,45
K2a c2 1,503 0,830 1,81 75,14
K2a c3 1,214 0,665 1,83 60,72
K2a c4 1,241 0,649 1,91 62,07
K2a c5 1,227 0,658 1,86 61,37
K2b c1 0,409 0,239 1,71 20,45
K2b c2 1,024 0,562 1,82 51,18
K2b c3
K2b c4 1,222 0,649 1,88 61,12
K2b c5 0,960 0,476 2,02 47,99

Performed a PCR with all of the Minis to confirm bands that are potentially depicting vector + insert

Name K1 c1 K1 c2 K1 c3 K1 c4 K1 c5 K2a c1 K2a c2 K2a c3 K2a c4 K2a c5 K2b c1 K2b c2 K2b c3 K2b c4 K2b c5
CHO-S fw 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25
CHO-S Primer re 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25
DNA 3,8 1,4 1,8 1,6 1,4 1,1 1,3 1,6 1,6 1,6 4,9 2 1,6 2,1
Water 6,2 8,6 8,2 8,4 8,6 8,9 8,7 8,4 8,4 8,4 5,1 8 8,4 7,9
Q5 Hifi MM 2x 12,5 12,5 12,5 12,5 12,5 12,5 12,5 12,5 12,5 12,5 12,5 12,5 12,5 12,5 12,5
Total 50 50 50 50 50 50 50 50 50 50 50 50 50 50 50

Primer for this PCR are stored at -20°C the original primer tubes have a concentration of 100µM. Besides them there are also Eppis with the ready to pcr concentration of 10µM;

Problems faced / possible error causes

Not a real problem but teh bands of corerct lenght were quite weak in comparison to what is probably just oure vector at 5,7kb;
To tackle this issue PCR was performed. If the PCR is successfull, then Samples with vector + Insert will show a band of the corresponding inserts length in a gel;

Materials used

Q5 Hifi Polymerase empty!

Pending tasks, next steps, comments for next people in the Lab

  • Perform Gel with PCR Products;
  • Send positive ones to Kiel (call Matthias Peipp beforehand)
  • Furhter try to make the Measurement Part work –Puc digest or also possible to open any other vector blunt and clone the part behind a promotor
  • If successfull heatshock
  • Plate out

Aim / Task Gel of PCR, Digest of pUC19 (EcoRI, HindIII), ON cultures, Gel electrophoresis of unclear samples
Protocol -
Participants Felix Pfister, Leon Altmann

Protocol

Notes

The PCR samples from 28.8 were applied to two 0,8% Agarose Gel and ran at 130V for 1 hour.

Gel 1:

Ladder K1 c1 K1 c2 K1 c3 K1c4 K1 c5 K2a c1 K2a c2 K2a c3 K2a c4 K2a c5

Gel 2:

Ladder K2b c1 K2b c2 K2b c3 K2b c4 K2b c5 Control

The PCR was performed to see whether the desired Insert was implemented into the vector, as the Gel from 28.8 allowed no precise determination.

The PCR Water-control showed a clear band at approximately 1,2 kb. This band was also seen in every other samples, hinting towards contamination of the water or PCR mix.

Other than that, the PCR samples did not show bands that would allow the assumption of successful Gibson assembly. Probably all the vectors are empty.

Another Digestion of pUC19 was performed to exclude potential problems with the blunt cutting enzymes due to re-ligation.

EcoRI and HindIII were used and were expected to yield a 2,6 kb and a 0,1 kb fragment.
Of EcoRI and HindIII a 1:10 Dilution was performed for easier pipetting.

Digestion Mix:

Name Amount [µl]
DNA (pUC Midi 467,5 ng/µl) 4,27
CutSmart x10 5
EcoRI 1
HindIII 2
Water 37,73
Total Amount 50

Digestion @ 37°C for 1h;

Inactivation @ 80°C for 20min;

Samples applied to a 0,8% Agarose Gel: Ladder pUC19

The digest gave the same result as every other digest with blunt end enzymes, indicating that the pUC19 sample we use is in fact contaminated, or no pUC19 at all!

To clarify which of the stored samples in the freezer @ -20°C and in the refrigerator @ 4°C are still of use Gel electrophoresis was performed.

Gel 1 part 1
DNA Ladder Gelex K2a 16.8 Gelex K3b 16.8 Gelex K4a 16.8 Gelex K4b 16.8 Gelex K2b 09.7 Gelex K3b 09.7 Gelex K4b 09.7 Gelex K4b 09.7 PC Vector 4b/a 3ab pACYC184 p3 Psec digested Gelex 21.08 Attempt 3 pSec
Gel 1 part 2
DNA Ladder K2ab 2 30.07 K2a 1 30.07 K2a 2 30.07 K2b 2 30.07 K2b 1 30.07 Gelex K1 CHO-S 8.8 Gelex K2a CHO-S 8.8 Gelex K2b CHO-S 8.8 Purified CFP 9.8 / 3ab 1 3ab 2 3ab 3
Gel 2
DNA Ladder 25.8 K1 c1 25.8 K1 c2 25.8 K1 c3 25.8 K1 c4 25.8 K2a c1 25.8 K2a c3 25.8 K2a c4 25.8 K2a c6 DNA CHO K2b c1 25.8 DNA CHO K2b c2 25.8 DNA CHO K2b c3 25.8 DNA CHO K2b c4 25.8
Gel 3
Ladder 2a 2a 1 2a 2 2b 2b 1 4ab 3ab

Pending tasks, next steps, comments for next people in the Lab

  • Decision which of the tested samples remains usefull and should therefore be kept in storage;
  • Find alternative to pUC19 for the measurement part
    • One can not perform blunt end cloning with the ordered gBlocks
    • However it should be possible with the PCr products of the gBlocks as the have 5’ phosphate groups

<Beschreibung> <Beschreibung> <Beschreibung> <Beschreibung>

Aim / Task MiniPrep of Gibson Assembly Colonies from 23.08 / MiniPrep of sequenced CHO K1 E1-2 and CHO K2a E1-3 (16.08)
Protocol -
Participants Felix Pfister, Jannik Wagner

Protocol

Notes

  • MiniPrep of Gibson Assembly Colonies K1, K2a, K2b (5 each)
    • Eluated in 50 µl dH2O
  • MiniPrep of CHO K1 E1-2 and K2a E1-3
    • Lost K1 CHO K1 E1-2
    • Eluated in 50 µl dH2O

  • Control per Electrophoresis

    • Loading
      • DNA Ladder/K1-1/K1-2/K1-3/K1-4/K1-5/empty/K2a-1/K2a-2/K2a-3/K2a-4/K2a-5/
      • DNA Ladder/K2b-1/K2b-2/K2b-3/K2b-4/K2b-5/empty/CHO K2a E1-3

  • Control digestion of both MP

<Beschreibung> <Beschreibung>

Aim / Task MiniPrep of Gibson Assembly Colonies from 23.08 / MiniPrep of sequenced CHO K1 E1-2 and CHO K2a E1-3 (16.08)
Protocol -
Participants Felix Pfister, Jannik Wagner

Protocol

Notes

  • MiniPrep of Gibson Assembly Colonies K1, K2a, K2b (5 each)
    • Eluated in 50 µl dH2O
  • MiniPrep of CHO K1 E1-2 and K2a E1-3
    • Lost K1 CHO K1 E1-2
    • Eluated in 50 µl dH2O

  • Control per Electrophoresis

    • Loading
      • DNA Ladder/K1-1/K1-2/K1-3/K1-4/K1-5/empty/K2a-1/K2a-2/K2a-3/K2a-4/K2a-5/
      • DNA Ladder/K2b-1/K2b-2/K2b-3/K2b-4/K2b-5/empty/CHO K2a E1-3

  • Control digestion of both MP

<Beschreibung> <Beschreibung>

Aim / Task PCR of measurment part, gel-electrophoresis of measurment part, gel extraction of measurment part
Protocol Q5 high fidelity PCR Kit, qiagen gel extraction kit
Participants Lena Schorr

Protocol

Notes

  • PCR of the iGEM measurment part CFP
    • 50µl reaction
1x 2,5x
Q5 high fidelity master mix 25µl 62,5µl
10µM forward primer 1,25 µl 3,125µl
10µM reverse primer 1,25µl 3,125µl
template DNA (CFP) 0,5µl (=25ng) -
nuclease free water 22µl 55µl
  • with melting temperature of 56°C
  • program from the protocol
  • PCR products are stored at -20°C
    • M = measurement part
    • C = control with water
  • gel electrophoresis
    • 1% gel
    • run for 1h, 120V

Scheme:

ladder (7µl) - measurment part (25µl –>10 µl PCR product + 10µl MilliQ + 5µl loading Dye) - water control (“”)

–>see below

  • gel extraction via qiagen gel extraction kit
    • band of CFP at 723 bp (see picture below)
    • elution with 50µl MiliQ water
    • storage at -20°C

Protocol

Q5 high fidelity PCR Kit, qiagen gel extraction kit

Pending tasks, next steps, comments for next people in the Lab

Measurement of DNA concentration of extracted PCR product (CFP –stored at -20°C)

<Beschreibung>

September

Aim / Task CHO parts of 30.07. sequencing + ampicilin aliquots
Protocol Lsg Sequenzing Anleitung
Participants Lena Schorr, Leon Altmann

Notes

Total volume should be 15 µL with a concentration of 100 µg/µL

Accodring to the nanodrop results of 30.08. following DNA volumes were used:

K 1-1: 5,9 µL (C159961)

K 1-2: 6,2 µL (C159960)

K 1-3: 2,96 µL (C159959)

K 1-4: 2,8 µL (C159958)

K 1-5: 4,2 µL (C159957)

K 2a-1: 2,44 µL (C159956)

K 2a-2: 5,3 µL (C159955)

K 2a-3: 3,2 µL (C159954)

K 2a-4: 2,9 µL (C159953)

K 2a-5: 2,66 µL (C159952)

K 2b-1: 6,82 µL (C159951)

K 2b-2: 3,5 µL (C159950)

K 2b-3: 4,5 µL (C159949)

K 2b-4: 4,52 µL (C159948)

K 2b-5: 4,1 µL (C159947)

ad 15 µL with milliQ water

Amp aliqouts:

50 mg/ mL

Aim/Task: Ligation of pUC19 with Measurement Part, Heatshock into DH5a, PCR of Measurement Part
Protocoll used: Heat Shock Protocol, NEB Ligation Protocol (M0202), Q5 Hifi PCR Protocol
Date, Site: 03.09.19 , nfz
Name: Felix, Eva

Notes

Ligation of Measurement Part with cut pUC19 from earlier of the day;

Relative amounts of vector 1 : Insert 4;

10 ng of Vector and approx. 10 ng of Insert as the Insert is only 700 bp long;

Ligation with T4 DNA Ligase Protocol (M020) from NEB;

Reaction set up on ice;

Name Amount [µl]
T4 DNA Ligase Buffer x10 3
Vector DNA 20
Insert DNA 0,5
Water 5,5,
T4 Ligase 1
Total amount 30 µl

Incubate @RT for 10 min;

Heat inactivate @65 for 10 min;

With the sample Heat shock was performed after Evas Protocol;

The transformed cells were seeded on an Amp+ plate and placed into the incubator @ 37 °C;

For the case of a failed ligation a PCR with the measurement part was set up;

|| | — || — | | Name | Amount [µl] | | ——————————- | ————— | | DNA | 0,5 | | Q5 Hifi Polymerase MasterMix x2 | 12,5 | | Measurement Primer fw | 1,25 | | Measurement Primer re | 1,25 | | Nuclease free water | 9,5 | | Total amount | 30 µl |

Problems faced / possible error causes

Q5 Hifi Polymerase is stored in the topmost drawer in the -20 °C fridge in the winkler lab and no longer in the Plastik Box in drawer 2;

Pending tasks, next steps, comments for next people in the Lab

- Check Plate in the Incubator and pick colonies if transformation was successful;

- IF not new digestion with the ordered pUC19 (XbaI/EcoRI)

Aim / Task Digestion of pUC19 and CFP with Xbal and EcoRI
Protocol Heat Shock Protocol, NEB Ligation Protocol (M0202), Q5 Hifi PCR Protocol
Participants Eva Neugebauer, Felix Pfister

Notes

Nanodrop measurement of gelextraction from 31.07. and and 01.08.

CFP PCR gel extr. 31.07.: 13,6

CFP PCR gel extr. 01.08. 1: 21,82

CFP PCR gel extr. 01.08. 2: 16,96

Digest of gelextr. (31.08.) and pUC19 with EcoRI and XbaI:

43µL DNA + 5 µL cutsmart + 1 µL of each enzyme (HF)

15 min 37°C

20 min 65°C heat inactivation

Pending tasks, next steps, comments for next people in the Lab

Ligation of puc19 and CFP

New PCR of CFP

Aim / Task Ligation of pUC19 with Measurement Part, Heatshock into DH5a, PCR of Measurement Part
Protocol Heat Shock Protocol, NEB Ligation Protocol (M0202), Q5 Hifi PCR Protocol
Participants Eva Neugebauer, Felix Pfister

Notes

Ligation of Measurement Part with cut pUC19 from earlier of the day;

Relative amounts of vector 1 : Insert 4;

10 ng of Vector and approx. 10 ng of Insert as the Insert is only 700 bp long;

Ligation with T4 DNA Ligase Protocol (M020) from NEB;

Reaction set up on ice;

Name Amount [µl]
T4 DNA Ligase Buffer x10 3
Vector DNA 20
Insert DNA 0,5
Water 5,5,
T4 Ligase 1
Total amount 30 µl

Incubate @RT for 10 min;

Heat inactivate @65 for 10 min;

With the sample Heat shock was performed after Evas Protocol;

The transformed cells were seeded on an Amp+ plate and placed into the incubator @ 37 °C;

For the case of a failed ligation a PCR with the measurement part was set up;

Name Amount [µl]
DNA 0,5
Q5 Hifi Polymerase MasterMix x2 12,5
Measurement Primer fw 1,25
Measurement Primer re 1,25
Nuclease free water 9,5
Total amount 30 µl

Problems faced / possible error causes

Q5 Hifi Polymerase is stored in the topmost drawer in the -20 °C fridge in the winkler lab and no longer in the Plastik Box in drawer 2;

Pending tasks, next steps, comments for next people in the Lab

  • Check Plate in the Incubator and pick colonies if transformation was successful;

  • IF not new digestion with the ordered pUC19 (XbaI/EcoRI)

Aim / Task Digestion of pUC19 and CFP with Xbal and EcoRI
Protocol Lsg Sequenzing Anleitung
Participants Eva Neugebauer, Felix Pfister

Notes

Nanodrop measurement of gelextraction from 31.07. and and 01.08.

CFP PCR gel extr. 31.07.: 13,6

CFP PCR gel extr. 01.08. 1: 21,82

CFP PCR gel extr. 01.08. 2: 16,96

Digest of gelextr. (31.08.) and pUC19 with EcoRI and XbaI:

43µL DNA + 5 µL cutsmart + 1 µL of each enzyme (HF)

15 min 37°C

20 min 65°C heat inactivation

Pending tasks, next steps, comments for next people in the Lab

Ligation of puc19 and CFP

New PCR of CFP

Aim / Task CHO parts of 30.07. sequencing + ampicilin aliquots
Protocol Lsg Sequenzing Anleitung
Participants Eva neugebauer, Leon Altmann

Notes

Total volume should be 15 µL with a concentration of 100 µg/µL

Accodring to the nanodrop results of 30.08. following DNA volumes were used:

K 1-1: 5,9 µL (C159961)

K 1-2: 6,2 µL (C159960)

K 1-3: 2,96 µL (C159959)

K 1-4: 2,8 µL (C159958)

K 1-5: 4,2 µL (C159957)

K 2a-1: 2,44 µL (C159956)

K 2a-2: 5,3 µL (C159955)

K 2a-3: 3,2 µL (C159954)

K 2a-4: 2,9 µL (C159953)

K 2a-5: 2,66 µL (C159952)

K 2b-1: 6,82 µL (C159951)

K 2b-2: 3,5 µL (C159950)

K 2b-3: 4,5 µL (C159949)

K 2b-4: 4,52 µL (C159948)

K 2b-5: 4,1 µL (C159947)

ad 15 µL with milliQ water

Amp aliqouts:

50 mg/ mL

Aim / Task Digestion of pUC19 and CFP with Xbal and EcoRI
Protocol -
Participants Felix Pfister, Eva Neugebauer

Notes

Nanodrop measurement of gelextraction from 31.07. and and 01.08.

CFP PCR gel extr. 31.07.: 13,6

CFP PCR gel extr. 01.08. 1: 21,82

CFP PCR gel extr. 01.08. 2: 16,96

Digest of gelextr. (31.08.) and pUC19 with EcoRI and XbaI:

43µL DNA + 5 µL cutsmart + 1 µL of each enzyme (HF)

15 min 37°C

20 min 65°C heat inactivation

Pending tasks, next steps, comments for next people in the Lab

Ligation of puc19 and CFP

New PCR of CFP

Aim / Task Digest of new pUC19 (XbaI/EcoRI), PCR clean up of CFP, Gel extraction of CFP, Digest purified CFP, Ligation of pUC19 with CFP, Heatshock
Protocol
Participants Leon Altmann

Notes

Digestion of pUC19 with EcoRI and XbaI:

Name Amount [µl]
pUC19 DNa 2 (= 2µg –DNA conc. 100ng/µl)
EcoRI-HF 1
XbaI 1
Cut Smart Buffer x10 2
Water 14
Total amount 20 µl

Incubate @37 °C for 20 min;

Inactivate @ 65 °C for 20 min;

PCR Purification:

Purified PCR sample from 3.9.19 with the PCR Purification Kit and followed the Kits protocol;

Eluted DNA in 30 µl Buffer EB;

Gel Extraction:

Extracted DNA from the Gel prepared earlier this DNA to extract the Measurement Part;

Used the QiaQuick Gel Extraction Kit and followed its protocol;

Weight of Gel fragment ~0,02g –60 µl Buffer QG for Gel solving;

Eluted the DNA in 35 µl Buffer EB;

DNA Measuring at Nanodrop:

Name ng/µl 260 280 260⁄280 260⁄230
PCR Clean Up CFP 45,6 0,912 0,500 1,82 1,69
Gel Ex CFP 8,5 0,169 0,085 2,00 0,02

Digestion of the purified samples (Gel Ex CFP, PCR Clean Up CFP):

Name Amount [µl] PCR Purified Amount [µl] Gel Ex CFP
CFP DNA 30 (= 1,37 µg) 35 (= 927,5 ng)
EcoRI-HF 1 1
XbaI 1 1
Cut Smart Buffer x10 5 5
Water 15 8
Total amount 50 µl 50 µl

Incubate @37 °C for 20 min;

Inactivate @ 65 °C for 20 min;

Ligation of Measurement Part with the digested pUC in different ratios

Ligation with T4 DNA Ligase Protocol (M020) from NEB;

Reaction set up on ice;

Name Amount [µl] CFP PCR 1:5 Amount [µl] CFP PCR 1:7 Amount [µl] CFP PCR 1:10 Amount [µl] Gel Ex 1:5
T4 DNA Ligase Buffer x10 2 2 2 2
Vector DNA 0,5 0,5 0,5 0,5
Insert DNA 1,4 2 2,84 11,76 ( = 100 ng)
Water 15,1 14,5 13,7 5,24
T4 Ligase 1 1 1 1
Total amount 20 µl 20 µl 20 µl 20 µl

Incubate @RT for 10 min;

Heat inactivate @65 for 10 min;

With the sample Heat shock was performed after Evas Protocol;

The transformed cells and one sample being ony digested vector were seeded on Amp+ plates and placed into the incubator @ 37 °C overnight;

Placed the colonies picked earlier by Eva into the Shaker overnight @ 37 °C and 230 rpm;

Files used / generated

In parallel designed Primer for the restriction digestion of the Constructs for the E. coli expression;

Designed them with the intent to open the pACYC184 vector with ClaI and SalI and accordingly also digest the constructs with the same enzymes to perform classical cloning;

Pending tasks, next steps, comments for next people in the Lab

  • Mini Prep from the Overnight cultures in the Shaker (one could also look at the cells under the fluorescence microscope as successfull ligation + transformation should produce CFP);

  • Gel electrophoresis thereof

  • Heatshock into other Strains BL21, Star, Tuner;

  • Check Plates in the Incubator, prepare ON cultures;

Aim / Task CFP ligation with restrictionenzyme digest
Protocol
Participants Eva Neugebauer

Notes

Plates of Heatshock (03.09.2019)

4 small clonies detactable

Plate back to incubator

Later clone picking

1% Agarose gel

M (5µL) | 1 CFP 01. | 2 CFP 01. | puc19 Verdau 3.9. | - | - | M(5µL) | PCR L | PCR R

For: 1 CFP 01., 2 CFP 01., puc19 Verdau 3.9. following composition was used:

5µL DNA, 3,3 µL Loading Dye, 11,7 µL MilliQ water

For: PCR R and PCR L following composition was used:

12µL DNA, 3,3 µL Loading Dye, 4,7 µL MilliQ water

Gel running running at 110V

No band for the vector -probably not enough DNA loaded on the gel

Cutting out the CFP from the PCR (PCR L was the probe and R the control)

Problems faced / possible error causes

Plate was the wrong way round in the incubator

No negative control for the heatshock -clones might be just undigested vector

insert picture from 4.9.19 here; name on labfolder:TS05405Sept42019.jpg

Aim/Task: Digest of new pUC19 (XbaI/EcoRI), PCR clean up of CFP, Gel extraction of CFP, Digest purified CFP, Ligation of pUC19 with CFP, Heatshock
Protocoll used:
Date: 2019-09-04
Name: Leon

Notes

Digestion of pUC19 with EcoRI and XbaI:

Name Amount [µl]
pUC19 DNa 2 (= 2µg –DNA conc. 100ng/µl)
EcoRI-HF 1
XbaI 1
Cut Smart Buffer x10 2
Water 14
Total amount 20 µl

Incubate @37 °C for 20 min;

Inactivate @ 65 °C for 20 min;

PCR Purification:

Purified PCR sample from 3.9.19 with the PCR Purification Kit and followed the Kits protocol;

Eluted DNA in 30 µl Buffer EB;

Gel Extraction:

Extracted DNA from the Gel prepared earlier this DNA to extract the Measurement Part;

Used the QiaQuick Gel Extraction Kit and followed its protocol;

Weight of Gel fragment ~0,02g –60 µl Buffer QG for Gel solving;

Eluted the DNA in 35 µl Buffer EB;

DNA Measuring at Nanodrop:

Name ng/µl 260 280 260⁄280 260⁄230
PCR Clean Up CFP 45,6 0,912 0,500 1,82 1,69
Gel Ex CFP 8,5 0,169 0,085 2,00 0,02

Digestion of the purified samples (Gel Ex CFP, PCR Clean Up CFP):

Name Amount [µl] PCR Purified Amount [µl] Gel Ex CFP
CFP DNA 30 (= 1,37 µg) 35 (= 927,5 ng)
EcoRI-HF 1 1
XbaI 1 1
Cut Smart Buffer x10 5 5
Water 15 8
Total amount 50 µl 50 µl

Incubate @37 °C for 20 min;

Inactivate @ 65 °C for 20 min;

Ligation of Measurement Part with the digested pUC in different ratios

Ligation with T4 DNA Ligase Protocol (M020) from NEB;

Reaction set up on ice;

Name Amount [µl] CFP PCR 1:5 Amount [µl] CFP PCR 1:7 Amount [µl] CFP PCR 1:10 Amount [µl] Gel Ex 1:5
T4 DNA Ligase Buffer x10 2 2 2 2
Vector DNA 0,5 0,5 0,5 0,5
Insert DNA 1,4 2 2,84 11,76 ( = 100 ng)
Water 15,1 14,5 13,7 5,24
T4 Ligase 1 1 1 1
Total amount 20 µl 20 µl 20 µl 20 µl

Incubate @RT for 10 min;

Heat inactivate @65 for 10 min;

With the sample Heat shock was performed after Evas Protocol;

The transformed cells and one sample being ony digested vector were seeded on Amp+ plates and placed into the incubator @ 37 °C overnight;

Placed the colonies picked earlier by Eva into the Shaker overnight @ 37 °C and 230 rpm;

Files used / generated

In parallel designed Primer for the restriction digestion of the Constructs for the E. coli expression;

Designed them with the intent to open the pACYC184 vector with ClaI and SalI and accordingly also digest the constructs with the same enzymes to perform classical cloning;

Pending tasks, next steps, comments for next people in the Lab

- Mini Prep from the Overnight cultures in the Shaker (one could also look at the cells under the fluorescence microscope as successfull ligation + transformation should produce CFP);

- Gel electrophoresis thereof

- Heatshock into other Strains BL21, Star, Tuner;

- Check Plates in the Incubator, prepare ON cultures;

<CFP_gel>

Aim / Task CFP ligation with restrictionenzyme digest
Protocol
Participants Eva Neugebauer

Notes

Plates of Heatshock (03.09.2019)

4 small clonies detactable

Plate back to incubator

Later clone picking

1% Agarose gel

M (5µL) | 1 CFP 01. | 2 CFP 01. | puc19 Verdau 3.9. | - | - | M(5µL) | PCR L | PCR R

For: 1 CFP 01., 2 CFP 01., puc19 Verdau 3.9. following composition was used:

5µL DNA, 3,3 µL Loading Dye, 11,7 µL MilliQ water

For: PCR R and PCR L following composition was used:

12µL DNA, 3,3 µL Loading Dye, 4,7 µL MilliQ water

Gel running running at 110V

No band for the vector -probably not enough DNA loaded on the gel

Cutting out the CFP from the PCR (PCR L was the probe and R the control)

Problems faced / possible error causes

Plate was the wrong way round in the incubator

No negative control for the heatshock -clones might be just undigested vector

<Agarose_gel_cfp>

Aim / Task CFP ligation with restrictionenzyme digest
Protocol
Participants Leon Altmann

Notes

ON Plates in the incubator were checked. The Control plate with only cut vector had 4 very small colonies on it. Whereas all Plates with Vector + Insert (pUC19 + CFP) had much higher numbers. Comparing the plates, the Gel Ex. Plate 1:5 had the least amount and within the PCR Plates a nice gradient was visible. The higher the Vector: Insert ratio was, the larger was the number of colonies.

With the fluorescence microscope of the AG Behrens the plates were screened for positive clones.

Number of CFp pos. clones was rather low <10 %, positive clones were marked and later on picked to set up ON cultures;

Incubated ON cultures @ 37 °C, 230 rpm;

The ON cultures in the shaker were split. A 500 µl Aliquot was stored at -20 °C after spinning down the cells. The other 4,5 ml were spinned down in the Megafuge (Mouse Lab), Miniprep not performed due to the low efficycy of the better ligation approach, so it is to be assumed that these clones are all negative;

Sequencing data was analyzed –all the sent in clones seem to be positive

Aim / Task estriction OH PCR with the E. coli Parts, Mini Prep of the CFP clones, Control Digest
Protocol
Participants Eva Neugebauer, Leon Altmann

Notes

To generate the necessary restriction sites for the classical cloning PCr was performed with the constructs for the E. coli expression.

Restriction OH PCR K1 K2a K2b K3a K3b K4a K4b Water Control
Q5 MM x2 12,5 12,5 12,5 12,5 12,5 12,5 12,5 12,5
fw Primer 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25
re Primer 1,25 1,25 1,25 1,25 1,25 1,25 1,25 1,25
DNA 0,5 0,5 2,5 0,5 0,5 0,5 0,5 0
Water 9,5 9,5 7,5 9,5 9,5 9,5 9,5 10
Total 25 25 25 25 25 25 25 25

The normal PCR Amplification Program was used in the PCR cycler, Samples were stored @ 4 °C;

Miniprep of the ON cultures that were now for 4 more hours in hte Shaker was performed;

Eluted the samples in 50 µl of EB Buffer;

DNA concentration of the samples was measured with the Nanodrop;

Name ng/µl 260 280 260⁄280 260⁄230
C1 77,25 1,545 0,706 2,19 2,28
C2 166,47 3,329 1,552 2,14 2.25
C3 50,54 1,011 0,519 1,95 1,82
C4 34,62 0,692 0,356 1,95 1,91
C5 80,80 1,616 0,782 2,07 1,99
C6 24,83 0,497 0,227 2,19 2,04
C7 33,04 0,661 0,308 2,14 2,08
C8 22,01 0,440 0,217 2,02 1,88

Test Digest

Miniprep samples were digested with EcoRI HF and SalI

Name C1 C2 C3 C4 C5 C6 C7 C8
SalI 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5
EcoRI HF 0,5 0,5 0,5 0,5 0,5 0,5 0,5 0,5
Cut Smart x 10 2,5 2,5 2,5 2,5 2,5 2,5 2,5 2,5
DNA 6,5 3 10 14,5 6,2 20 15,2 0
Water 15 18,5 11,5 7 15,3 1,5 6,3 21,5
Total 25 25 25 25 25 25 25 25

Incubation @ 37 °C for 15 min;

Inactivation @ 65 °C for 20 min;

Problems faced / possible error causes

During the Miniprep a washing step was forgotten so the eluted DNA presumable also contained genomic DNA; To assay this the samples were once more transferred onto purifiation säulchen to be washed and finally aeluted again;

Pending tasks, next steps, comments for next people in the Lab

  • Run a Gel of the digested Miniprep samples

  • Run a Gel of the PCR samples

  • Prepare new competent Cell ( DH5a, Tuner, BL21, Star)

  • Heat Shock with the PCR samples (Tuner, Bl21, Star) and positive Miniprep Samples (Tuner, BL21, Star)

Aim / Task Control Gel of PCR and Mini, PCR Purification/Gel Extraction, New Amp+ Plates, Heatshock of CFP c2 into Star/BL21/Tuner
Protocol
Participants Leon Altmann, Lena Schorr, Eva Neugebauer

Notes

Control Gel electrophoresis:

A 1% Agarose Gel was prepared and the samples from the digested Mini Prep (6.7.) and the E. coli Restriction PCR (6.7.) were applied;

Gel 1:

Ladder C1 C2 C3 C4 C5 C6 C7 C8

Gel 2:

Ladder K1 K2a K2b K3a H20 H20 K4a K4b K3b

Of the PCR 3 µl were used to create a 20 µl sample with loading dye and water;

Of the digested Mini Prep 25 µl were used together with 4,2 µl;

To the gel 15 µl of each sample were applied;

PCR Purification of K2b, K3a, K3b, K4a, K4b:

Performed PCR Purification according to the QiaQuick Purification Protocol and eluted the samples in 30 µl EB-Buffer;

Gel extraction K1, K2a:

For the Gel extraction another Gel of K1 and K2a with a higher sample concentration was prepared;

For K1 the band at approx 1,7 kb was extracted;

Construct 2a with the Sites for Restriction cloning is approx. 1400 bp long, to be safe though also the bad that was seen at the height of roughly 1200 bp was extracted and purified;

The samples were eluted in 50 µl EB Buffer;

Cast new Amp+ Plates:

New AMP Plates were necessary so 35g of LB-Agar Powder were mixed with 1l of MilliQ Water and mixed thoroughly. Mixture was autoclaved and after sufficient cooling Amp (50 mg/ml) was added to reach a concentration of 50 µg/ml;

With the Broth new Plates were casted;

Heatshock of CFP c2 into Star/Bl21/Tuner:

Heatshock with CFP+Puc19 clone 2 into Bl21, Star and Tuner with 2 µl DNA;

Eva Heatshock Protocol was used plus an extra Step with 200 µl SOC Medium after the 2 min incubation on ice;

Plated 200 µl of each sample onto Amp+ plates and stored the remaining 200 µl @ 4 °C;

Digestion of pACYC184 and purified samples with ClaI, SalI-HF:

pACYC184:

Name Amount [µl] pACYC184 Amount [µl] PCR purified K2b, K3b ,K4a, K4b Amount [µl] Gel Ex. K1, K2a
DNA 5,32 15 40
ClaI 1 1 1
SalI-HF 1 1 1
Cut Smart Buffer x10 5 2,5 5
Water 44,86 4,5 4
Total amount 50 µl 25 µl 50 µl

Incubate @37 °C for 15 min;

Inactivate @ 65 °C for 20 min;

To avoid uncut vector, that might reduce the efficacy of a following ligation Gel Extraction of the 3,6 kb long fragment was performed;

The sample was eluted in 30 µl Buffer EB;

Ligation of digested and gel extracted pACYC184 with the digested the digested E. coli constructs (K1-K4b) in a 1:7 ratio (vector:insert):

Before the Ligation the amount DNA amount was measured in the Nanodrop:

Name ng/µl 260 280 260⁄280 260⁄230
pACYC184 14,94 0,290 0,160 1,81 0,02
K1 dig 75,94 1,519 0,867 1,75 0,45
K2a 1 dig (oben) 127,04 2,541 1,624 1,56 0,44
K2a 2 dig (unten) 123,97 2,479 1,584 1,56 0,43
K2b dig 71,40 1,428 0,839 1,70 1,01
K3a dig 71,79 1,463 0,830 1,73 0,87
K3b dig 63,78 1,276 0,776 1,64 0,81
K4a dig 56.60 1,132 0,665 1,70 0,86
K4b dig 76,38 1,528 0,993 1,54 0,80

With the results from the Nanidrop Measurement Ligation with T4 DNA Ligase Protocol (M020) from NEB was performed;

Reaction set up on ice;

Name Amount [µl] K1 Amount [µl] K2a 1 Amount [µl] K2a 2 Amount [µl] K2b Amount [µl] K3a Amount [µl] K3b Amount [µl] K4a Amount [µl] K4b
T4 DNA Ligase Buffer x10 2 2 2 2 2 2 2 2
Vector DNA 3,5 3,5 3,5 3,5 3,5 2,7 2,7 2,7
Insert DNA 4,6 2,75 2,8 3,5 3,5 5,5 6,2 4,6
Water 8,9 10,75 10,7 10 10 8 7,3 8,9
T4 Ligase 1 1 1 1 1 1 1 1
Total amount 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl

Short Spin the Samples and incubate @RT for 10 min;

With the samples Heat shock was performed after Evas Protocol + SOC Step;

The 200µl of the ransformed cells were seeded on Amp+ plates and placed into the incubator @ 37 °C overnight;

Calculations and Results

Control Gel electrophoresis:

The control gel electrophoresis showed that the digestion in most of the samples were successfull. In some (4,6,7,8) uncut DNA was visible;

Sample 5 and 8 do also not show the expected band at approx 0,7 kb;

All PCR samples showed bands at their expected heights;

Just K1 and K2a had a strong unspecific bands. This is why the correct band was extracted out of the Gel instead of PCR Purification like with the other samples;

Problems faced / possible error causes

Forgot to inactivate the Ligase @ 65 °C for 10 min;

pirctures have to be inserted here:

7.9_Restriction_PCR,_Miniprep_Gel.jpeg

7.9_Restriction_K1,_K2a_for_Gel_Ex.jpeg

<Restriction_gel> <Restriction_PCR>

Aim / Task CFP ligation with restrictionenzyme digest
Protocol
Participants Leon Altmann

Notes

Checked the ON cultures in the Shaker –A 500 ml Aliquot was frozen away after centrifuging and getting rid of the supernatant;

The other 4,5 ml were spinned down in hte Megafuge for 5 min, 4000 rpm –All the pellets were rather small so the Samples were put into the Shaker under the same conditions for a couple more hours;

The ordered Primer for Restriction Overhang PCR were diluted in TE-Buffer, so that the concentration is 100 µM;

Name DNA [nmol] TE-Buffer [µl]
K1 restriction fw 31,9 319
K1 restriction re 32,4 324
Restriction fw 99,9 999
Restriction re 106,8 1068

Incubation @ 50 °C to elute the Primers;

Aim / Task CFP ligation with restrictionenzyme digest
Protocol
Participants Leon Altmann

Notes

Checked the Plates in the Incubator;

The Cells with the E. coli Restriction constructs were seeded on the wrong plates AMp+ Plates instead of CAM plates;

The Cells with the Measurement Part (CFP) have grown nicely; Most colonies grew with the Tuner strain;

2l New LB Medium with 20g LB powder per Liter were prepared and autoclaved;

New Heatshock with the Rest of the Ligation samples (15 µl) into Tuner;

200 µl of the Heatshock samples were plated on CAM+ Plates and Placed in the incubator @ 37 °C; (Rest stored @ 4 °C)

Made 3 ON cultures with LB Amp+ (300 ml) one for each of the Measurement Part Plates (BL21, Tuner, Star);

Put them in the Shaker @ 37 °C, 230 rpm

Pending tasks, next steps, comments for next people in the Lab

  • Prepare new competent cells (BL21, Star, Tuner, DH5a);

  • Check Plates in the Incubator and prepare cultures –Mini Prep;

  • Check Cultures in the Shaker and prepare for CFP Measuring;

Aim / Task colony PCR
Protocol
Participants Eva Neugebauer

Notes

Preparation of labled Eppis with 1 mL LB with Chloramphenicol.

Preparation of Mastermixes for the colony PCR with: Q5 MM, Primer, Water like following:

K1 All other constructs
Q5 MM 75 µL 475 µL
fw primer 7,5 µL 47,5 µL
re primer 7,5 µL 47,5 µL
H2O 60 µL 380 µL

25 µL per colony master mix in the according PCR tube.

Picking of clones with a pipet. Putting the pipette in the PCR tube and then into the LB.

PCR program: Konstrukt amplification

Running gel on a 1% agarose gel.

5 µL Loading dye was added to the 25µL PCR and then 15µL loaded on the gel.

Running at 130 V.

Loading scheme of the gel:

Gel 1:

Marker | K1.1 | K1.2 | K1.3 | K1.4 | K1.5 | K2a.1 | K2a.2 | K2a.3 | K2a.4 | K2a.5 | K2a.1obere |K2a.2obere| K2a.3obere

Gel 2:

Marker| K2a.4obere | K2a.5obere | K2b.1 | K2b.2 | K2b.3 | K2b.4 | K3a.1 | K3a.2 | K3a.3 | K3a.4 | K3a.5 | K3b.1 | K3b.2

Gel 3:

Marker| K3b.3 | K3b.4 | K3b.5 | K4a.1| K4a.2| K4a.3| K4a.4|K4b.1|K4b.2|K4b.3|K4b.3|K4b.4|h20 PCR|milliq

Aim / Task colony PCR
Protocol
Participants Martin Borgmann, Felix Pfister

Notes

Colony PCR

Preparation of labled Eppis with 1 mL LB with Chloramphenicol.

Preparation of Mastermixes for the colony PCR with: Q5 MM, Primers, Water as followed (3 colonies of each construct):

K1 (3 reactions) All other constructs (21 reactions)
Q5 MM 37,5 µL 262,5 µL
fw primer 3,75 µL 26,25 µL
re primer 3,75 µL 26,25 µL
H2O 30 µL 210 µL

Primers:

  • ​ K1: “K1 restriction fw” and “K1 restriction re”
  • ​ all other constructs: “restriction fw” and “restriction re”

25 µL per colony master mix in the according PCR tube.

Picking of clones with a pipet tip

PCR program: Konstrukt amplification

​ modifcation: 1st step at 90°C 10 min

Gel electrophores (1% agarose, 130 V)

2 µL Loading dye was added to the 10 µL PCR and then loaded on the gel.

Loading scheme of the gel (2 x 14 pockets, 1mm wide):

First Row:

1 kb Ladder | K1.1 | K1.2 | K1.3 |K2a1.1 | K2a1.2 | K2a1.3 | K2b.1| K2b.2 | K2b.3 | K3a.1 | K3a.2 | K3a.3

Second Row:

1 kb Ladder K3b.1 | K3b.2 | K3b.3 | K4a.1 | K4a.2 | K4a.3 | K4b.1 | K4b.2 | K4b.3 | K2a2.1 | K2a2.2 | K2a.3

Calculations and Results

Weak bands

​ might be repeated with with more cycles!

Problems faced / possible error causes

Didn’t prepare 1 replicate in the MM, therefore the last tube had a little less volume than the others.

Switched poles during electrophoresis, therefore lost the first row.

Mini Prep

Miniprep of ON cultures of the colonies that were incubated after colony PCR (09.09.19)

Assessment of success using gel electrophoresis (1% gel, 130 V)

Aim / Task Testing of miniprep from 10.09.
Protocol https://www.helmholtz-muenchen.de/fileadmin/PEPF/Protocols/Chemically-competent-cells.pdf
Participants Marie Wiedemann

Notes

  • Used prepared bacteria from -80 °C (DH5a, Star, BL21, Tuner)
  • defrosted them at RT, put in 125 ml LB Medium
  • incubated at 30 °C for 1h
  • checked oD until between 0.4 and 0.6
  • oD: DH5a: 0,564; Star: 0,654; BL21: 0,401; Tuner: 0,82
  • diluted Tuner to oD of 0,628
  • Following steps consitently on ice
  • split in 50ml falcons
  • incubate ion ice 10 min
  • spin in ultracentrifuge 10 min 4000rpm 4 °C
  • resuspend pellet in 25 ml CC-Puffer (already prepared)
  • incubate 10 min on ice
  • centrifuge 10 min 4000 rpm 4 °C
  • resuspend pellet in 4,65 ml CC-Puffer + 0,35 ml 15% DMSO per falcon
  • incubate on ice 10 min
  • aliquoted in 1,5 ml eppis 200 µl per eppi
  • freezed in -80 °C

Materials used

CC-Puffer

DMSO

LB-Medium

Aim / Task Testing of miniprep from 10.09.
Protocol
Participants Eva Neugebauer

Notes

Measurement of the miniprep samples from 10.09. at the nanodrop.

Concentration in ng/µL:

K1.1: 64,2; K1.2 168,7; K2a.1.1 61,1; K2a.1.2 179,4; K2a2.1 132,6; K2a2.2 88,9; K2b.1 201,4; K2b.2 152,0; K3a.1 191; K3a.2 173,1; K3b.1 189,5; K3b.2 175; K4a.1 91,9; K4a.2 72,5

K4b.1 73,5; K4b.2 166,2

Testdigestion with 500 ng DNA, 0,5 µL Cla-I and 0,5 µL SalI, 5 µL Cut smart ad 25 µL for 15 min at 37°C

Adding of 5 µL Loading Dye to the digest. Running a 0,8% gel with 5µL Marker and 15µL of the digestion samples at 130V.

Pipetting scheme was this:

Gel1:

M | K1.1 | K1.2 | K2a.1.1 | K2a.1.2 | K2a2.1 | K2a2.2 | K2b.1| K2b.2 | K3a.1| K3a.2 | K3b.1 | K3b.2 | K4a.1

Gel2:

M | K4a.2 | K4b.1 | K4b.2

Aim / Task Digestion of E.coli constructs, ligation of vector and constructs, Heat-Shock of CHO-constructs in DH5a, overhang PCR with bacterial constructs
Protocol
Participants Martin Borgmann, Lena Schorr, Leon Altmann

Notes

  • Digestion of paCYC and K1, K2a, K2b, K3a, K3b, K4a, K4b with ClaI and SmaI –wrong enzymes!!! (instead of SmaI, SalI should have been unsed)
  • Heat-Shock of constructs (K1-1, K1-2, K2a-1, K2a-2, K2b-1, K2b-2) in DH5a
    • with 0,5µl of Mini samples from 30.8.19
    • plating the constructs on Amp plates
    • storage in the incubator
  • Overhang PCR with K1, K2a, K2b, K3a, K3b, K4a, K4b (from initial IDT constructs) and K2a, K2b, K3a, K4b from PCR clean up from 13.08.
1x 16x
Q5 high fidelity 25µl 400µl
10µM forward primer 2,5µl 40µl
10µM reverse primer 2,5µl 40µl
DNA template 1µl -
nuclease free water 19µl 304µl

  • gel electrophoresis (0,8% and 1%)

    • 0,8%: ladder - K1 - K2a - K2b - K3a - K3b - K4a - K4b - H2O (from the initiall constructs)
    • 1%: ladder - K2a - K2b - K3a - K3b - K4a - K4b - H20

  • Preparation of overnight culture of heat shocked bacteria with LB and Amp

<Overhang_PCR_restriction> <Overhang_PCR_restriction2>

Aim / Task Midi Prep of ON cultures (K1-1, K1-2, K2a-1, K2a-2, K2b-1, K2b-2); Preparation of new LB Medium, PCR purification of PCR samples from the 12.9.; inoculation of clones from the heatshock from the 13.9. in 5ml LB-Amp
Protocol Midi prep protocol
Participants Martin Borgmann, Lena Schorr

Notes

  • PCR purification o

    • PCR samples from the 12.9

    • Elution in 150 µl MiliQ water

    • concentration:

  • Preparation of 3 1l bottles LB medium

  • Midi Prep of the overnight cultures of

K1-1, K1-2, K2a-1, K2a-2, K2b-1, K2b-2 (CHO constructs) in DH5a

  • protocol ion labfolder at the 20.8.

  • eluation in 100µl MilliQ water

  • Inoculation of clones from the heatshock (13.9.) in 5ml LB-Amb

    • over night in the shaker at 37°

Pending tasks, next steps, comments for next people in the Lab

–>Mini Prep of the overnight cultures

–>gel-electrophoresis of the midi prep + from the current mini-prep

Aim / Task iniprep of ON cultures from 14.09.19 + Gel electrophoresis + Gel electrophoresis from Midiprep from 14.09.19
Protocol ExtractMe Plasmid Mini Kit
Participants Martin Borgmann

Notes

  • Miniprep of over night cultures from 14.09.19 using “ExtractMe Plasmid Mini Kit”

    • Elution in 60 µL (50µL left after taking 10 µL for electrophoresis)

  • Preparation of Gel electrophoresis

    • Dilution of Midiprep samples from 14.09.19

    1:100

    to avoid extrem bright fluorescence that might disturb the picture of the upcoming gel electrophoresis (samples have a DNA conc. of up to 5700 ng/µL)

    • 10 µL sample + 990 µL H2O

    • Sample “Midi K2-a-2” diluted 1:10 since its DNA conentration was only 337 ng/µL)

    • 10 µL sample + 90 µL H2O

  • Gel electrophoresis in 0,8% agarose gel

    • Loading scheme: 1kb ladder | Mini K1-1 | Mini K1-2 | Mini K2a-1 | Mini K2a-2 | Mini K2b-1 | Mini K2b-2 | Midi K1-1 | Midi K1-2 | Midi K2a-1 | Midi K2a-2 | Midi K2b-1 | Midi K2b-2

<Miniprep_Gel_electrophoresis>

Aim / Task Control Digest of MidiPrep samples (14.9) and Mini Prep samples (15.9); Prepare samples for Kiel
Protocol
Participants Leon Altmann

Notes

Digest of MidiPrep samples (14.9) and Mini Prep samples (15.9) with EcoRI-HF and NHeI-HF to check properly check the samples;

Name Mini Prep samples [µl] Midi Prep samples [µl]
DNA 2 0,5
EcoRI-HF 0,5 0,5
NheI-HF 0,5 0,5
Cut Smart Buffer x10 2 2
Water 15 1,5
Total amount 20 µl 20 µl

Incubate @37 °C for 20 min;

Inactivate @ 65 °C for 20 min;

Prepare 0,8 % Agarose Gel to examine digested samples;

Loading order:

| Ladder | Midi K1-1 | Midi K1-2 | Midi K2a- 1 | Midi K2a-2 | Midi K2b-1 | Midi K2b-2 | Mini K1-1 | Mini K1-2 | Mini K2a-1 | Mini K2a-2 | Mini K2b-1 | Mini K2b-2 | Ladder |

Prepared 100 µg of the Midi Prep samples K1-2, K2a-1, K2b-1 for Kiel and sent them there via Post;

Aim / Task Splitting of the 293T cells
Protocol -
Participants Martin Borgmann, Eva Neugebauer

Notes:

-293T cells were not properly attached -Probably most of them died

Work done:

-Mediumchange of the cells

Pending tasks, next steps, comments for next people in the Lab

Check if cells are growing

Getting new cells and culture them proberly :)

Aim / Task Splitting 239T cells
Protocol
Participants Martin Borgmann, Eva Neugebauer

Notes

Check HEKs in the Incubator –All of them died

Pending tasks, next steps, comments for next people in the Lab

Get new HEKs

Proceed with Measurement Part

Aim / Task Splitting 239T cells
Protocol
Participants Martin Borgmann, Eva Neugebauer, Leon Altmann

Notes

Repeat the IPTG

Pending tasks, next steps, comments for next people in the Lab

Check if cells are growing

Getting new cells and culture them properly

CFP part:

Cheking plates in the incubator

Plenty colonies in Tuner but not BL21 and STAR (Those were the “new” competent cells)

=BL21 and STAR not properly competent!!

New heatshock with old stock of competent bacs (STAR and BL21) acording to Evas protocol

HEK:

Check the flasks in the incubator: Live and happy ~ 50% confluent

Seewing of 4 x 10^6 cells in a plate (10 mL) like following.

  1. Discard medium

  2. Washing with 3 mL Trypsin

  3. Incubation with 3 mL trypsin

  4. When cells were detached mixing with 7 mL Medium

  5. Counting in neubauer chamber (total cell number 10,2 x 10^6 cells)

  6. 8 mL cell mix in the plate + 2 mL medium

Rest of the cells back in the flask, ad 20 mL with medium

E. coli:

Digest of pACYC184: 5µL cutsmart, 11µL DNA, 1µL Cla-I, 1µL Sal-I

Water was not added! (WRONG)

Digest of the constructs which were already digested on the 07.09. but not for 1 h just 15 min.

1 h digest with Cla-I and Sal-I (each 0,5µL)

Ligation

Aim / Task HEK transfer, vector Digestion, Ligation, Heatshock
Protocol
Participants Eva Neugebauer, Leon Altmann

Notes

CFP part:

Checking plates in the incubator

Plenty colonies in Tuner but not BL21 and STAR (Those were the “new” competent cells)

=BL21 and STAR not properly competent!!

New heatshock with old stock of competent bacs (STAR and BL21) acording to Evas protocol

HEK:

Check the flasks in the incubator: Live and happy ~ 50% confluent

Seeding of 4 x 10^6 cells in a cell culture plate (10 ml) like following.

  1. Discard medium

  2. Washing with 3 ml trypsin

  3. Incubation with 3 ml trypsin

  4. When cells are detached mixing with 7 ml Medium

  5. Counting in neubauer chamber (total cell number 10,2 x 10^6 cells)

  6. 8 ml cell mix in the plate + 2 ml medium

Rest of the cells back in the flask, ad 20 ml with medium

E. coli:

Digest of pACYC184: 5µL cutsmart, 11µL DNA, 1µL Cla-I, 1µL Sal-I

Water was not added! (WRONG);

Incubation @37 °C for 1h and following inactivation @65 °C for 20 min;

Proceeded nonetheless and added water aftewards to have a convenient volume of 50 µl

Digest of the constructs (K1 - K4b) which were already digested on the 07.09.19. ClaI is not a time saver qualified enzyme and thus the digestion time of 15 minutes might have not been sufficient. 1 h digest with Cla-I and Sal-I (each 0,5µL) in a volume of 20 µl @37 °C;

Inactivation @65 °C for 20 min;

With the sample concentration determined on 7.9 the new sample concentration was calculated, as the digest altered the volume;

Sample Concentration:

K1 K2a K2b K3a K3b K4a K4b
67.5 ng 110 ng 58,4 ng 71,8 ng 63,8 ng 56,6 ng 62,9 ng

Only 6 Chloramphenicol plates were left in the fridge, so ligation was only performed with K1, K2a and K2b;

Amounts necessary for the ligation were calculated for all constructs;

Construct 1:10 ng 1:10 µl 1:15 ng 1:15 µl
K1 207,4 3 311,1 4,5
K2a 151,4 1,4 227,1 2,1
K2b 144,2 2,46 216,3 3,7
K3a 105,6 1,8 158,4 2,7
K3b 100 1,9 150 2,85
K4a 105,6 2,3 158,4 3,45
K4b 100,6 1,6 150,9 2,4

Ligation

K1 K2a K2b
1:10 1:15 1:10 1:15 1:10 1:15
DNA 3 4,5 1,4 2,1 2,46 3,7
Vector 1,25 1,25 1,25 1,25 1,25 1,25
Ligase Buffer x10 2 2 2 2 2 2
Ligase 1 1 1 1 1 1
H20 12,75 11,25 14,35 13,65 13,3 12,05
Total 20 µl 20 µl 20 µl 20 µl 20 µl 20 µl

Incubation @RT for 20 min;

Inactivation @65 °C for 20 min;

Perform Heatshock after Evas Protocol;

Heatshock samples were not plated on plates, as the remaining Chloramphenicol+ were found to be contaminated; Samples were therefor stored @4 °C;

Pending tasks, next steps, comments for next people in the Lab

  • Produce new Chloramph Plates

  • Test Transfection of HEK293T cells with K1

Aim / Task calciumchoride transfection of heks with K1
Protocol
Participants Eva Neugebauer, Leon Altmann

Notes

Take Plates with negative control for measurement Part out of the incubator, colonies grew on alle of them!

Picked clones and prepared ON cultures with 8 ml LB-Amp, put them in the fridge until later;

Medium Change 1h prior to Transfection of the HEKs, DMEM without Hepes (cells were already in Medium without Hepes);

Confluence ~ 70%;

Prepare new LB Agar for new Chloramphenicol Plates; 35 g LB Agar were weighed in and mixed thorougly with 1 l Water;

Autoclaved Medium;

Performed Calciumchlorid Transfection:

Perpared two mixes:

A) 1 ml of 2x HBS in a 15 ml Falcon;

B) DNA Mix:

Water 860 µl
Plasmid 20 µl (1 µg/µl)
2,5 M CaCl2 100 µl
2,5 mM Chloroquine 20 µl

Carefully apply the DNA mix to the 2x HBS over the time of 1 min, while constantly blowing air into the HBS with a Pipette boy;

Vortex the resulting Mix thorougly and apply it dropwise to the Cells;

After the prepared Medium has cooled sufficiently added Chloamphenicol (35 mg/ml) to reach a concentration of 25 µg/ml –735 µl;

Poured new Chloramph Plates; Seeded the Heatshock Samples from 25.9 that were stored in the fridge onto Chloramphenicol Plates;

Placed them in the Incubator;

Placed the earlier prepared ON cultures in the shaker;

Performed Medium Change 6 h post transfection with 10 ml Medium;

Aim / Task measuring measuring part
Protocol
Participants Eva Neugebauer, Leon Altmann

Notes

Measurement part:

Take ON cultures out of shaker

Measure the smaples with spectrometer

Bl21 puc19 neg: 1,098

STAR puC19 neg: 1,17

TUNER pUC19: CFP neg: 1,41

Prepared samples and induced them with IPTG

Culture [mL] LB Amp [mL] IPTG 1mM with LB amp [mL]
BL21 pos 5 0 5
STAR pos 5 0 5
TUNER pos 5 0 5
BL21 negative 2,5 2,5 5
STAR negative 2,5 2,5 5
TUNER negative 2 3 5

Incubation for 3 hours at 37°C

Sonification:

Measuring cell desnity at spectrometer

acording to this 10^7 cells/ mL were added for a 10 mL endvolume solution

centrifugation for 10 min 4000 g

Resuspend in 10 mL sonification buffer

3 10s pulses at 30W

Remove cell debris at 10.000g for 10 min

Measuring in ELISA reader:

Not possible ELISA has just OD detection no flourescence

E. Coli constructs:

Plates from heatshock out of the incubator

they were all overgrown

K1 293T:

Medium chance

1:5 splitting of the 293T cell flask

Aim / Task ITPG induction of ON culture
Protocol
Participants Eva Neugebauer

Notes

OD measurement of the ON cultures

Culture OD amount ON culture for OD of 0,5 in 5 mL [mL]
Tuner CFP pos 2,2075 1,13
Star CFP pos 2,59 0,96
BL21 CFP pos 1,757 1,48
Tuner CFP neg 1,97 1,25
Star CFP neg 1,769 1,43
BL21 CFP neg 1,672 1,5

5mL induction culture for each ON culture with

  1. 0,5 mM IPTG (2,5mL of 1mM IPTG solution in LB Amp)

  2. 0,3 mM IPTG (1,5mL of 1mM IPTG solution in LB Amp)

ad 5 mL with LB

ON cultures filled up to ca 5 mL with LB amp

Aim / Task transfection
Protocol -
Participants Eva Neugebauer, Leon Altmann

Protocol

Notes

Medium changen of the HEK plates (K2a, K2b) 1 h pre-transfection;

Harvesting supernatant from last weeks K1;

Splitting of the 5 flasks with HEK cells (ratio 1:5);

DNA for transfection stems from the Midi-Prep of 14th of September;

20 µg of DNA were brought into a 1 µg/µl concentration for the transfection;

Transfection with K2a, K2b according to Annes’ protocol, time of transfection approx. 11:00;

Changed Medium 8 h post-transfection with 10 ml DMEM;

Aim / Task transfection
Protocol -
Participants Eva Neugebauer, Leon Altmann

Protocol

Notes

Medium changen of the HEK plates (K2a, K2b) 1 h pre-transfection;

Harvesting supernatant from last weeks K1;

Splitting of the 5 flasks with HEK cells (ratio 1:5);

DNA for transfection stems from the Midi-Prep of 14th of September;

20 µg of DNA were brought into a 1 µg/µl concentration for the transfection;

Transfection with K2a, K2b according to Annes’ protocol, time of transfection approx. 11:00;

Changed Medium 8 h post-transfection with 10 ml DMEM;

October

Aim / Task Western blot
Protocol -
Participants Eva Neugebauer

Protocol

Notes

  1. Preperation of a 10% Bis-tris Gel.

  2. Mixing of 10 µL harvest of K1 day1, K1 day2, K2a, and K2b with 2x loading buffer

  3. 95°C for 5 min

  4. Loading of the gel with 5µL marker |K1 day1|K1 day4|K2a|K2b (samples 15µL)

  5. Running gel for 1 h at 130V in 1x MOPS buffer

  6. Blotting to the membrane at 65mA for 1h30

  7. Blocking membrane in 10% milkpowder for 1h30

  8. Washing with PBST

  9. Adding primary antibody (mouse anti-Histag in 1:2000 dilution) 10mL in 10% milkpowder

  10. Shaking overnight at 4°C

  11. 3 times washing for 10 min with PBST

  12. Adding secondary antibody (HRP anti mouse in 1:2000 dilution) 10 ml in 10% milkpowder

  13. Shaking at RT for 1h

  14. 3 times washing for 10 min with PBST

  15. Adding 2mL of the reaction mix directly before taking the picture of the blot

<Westernblot_02_10_2019>

Aim / Task Harvesting K2a and K2b supernatant
Protocol -
Participants Eva Neugebauer, Leon Altmann

Protocol

Notes

Harvesting K2a and K2b supernatant, reapply 4 ml of Medium to the plates;

Prepared new plates for transfection;

Took 3 flasks with HEk Cells and collected the cells in a 50 ml Falcon after making them loose with trypsin and inactivating it again with Medum;

Cells were counted using a Neubauer Chamber in a 1:1 Dilution with Trypan Blue;
Counted: 115/131/119 –Mean is 121,6 Cells per quadrant –Therefore 2,432*106 cells/ml;

Seed 4*106 cells per Plate for the transfection tomorrow (1,64 ml of the cell suspension);

The remaining 5,4 ml (~13*106 cells) were applied to a new cell culture flask;
Everything was put in the incubator @37 °C, 5% Co2;

Aim / Task Harvesting supernatant
Protocol -
Participants Eva Neugebauer, Leon Altmann

Protocol

Notes

Change Medium 1h pre-transfection on the 6 prepared plates;

Harvesting supernatant from yesterdays transformation (K2a, K2b);

DNA for transfection stems from the Midi-Prep of 14th of September (K1-2, K2a-1, K2b-1);

20 µg of each DNA were brought into a 1 µg/µl concentration for the transfection;

With each Construct two plates were transfected (K1, K2a, K2b) according to Annes’ protocol, time of transfection approx. 11:30;

Aim / Task Test Miniprep of used strains for Measurement Part
Protocol Miniprep
Participants Leon Altmann

Protocol

Notes

Check XGal/IPTG/Amp+ Plates –overgrown, all colonies blue (on negative and positive plates!) Induced the ON cultures with 0 mM, 0,33 mM and 0,5 mM IPTG; Placed them in the incubator Meanwhile Miniprep with 1 ml of each of the uninduced CFP pos cultures was performed. Applied 10 µl sample + 3 µl 6xDye + 5 µl H2O to a 0.8% Agarose Gel

Gel: Ladder/Bl21/Star/Tuner

–Tuner does not contain the desired insert. This notion is corraborated by the pellets colour.

Aim / Task Harvesting
Protocol -
Participants Eva Neugebauer

Protocol

Notes

Harvesting of all transfected cells:

collection of the medium and centrifugation at max for 10 minutes.

The supernatant still needs to be filtrated.

Aim / Task Westernblot
Protocol -
Participants Eva Neugebauer, Leon Altmann

Protocol

Notes

  1. Removal of of the storage buffer by centrifugation with 100 g for 1 min

  2. Adding 600 µL binding buffer and centrifugation with 100 g for 1 min

  3. Adding the whole sample in multiple steps to the colume with each time centrifugation for 30s at 100g (Keeping the flow through)

  4. Washing once with 600 µL of binding buffer

  5. First Elution step: Adding 200µL Elution buffer to the colum

  6. Second Elution step: Adding 200 µL Elution buffer to the colum

Running 15% SDS Gel at 300V for 30 min

10 µL of the samples were applied and 7 µL of the ladder

Pipetting scheme:

Ladder | Wash K1 | Wash K2a | Wash K2b | Elution1 K1 | Elution 1 K2a | Elution 1 K2b | Elution 2 K1 | Elution 2 K2a | Elution 2 K2b

Stain the finished Gel with Coomasie and unstain it sufficiently to see clear bands;

Measure the samples in the Nanodrop A280:

Determine the Extinction Coefficient for each of the constructs with ProtParam (OnlineTool);

1 2 3 Mean
Elution 1 K1 25,54 25,08 27,04 25,9
K2a 75,6 72,57 72,02 73,22
K2b 47,28 48,16 48,99 48,14
Elution 2 K1 13,0 12,02 12,41 12,47
K2a 11,74 11,58 12,53 11,95
K2b 25,09 24,75 24,78 24,87

For a 1:1 ratio of K2a and b following volumes were added together:

E1: K2b 100µL with K2a 65,9µL

E2: K2a 100µL with K2b 48µL

Samples were sended to Kiel for killing assay.

10µL of each sample was kept for a westernblot to verify that the tag and catcherreaction happend.

Aim / Task Validate data from 3.10.19
Protocol -
Participants Leon Altmann

Protocol

Notes

Notes

Performed restriction digest with Tuner and BL21 (control) to check the result from 3rd October. Took Miniprep samples from 3.10.19 and digested the samples with SalI and EcoRI; Incubate @ 37 °C for 45 min; Inactivation fro 20 min @ 65 °C;

Applied digested samples to 0.8% Agarose Gel: Ladder/Bl21/Tuner

Aim / Task Westernblot of the purified proteins
Protocol -
Participants Eva Neugebauer

Protocol

10% BisTris Gel

10µL purified samples were mixed with 2x loading dye.

95°C for 5 min

Running gel for 1 h 130V:

Pipetting scheme of the gel: 5µL each

Ladder | E1 K1 | E1 K2a | K1 K2b | K1 K2ab | E2 K1 | E2 K2a | E2 K2b | E2 K2ab

Blotting for 1:30 h at 65mA

Blocking the membrane with 10% milkpowdersolution at 4°C for 5h

1x washing with PBST

Adding his-Ab (1:5000 in milkpowdersolution) overnight at 4°C

Aim / Task New Transfection
Protocol -
Participants Eva Neugebauer

Protocol

Notes

Splitting HEK293T cells in 3 wells of a 6 well plate

Aim / Task New Transfection, Purification, Western blot
Protocol -
Participants Eva Neugebauer

Protocol

  • Medium change 1 h before transfection
  • For transfection 2 µg DNA were used of K1-1, K2a-1 and K2b-1: Diluted 500 µL medium with nothing added
  • Mixed with 500µL Medium with nothing added with 4µL genjet
  • Incubation for 10 min at RT

Aim / Task New Transfection, Purification, Western blot
Protocol -
Participants Eva Neugebauer

Protocol

Harvesting of the cells Centrifuagtion at 2500 rpm for 5 min 600µL of supernatant were used for the purification via Histrap

Histrap Purification: centrifuge at 4°C open columns a quater and placing it in a 2 mL eppi Centrifuation at 500 rpm for 1 min Adding 600 µL Binding buffer Centrifugation at 500 rpm for 1 min Adding 600µL sample Centrifugation at 500 rpm for 1 min Washing with 600µL binding buffer Centrifugation at 500 rpm for 1 min 3x Elution step: Adding 100µL Elution buffer and centrifugation at 500 rpm for 1 min

Tag-Catcher reaction: Adding same colume K2a and K2b and incubate them 20 min at RT For this samples: Supernatant (SN) harvest, Elution (E) 1-3

Gel running :

at 130V for 1 h with following pipetting scheme

Gel1 M, SN K1, SN K2a, SN K2b, SN K2ab, E1 K1, E1 K2a, E1 K2b, E1 K2ab, E2 K1, E2 K2a, E2 K2b, E2 K2ab

Gel 2 M, SN K1, SN K2a, SN K2b, SN K2ab, E3 K1, E3 K2a, E3 K2b, E3 K2ab

Marker 5µL, Samples 20µL (10µL sample+10µL loading dye) After loading dye adding samples were cooked for 2 min at 95°C except the SN samples of the gel 2

Blotting: Both gels were blotted semidry for 1:23 h at 130mA

Blocking: Membranes were blocked for 1h 45 with 10% milkpowdersolution

Primary Ab: 2 washing steps with PBST anti-Histag ab was diluted 1:2000 in 10mL 10% milkpowdersolution and added to the membrane Incubation overnight at 4°C

Aim / Task Westernblot
Protocol -
Participants Eva Neugebauer

Protocol

Notes

3x washing with PBST for each time 10 min at RT Adding primary antibody which was diluted 1:2000 in 10% milkpowdersolution 1h RT incubation 3x washing with PBST for wach time 10 min at RT Adding for each blot directly for taking the picture the solution for HRP (2mL)

Aim / Task Test Miniprep of used strains for Measurement Part
Protocol Miniprep
Participants Leon Altmann

Protocol

Notes

3x washing with PBST for each time 10 min at RT Adding primary antibody which was diluted 1:2000 in 10% milkpowdersolution 1h RT incubation 3x washing with PBST for wach time 10 min at RT Adding for each blot directly for taking the picture the solution for HRP (2mL)