11.6.3. Bisulfite PCR

Bisulfite amplicons are amplified using a nested PCR protocol (Wang et al., 2006; Foret et al., 2009). Nested primers contained an additional 9 nucleotide-long linkers with EcoRI or HindIII recognition sequences allowing directional cloning of the amplicons.

PCR reactions are performed in 25 ml volume containing:
   1x PCR buffer, 
   mM MgCl2,
   mM dNTP,
   50 pmol each forward and reverse primer,
   5 units Taq polymerase.

Reaction efficiencies are optimized via annealing temperature gradients (Mastercycler gradient PCR machine, Eppendorf) and testing multiple Taq polymerases such as GoTaq (Promega) or FastStart Taq (Roche).

Cycling profile is as follows:
    Initial denaturation at 940C for 2 min,
    Followed by 40 cycles of:
       15 sec denaturation at 940C,
       15 sec annealing at primer-specific optimal temperature,
       60 sec extension at 720C,
    A final extension cycle at 720C for 5 min.

When using FastStart Taq polymerase, the denaturation temperature was increased to 950C, initial denaturation time to 5 min and cycling denaturation and annealing times to 30 sec.