Pfama-1 Gene Amplification Protocol

All the PCR protocols and primers were used as previously described with minor modifications [41 (link)]. Primary PCR for Pfama-1 was performed using 1 µl of genomic DNA and 1 µM of forward and reverse primers in a 10 µl reaction volume containing 5 µl of 2X Promega master mix. Secondary PCR was performed using 2 µl of primary PCR product as template with 1 µM of each primer and 25 µl of 2X Promega master mix in 50 µl reaction volumes. The PCR amplification conditions were: initial denaturation at 95 °C for 5 min followed by (30 cycles for primary PCR and 35 cycles for secondary PCR) denaturation at 95 °C for 2 min, annealing at 52 °C for 30 s, extension at 68 °C for 45 s and a final extension of 5 min at 68 °C. The PCR product of Pfama-1 was analysed on 2% agarose gel and expected positive amplicon size was 500 bp.