Protocol for a Routine Vent (exo-) PCR
All components should be mixed and spun down prior to pipetting. These recommendations serve as a starting point; in order to maximize amplification the reaction conditions may require optimization (see Guidelines for PCR Optimization for Vent DNA Polymerase).
- Prepare the following 50 µl reaction in a 0.5 ml PCR tube on ice:
COMPONENT VOLUME (μl)
ThermoPol Reaction Buffer (10X)
Deoxynucleotide (dNTP) Solution Mix (10 mM)
Upstream Primer (10 μM stock)
Downstream Primer (10 μM stock)
determined by user
Vent (exo-) DNA Polymerase*
Bring reaction to a final volume of 50 μl
* Due to the difficulties in pipetting small volumes of enzyme, Vent (exo-) DNA Polymerase can be diluted in Diluent D (NEB #B8004S) or 1X reaction buffer. For example, 1 µl of Vent (exo-) DNA Polymerase is mixed with 4 µl of diluent and 1 µl of that mixture is added to the reaction. Enzyme diluted in Diluent D can be stored at -20°C for future use.
- 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 reaction:
STEP TEMP TIME Initial Denaturation
2-5 minutes 20-30 Cycles
1 minute per kb
72°C 5 minutes Hold 4-10°C