PCR AND RT-PCR

PCR amplification of 185/333 sequences was carried out in 50 μl reactions using the Advantage 2 PCR system (Clontech) or Phusion DNA polymerase (Finnzymes). Each reaction consisted of 0.2 μM dNTPs, 0.4 M of each primer (Table I) and 1 l of RT-PCR reaction or gDNA as template.

Cycling parameters:

Advantage 2,

  • An initial denaturation at 94C for 2 min.
  • 35 cycles of denaturation at 94C for 30 s, annealing at 60C for 30 s and extension at 72C for 3 min.
  • Final extension at 72C for 5 min.

Phusion DNA polymerase

  • An initial denaturation at 98C for 1.5 min,
  • 35 cycles of denaturation at 98C for 15 s, annealing at 60C for 20 s and extension at 72C for 20-40 s, followed by a
  • Final extension at 72C for 3 min.


Sham Nair 2014