PCR - Polymerase Chain Reaction

Materials:
  1. Template DNA, 1-10ng/ul
  2. Primers, dissolve primers with sterile T108 to 20 pmol/ul and keep aliquot at -80oC
  3. 10X buffer containing MgCl2, pH7.8 (Usually come with Taq polymerase. If the concentration is not 10X, adjust the amount of buffer and H2O in the table below)
  4. Taq polymerase
  5. 2mM dNTPs (2mM of each dNTP (dATP, dCTP, dGTP, and dTTP)
  6. Sterile MilliQ H20
  7. PCR tubes (250ul)
  8. Pipettes
  9. Aerosol or replacement tips
  10. gloves
  11. Ice
  12. PCR machine (Oil-free)
Procedure
  1. Set up reactions on ice as following:
 Total 50 ml
H2O (ul)
10X Buffer
(ul)
2mM dNTP (ul)
5' Primer (ul)
3' Primer (ul)
Template DNA (ul)
Taq polymerase (ul)
Reaction
28.5
5
5
5
5
1
0.5
control
29.5
5
5
5
5
0
0.5
  1. Set up PCR program on PCR machine:
    2. Step 1 Pre-Denaturation 95oC for 5min
    Step 2 Denaturation 95oC for 5min
    Step 3 Annealing 2oC below primers' annealing temperature,  1 min
    Step 4 Synthesis 72oC for 2 min (Note that polymerase from some suppliers have a different optimum temperature)
    Step 5 Go to step 2 for 20 - 40 cycles.
    Step 6 Hold at 72oC for 15 min
    Step 7 Hold at 4oC until taking out

     

    Notes and tools:

    • As indicated in Molecular Biology Techniques Manual, the following rules should be considered(1-7):
      1. Primers should be 17-28 bases in length;
      2. Base composition should be 50-60% (G+C);
      3. Primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming;
      4. Tms between 55-80oC are preferred;
      5. Runs of three or more Cs or Gs at the 3'-ends of primers may promote mispriming at G or C-rich sequences (because of stability of annealing), and should be avoided;
      6. 3'-ends of primers should not be complementary (ie. base pair), as otherwise primer dimers will be synthesised preferentially to any other product;
      7. primer self-complementarity (ability to form 2o structures such as hairpins) should be avoided.
      8. Design the Tm of 5' and 3' primers as close as possible.
    • The selection of DNA polymerase:
      1. To PCR something you want to use for cloning, use proof-reading DNA polymerase like Pfu from Stratagene or Tgo from Roche. To PCR something you don’t care so much about the fidelity, you can use Taq Polymerase. Taq polymerase is faster and cheaper, but is 10 times LESS accurate than Pfu polymerase. But Pfu from Stratagene has a strict requirement on salt concentration. When doing RT-PCR, you have to purify the RT solution before PCR. We use Tgo DNA polymerase which has high fidelity and easy to handle and this enzyme prefers lower anealing temperature. Invitrogen also has different kind of DNA polymerase. But remember, some DNA polymerase from Invitrogen the optimam enlongation temperature is at 68oC instead of commonly used at 72oC.
    • Primer design tool/softwares:
      1. NCBI (sequence search, BLAST)
      2. Vector NTI
      3. Primer Premier
      4. Jellyfish
      5. Visual Cloning
      6. or many online programs

     

     
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