All components should be mixed and spun down prior to pipetting. These recommendations serve as a starting poin; 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)
Diluent D (NEB# B8004)
- Prepare the following 50 μl reation in a 0.5 ml PCR tube on ice:
*Due to the difficutlies in pipetting small volumes of enzyme, Vent DNA Polymerase can be diluted in Diluent D (NEB# B8004S) or 1X reaction buffer. For example, 1 μl of Vent 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.
|ThermoPol Reaction Buffer (10X)
|Deoxynucleotide (dNTP) Solution Mix (10 mM)
|Upstream Primer (10 μM stock)
|Downstream Primer (10 μM stock)
||determined by user
|Vent DNA Polymerase*
|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:
1 minute per kb