We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (94°C).
| COMPONENT | 25μl REACTION |
50μl REACTION |
FINAL CONCENTRATION |
| 5X OneTaq Quick-Load Buffer* | 5 μl | 10 μl | 1X |
| 10 mM dNTPs | 0.5μl | 1μl | 200μM |
| 10 μM Forward Primer | 0.5μl | 1μl | 0.2μM (0.05-1μM) |
| 10 μM Reverse Primer | 0.5μl | 1μl | 0.2μM (0.05-1μM) |
| OneTaqQuick-Load DNA Polymerase** | 0.125 μl | 0.25μl | 1.25 units/50 μl PCR |
| Template DNA | variable | variable | <1,000 ng |
| Nuclease-Free Water | to 25 μl | to 50μl |
*OneTaq Standard Reaction Buffer can also be used
**For amplicons between 3–6 kb, use 2.5–5 units/50 µl rxn
Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
Transfer PCR tubes from ice to a PCR machine with the block preheated to 94°C and begin thermocycling:
| STEP | TEMP | TIME |
| Initial Denaturation | 94°C | 30 seconds |
| 30 Cycles |
94°C 45-68°C 68°C |
15-30 seconds 15-60 seconds 1 minute/kb |
| Final Extension | 68°C |
5 minutes |
| Hold | 4-10°C |
