PCR reagent final concentrations (50 uL total per reaction):

10X ThermoPol Reaction Buffer - 1X (in this case we put 5 uL in to the reaction)

dNTPs - 0.75 mM 

F primer - 2 uM 

R primer - 2 uM 

template (pAY196) - ~4 ng/uL 

additional MgSO4 - 2 mM (1 uL from 100 mM stock, which typically comes with Vent Pol)

Vent DNA Polymerase (NEB, cat # M0254S) - 2 units 

H20 - mark up to 50 uL

 

25 cycles of:

initial denaturation - yes

denaturing temp - 94 °C - 30 seconds

annealing temp - 55 °C - 30 seconds

extension temp - 72 °C - 3 minutes

final extension - yes

 

check that PCR worked on a 0.8% agarose gel with 1 kb DNA ladder for reference. look for band ~2862 bp in length.