Standardization of annealing temperature for
Single-variant specific PCRs
A gradient PCR (SimpliAmp Thermal Cycler, Applied Biosystems, USA) was
performed for each of the variant separately with freshly prepared cDNA
template to standardize the annealing temperature. Couple of distinct
tubes was prepared for each of the variant using the respective primer
pairs to differentiate between the wild type and the mutant. The PCR was
carried out in 10µl reaction volume containing 3-5 ng/µl DNA, 5µl master
mixture (GoTaq® G2 Green Master; Promega, USA), 0.2µM of each forward
and reverse primer and 2.8 µl nuclease free water. The thermocycling
conditions were as follows: initial denaturation at 95°C for 1 min
followed by 30 cycles at 95°C for 30s, annealing at a range of 55-65°C
for 30s and 72°C for 30s followed by a final extension at 72°C for 5min.
The PCR products were electrophoresed on a 1% (w/v) agarose gel stained
with ethidium bromide (UltraPure™ Ethidium Bromide, 10 mg/mL; Thermo
Fisher, USA) and visualized using a gel documentation system (Bio-Rad,
USA).