Protocol for a Routine Vent 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 Vent DNA Polymerase Guidelines for PCR Optimization Protocol).
ThermoPol Reaction Buffer (NEB# B9004)
- Prepare the following 50 μl reaction in a 0.5 ml PCR tube on ice:
COMPONENT VOLUME (μl)
ThermoPol Reaction Buffer (10X)
5 μl 1X Deoxynucleotide (dNTP) Solution Mix (10 mM) 1 μl 200 μM
Upstream Primer (10 μM stock) 0.5-2.5 μL 0.1-0.5 μM
Downstream Primer (10 μM stock) 0.5-2.5 μl
DNA Template determined by user Vent DNA Polymerase* 0.25-0.5 μl 0.5-1 unit
MgSO4 (optional) 1-6 mM
Bring reaction to a final volume of 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:
STEP TEMP TIME Initial Denaturation
2-5 minutes 20-30 Cycles
1 minute per kb
72°C 5 minutes Hold 4-10°C