General PCR methods

Polymerase Chain Reaction (PCR)

(adapted from Bruce A. Roe, Department of Chemistry and Biochemistry, The University of Oklahoma, Norman, Oklahoma 73019 broe@ou.edu)

The amplification of DNA fragments using the polymerase chain reaction (33) is performed in either the Perkin-Elmer Cetus DNA Thermal Cycler or the Perkin-Elmer Cetus Cycler 9600, by adding the following reagents to either a 0.2 ml thin-walled tube or a 1.5 ml tube, respectively: a small amount of the template DNA molecule (typically cosmid, plasmid, or genomic DNA ), the two primers flanking the region to be amplified, nucleotides, buffer, and Taq DNA polymerase. The cycling protocol consisted of 25-30 cycles of three-temperatures: strand denaturation at 95degC, primer annealing at 55degC, and primer extension at 72deg C, typically 30 seconds, 30 seconds, and 60 seconds for the DNA Thermal Cycler and 4 seconds, 10 seconds, and 60 seconds for the Thermal Cycler 9600, respectively. For reactions performed in the DNA Thermal Cycler, the reaction mixtures are overlaid with two drops of mineral oil prior to temperature cycling to eliminate liquid evaporation and condensation. This is not necessary for the Thermal Cycler 9600, which is equipped with a heated lid, maintained at 100degC, that closely contacted the sample tube caps and eliminated liquid evaporation and condensation. After PCR, aliquots of the mixture typically are loaded onto an agarose gel and electrophoresed to detect amplified product. In some instances where the yield from a single PCR is insufficient, the reaction is ethanol precipitated, resuspended, and an aliquot is used as template for a second round of PCR amplification.

Protocol

1. Add the following reagents to a 0.5 ml flat-topped microcentrifuge tube :

1 ul target DNA (10-20 ng)

2.5 ul each primer (40 uM )

1 ul AmpliTaq DNA Polymerase (5 U)

10 ul 2 mM dNTPs (2 mM each dNTP)

10 ul 10X PCR buffer

73 ul ddH20

100 ul

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