Team:IIT Madras/Notebook/Protocols

Ultracompetent Cell Preparation

1. Materials/Buffers
• SOB SOLUTION FOR COMPETENT CELL PREPARATION
1. 0.5% yeast Extract
2. 2% Tryptone
3. 10mM NaCl
4. 2.5mM KCl
5. 10mM MgCl2
6. 10mM MgSO4.
7. Dissolve all in nanopure water and autoclave
• TRANSFORMATION BUFFER FOR COMPETENT CELL PREPARATION
1. 10mM PIPES
2. 15mM CaCl2
3. 250mM KCl
4. Dissolve in nanopure water and adjust pH to 6.7 with KOH or HCl. The add MnCl2 to 55mM and adjust final volume. Sterilize by filtration with 0.45 µm filter. Store at 4C
2. Cells were cultured on 2xYT agar plate overnight at 370C.
3. 10-12 colonies were cultured in 250ml SOB medium.
4. It was incubated at 37C for 1hour. Then the flasks were transferred to 19C. It was incubated till the OD600 reached 0.5
5. Flask was placed in ice for 10min.
6. The cells were pelleted by spinning at 4000rpm for 10min at 4C.
7. Cells were resuspended in 80ml ice cold TB(Transformation Buffer) and stored on ice for 10min.
8. It was centrifuged again at 4000rpm for 10min at 4C.
9. Pellet was resuspended in 20ml of TB with 1.5ml DMSO.
10. Final volume was aliquoted into microcentrifuge tubes (100-500µl) and stored at -80C

Transformation

1. 100µl competent cells taken.
2. 2 µl Plasmid DNA added to the tube and shaken gently.
3. Mixture left on ice for 30 min.
4. Heat shock given at 420C for 2min.
5. Incubated on ice for 3-5 min.
6. 800 µl of LB broth added.
7. Flasks were shaken at 370C for 1hr.
8. They were centrifuged at 3000rpm for 5min.
9. 200 µl of the transformation mix was plated.
10. Plates were incubated at 370C overnight.

Miniprep

1. Overnight cultures were harvested.
2. They were centrifuged at 13000rpm for 1min.
3. The pellet was resuspended in 250 µl of HP1 solution.
4. The cells were lysed by adding 250 µl of lysis solution i.e. HP2. Tubes were inverted 5-6 times.
5. 350 µl of neutralization solution i.e. HN3 was added. Tbes were inverted 5-6 times to mix the solutions.
6. They were centrifuged at 13000rpm for 10 mins to get a white pellet.
7. The supernatant was carefully transferred to a HiElute Miniprep spin column.
8. It was centrifuged at 13000rpm for 1 min. Flow through was discarded.
9. 500 µl of wash solution i.e. HPB was added to the column.
10. It was centrifuged at 13000rpm for 1 min. Flow through was discarded.
11. 700 µl of wash solution i.e. HPE was added to the column.
12. It was centrifuged at 13000rpm for 1 min. Flow through was discarded.
13. It was centrifuged at 13000rpm for 1 min.
14. The column was transferred to a fresh tube.
15. 50 µl of elution buffer was added carefully to the column.
16. It was stood for 1 min.
17. It was centrifuged at 13000rpm for 1 min.
18. The DNA was collected in the fresh tube.

Restriction Digest (3A as well as Standard assembly)

1. Mix contains:
1. 9 µl MilliQ water.
2. 3 µl Upstream Digest.
3. 3 µl Downstream Digest
4. 2 µl Plasmid backbone.
5. 2 µl Ligase Buffer.
6. 1 µl Ligase.
2. Mix in incubated at Room Temperature for 15 min.
3. It is then heat inactivated at 650C for 20 min.

Ligation (3A Assembly)

1. Mix contains:
1. 9 µl MilliQ water.
2. 3 µl Upstream Digest.
3. 3 µl Downstream Digest.
4. 2 µl Plasmid backbone.
5. 2 µl Ligase Buffer.
6. 1 µl Ligase.
2. Mix in incubated at Room Temperature for 15 min.
3. It is then heat inactivated at 650C for 20 min.

Ligation (Standard Assembly)

1. Mix contains:
1. 12 µl MilliQ water.
2. 3 µl Insert.
3. 2 µl Plasmid backbone.
4. 2 µl Ligase Buffer.
5. 1 µl Ligase.
2. Mix in incubated at Room Temperature for 15 min.
3. It is then heat inactivated at 650C for 20 min.

PCR with Deep Vent Polymerase

1. Mix contains:
1. 1 µl Deep Vent polymerase.
2. 5 µl Bffer (10X).
3. 2 µl dNTPs.
4. 1 µl forward Primer.
5. 1 µl backward Primer.
6. 2 µl Template.
7. 38 µl MilliQ water.
2. Program used:
1. 960C for 2 min.
2. 960C for 30sec.
3. (Tm-5) 0C for 30sec.
4. 720C for ‘x’min (1min per kb).
5. GOTO 2 30 times.
6. 720C for 30min.
7. 40C for storage.

DpnI Digestion

1. 1 µl of DpnI was added to 1 tube of PCR product (<50 µl).
2. Incubated at 370C for 2hrs.
3. Heated at 800C for 15-20mins.

PCR with Phusion® Polymerase

1. Mix contains:
1. 10 µl Phusion® HF Buffer (5X).
2. 1 µl 10mM dNTPs.
3. 0.25 µl forward primer (0.5 µM final conc).
4. 0.25 µl reverse primer (0.5 µM final conc).
5. x µl template (add as needed).
6. 0.5 µl Phusion® DNA Polymerase.
7. Add MilliQ water to make p 50 µl volume.
2. Program used:
1. 980C for 2min.
2. 980C for 30sec.
3. (Tm+3) 0C for 30sec.
4. 720C for x sec(15sec per kb).
5. GOTO 2 30 times.
6. 720C for 30 min.
7. 40C for storage.

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