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.

2. Place tubes in the Thermal cycler and execute the following program: <ol> <li> 94&deg;C for 5 min <li> 94&deg;C for 1 min <li> 50-60&deg;C for 45 s <li> 72&deg;C for 1 min / 1kb <li> Go to step 2, repeat 29 times <li> 72&deg;C for 10 min <li> 4&deg;C hold </ol>

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.

2. Place tubes in the Thermal cycler and execute the following program: <ol> <li> 98&deg;C for 30 s <li> 98&deg;C for 10 s <li> 50-60&deg;C for 20 s <li> 72&deg;C for 30 s / 1kb <li> Go to step 2, repeat 29 times <li> 72&deg;C for 10 min <li> 4&deg;C hold </ol>

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