Team:IIT Madras/Protocols

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PCR Protocol

Composition of PCR reaction:

PHusion Phusion® High-Fidelity DNA Polymerase(from Finnzymes) - 0.2 mul
5x Phusion HF Buffer (from Finnzymes) - 4 mul
dNTP – 1 mul
DNA Template – 1 mul
Primers(from Bioserve) – 1 mul each.
Water – 12.8 mul

Total volume for the reaction is 20 mul.

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.

Digestion Protocol

Composition of the digestion mixture:

DNA - 4 mul
Enzyme 1 - 1.2 mul
Enzyme 2 - 1.2 mul
Buffer - 2 mul
Water - 11.6 mul

Total volume for the reaction is 20 mul.

Steps:

For EcorRI, PstI – The reaction mixture is kept at 37 degrees for 1.5 hours. For other Enzymes – The reaction mixture is kept at 37 degrees for 5 hours.
Inactivate enzyme by heating at 80 degrees for 20 mins.

T4 Ligation Protocol

Composition of Ligation mixture:

T4 Ligase Buffer - 1mul
6:1 molar insert:vector (vector~10ng)
miliQ water - (8.5 - DNA volume) mul
T4 ligase – 0.5 mul

Steps:

Leave reaction at 22.5degC for 30min
Denaturate ligase at 65degC for 10min
Store at -20degC

Ultracompetent Cell Preperation 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 LB 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

CAUTION!

   * 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.

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 LB 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 LB agar plates.
 10. Plates were incubated at 37C overnight.

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.

PCR Protocol with Taq Polymerase

  1. Mix contains:
        1. 0.4 µl Taq polymerase.
        2. 2 µl Bffer (10X).
        3. 0.8 µl dNTPs.
        4. 0.4 µl forward Primer.
        5. 0.4 µl backward Primer.
        6. 0.8 µl Template.
        7. 15.2 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.