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
 

2. Cover the reaction with 2 drops of mineral oil, add a drop of oil to the heat-block well to ensure good contact between the heat-block and the tube, and place the tube in the wells of a Perkin-Elmer Cetus DNA Thermal Cycler which has been pre-heated to 95degC (Soak file #19).

3. Abort the soak file program and begin thermal-cycle program #43 for the amplification. This program has 25 cycles of a three temperature program and is linked to a 4degC soak file, which will hold indefinitely:

95degC for 1 minute
55degC for 1 minute
72degC for 2 minutes
4. Analyze a 10 ul aliquot on a 1-2% agarose gel.