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[list="margin-right: 0px; margin-bottom: 1em; margin-left: 1em; padding-right: 0px; padding-left: 1em; border: 0px; color: rgb(76, 76, 76); font-family: Arial, Helvetica, sans-serif; line-height: 11.5556px; background-color: rgb(255, 255, 255);"][*]Prepare the following 50 µl reaction in a 0.5 ml PCR tube on ice:
* Due to the difficulties in pipetting small volumes of enzyme, Taq DNA Polymerase can be diluted in 1X reaction buffer. For example, 1 µl of Taq DNA Polymerase is mixed with 4 µl of 1X reaction buffer and 1 µl of that mixture is added to the reaction. Enzyme diluted 1X reaction buffer should not be stored for future use.
| Component | 25 μl reaction | 50 μl reaction | Final Concentration |
| 10X ThermoPol or Standard Taq Reaction Buffer | 2.5 µl | 5 μl | 1X |
| 10 mM dNTPs | 0.5 µl | 1 μl | 200 µM |
| 10 µM Forward Primer | 0.5 µl | 1 μl | 0.2 µM (0.05–1 µM) |
| 10 µM Reverse Primer | 0.5 µl | 1 μl | 0.2 µM (0.05–1 µM) |
| Template DNA | variable | variable | <1,000 ng |
| Taq DNA Polymerase* | 0.125 µl | 0.25 µl | 1.25 units/50 µl PCR |
| Nuclease-free water | to 25 µl | to 50 µl |
[*]Gently mix the reaction and spin down in microcentrifuge.
If the thermocycler does not have a heated cover, add one drop of mineral oil to the reaction tube to prevent evaporation.
[*]Cycling Conditions for a Routine PCR:
| CYCLE STEP | TEMP | TIME | CYCLES |
| Initial Denaturation | 95°C | 30 seconds | 1 |
| Denaturation Annealing Extension | 95°C 45-68°C 68°C | 15-30 seconds 15-60 seconds 1 minute per kb | 30 |
| Final Extension | 72°C | 5 minutes | 1 |
| Hold | 4°C | ∞ |
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