Team:University of Ottawa/Polymerase Chain Reaction

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Polymerase Chain Reaction (PCR)

This protocol serves as a guideline for setting-up a Polymerase Chain Reaction (PCR) for the amplification and manipulation of specific DNA templates. Optimization can be achieved by varying the composition and concentration of the reaction components, as well as altering the temperature and duration of the different thermal cycling steps. Consult the product information sheet specific to the polymerase employed. For an overview of the theoretical basis and the various applications of PCR consult: Short Protocols in Molecular Biology, Chapter 15 and Molecular Cloning: A Laboratory Manual, Chapter 8. Successful production of the desired DNA fragment by PCR is sensitive to contamination. Ensure that the work area is clean and use pipette tips and tubes reserved for PCR. To prevent cross-contamination of reagents, “double-dip” pipetting should be avoided.

Here are two examples that have resulted in successful amplification of yeast genomic DNA and plasmid DNA achieved using Taq polymerase (NEB) and Phusion High Fidelity Polymerase (NEB).


Taq polymerase

1. On ice, mix the following PCR reaction components with the polymerase added last. For convenience, a master

mix can be made.


Reaction components1x Vol (μl)
10X Reaction buffer2.5
10mM each dNTP0.5
Forward primer (10pmol/μl)1.25
Reverse primer (10pmol/μl)1.25
DNA template2
Taq polymerase0.5
Filter sterile ddH2O17
Total25



2. Place tubes in the Thermal cycler and execute the following program:

  1. 94°C for 5 min
  2. 94°C for 1 min
  3. 50-60°C for 45 s
  4. 72°C for 1 min / 1kb
  5. Go to step 2, repeat 29 times
  6. 72°C for 10 min
  7. 4°C hold

Phusion High Fidelity Polymerase

1. On ice, mix the following PCR reaction components with the polymerase added last. For convenience, a master mix can be made.


Reaction components1x Vol (μl)
5X Reaction buffer10
10mM each dNTP1
Forward primer (10pmol/μl)2.5
Reverse primer (10pmol/μl)2.5
DNA template4
Phusion polymerase0.5
Filter sterile ddH2O29.5
Total50



2. Place tubes in the Thermal cycler and execute the following program:

  1. 98°C for 30 s
  2. 98°C for 10 s
  3. 50-60°C for 20 s
  4. 72°C for 30 s / 1kb
  5. Go to step 2, repeat 29 times
  6. 72°C for 10 min
  7. 4°C hold


Note: Typically, 20 ng and 100 ng of plasmid or genomic DNA respectively has been used as template.