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Team:IIT Madras/Notebook/Protocols

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Contents 1 Ultracompetent Cell Preparation 1.1 Protocol 1.2 Variant 2 Transformation 2.1 Protocol 2.2 Variant 3 Miniprep 3.1 Protocol 4 Restriction Digest (3A as well as Standard assembly) 4.1 Protocol 4.2 Variant 5 Ligation (3A Assembly) 5.1 Protocol 5.2 Variant 6 Ligation (Standard Assembly) 6.1 Protocol 6.2 Variant 7 PCR with Deep Vent Polymerase 7.1 Protocol 8 DpnI Digestion 8.1 Protocol 9 PCR with Phusion® Polymerase 9.1 Protocol

Ultracompetent Cell Preparation

Protocol

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 37C.
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

Variant

• Caution: The whole procedure after the cells are pelleted out needs to be carried out in ice.
• Caution: TB buffer is heat sensitive, never take it out of ice.

 

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Transformation

Protocol

1. 100µl competent cells were thawed on ice
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 42C for 2min.
5. Incubated on ice for 3-5 min.
6. 800 µl of 2xYT broth added.
7. Flasks were shaken at 37C for 1hr.
8. They were centrifuged at 3000rpm for 5min and the pellet was resuspended into 100ul of the supernatant.
9. The 100 µl of the transformation mix was plated on 2xYT agar plates.
10. Plates were incubated at 37C overnight.

Variant

1. The amount of cells used can be varied greatly from 30ul to100ul.
• Caution: The amount of DNA added should not exceed 10% of the total volume (it generally doesn’t work, don’t flood the cells with DNA)
2. The heat shock step greatly varies from one lab to another (anywhere from 30s to 2 mins). For us 2 mins worked fine.
3. The cooling step after heat shock can also vary from 2-5 mins.

• Tip: Spinning the cells down reduces the chances of losing transformed cells.

 

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Miniprep

Protocol

1. Overnight cultures were harvested (2-3ml broth cultures).
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 center of the column.
16. Incubate for 1 min
17. It was centrifuged at 13000rpm for 1 min by placing it in a fresh tube.

Restriction Digest (3A as well as Standard assembly)

Protocol

1. Mix contains:
1. 5 µl NEB buffer 2.
2. 0.5 µl BSA
3. 1 µl Forward Enzyme
4. 1 µl Backward Enzyme.
5. 5 µl DNA part
6. 37.5 µl MilliQ water(DNAase free)
2. Reaction mix is incubated at 37C for 30min
3. It is then heat inactivated at 80C for 20 min.

Variant

For the digestions, enzymes from NEB were used.

1. The digestion time can be varied from 15-45 mins depending on the biobrick part. Some parts seem to require more than the others for complete digestion.
2. The amount of DNA added will alter the amount of water that needs to be added up to make the total reaction volume to 50ul. As much as 42.5ul of DNA can be digested in one reaction (the rest 7.5ul are enzymes and buffers).
3. In case of the PCR assembly procedure, only a single digest is required (instead of the regular double digest for standard and 3A assembly).

• Tip:Before proceeding to the ligation step, chopping off the phosphate ends of the loose ends with alkaline phosphatise in one of the 2 or 3 pieces in the ligation would help in preventing ligation of unneccesary fragments.
• Tip: Gel eluting the required product can increase the yield of the ligation product.
• Tip: Double digests with S,P or E,X followed by purifying the reaction mix using a PCR column can help in eliminating the tiny fragments that are released into the solution which helps reduce unwanted ligation products during the ligation step.
• Warning: Do not digest for too long, else the enzymes might show star activity.

Ligation (3A Assembly)

Protocol

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 65C for 20 min.

Variant

For the ligations, NEB T4 ligase was used

1. The incubation time can vary from 10 mins to 1 hour. The reaction went to completion in about 20 mins. There is not much difference with a 20 min ligation and an overnight 16C ligation.
2. The amount of water can be reduced to accommodate more DNA in the reaction mix.
3. Heat inactivation is required as the intact ligase tends to reduce the transformation efficiency.

Ligation (Standard Assembly)

Protocol

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 65C for 20 min.

Variant

For the ligations, NEB T4 ligase was used

1. The incubation time can vary from 10 mins to 1 hour. The reaction went to completion in about 20 mins. There is not much difference with a 20 min ligation and an overnight 16C ligation.
2. The amount of water can be reduced to accommodate more DNA in the reaction mix.
3. Heat inactivation is required as the intact ligase tends to reduce the transformation efficiency.

PCR with Deep Vent Polymerase

Protocol

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. 96C for 2 min.
2. 96C for 30sec.
3. (Tm-5)C for 30sec.
4. 72C for ‘x’min (1min per kb).
5. GOTO 2 30 times.
6. 72C for 30min.
7. 4C for storage.

DpnI Digestion

Protocol

1. 1 µl of DpnI was added to 1 tube of PCR product (<50 µl).
2. Incubated at 37C for 2hrs.
3. Heated at 80C for 15-20mins.

PCR with Phusion® Polymerase

Protocol

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. 98C for 2min.
2. 98C for 30sec.
3. (Tm+3)C for 30sec.
4. 72C for x sec(15sec per kb).
5. GOTO 2 30 times.
6. 72C for 30 min.
7. 4C for storage.

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