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Team:University of Ottawa/Polymerase Chain Reaction
From 2008.igem.org
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 components 1x Vol (ul)
10X Reaction buffer 2.5 10mM each dNTP 0.5 Forward primer (10pmol/ul) 1.25 Reverse primer (10pmol/ul) 1.25 DNA template 2 Taq polymerase 0.5 Filter sterile ddH2O 17
Total 25
2. Place tubes in the Thermal cycler and execute the following program: 1) 94oC for 5 min 2) 94oC for 1 min 3) 50-60oC for 45 s 4) 72oC for 1 min / 1kb 5) Go to step 2, repeat 29 times 6) 72oC for 10 min 7) 4oC 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 components 1x Vol (ul)
5X Reaction buffer 10 10mM each dNTP 1 Forward primer (10pmol/ul) 2.5 Reverse primer (10pmol/ul) 2.5 DNA template 4 Phusion polymerase 0.5 Filter sterile ddH2O 29.5
Total 50
2. Place tubes in the Thermal cycler and execute the following program: 1) 98oC for 30 s 2) 98oC for 10 s 3) 50-60oC for 20 s 4) 72oC for 30 s / 1kb 5) Go to step 2, repeat 29 times 6) 72oC for 10 min 7) 4oC hold
Note: Typically, 20 ng and 100 ng of plasmid or genomic DNA respectively has been used as template.
