Invitrogen platinum 2 hot start green pcr master mix 2x

The detection and genotyping of high-risk HPVs (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, and 58) and EBV were performed as previously described [22 (link)]. In addition, PCR was performed for MMTV-like virus (forward primer: 5′-GATGGTATGAAGCAGGATGG-3′ and reverse primer: 5′-CCTCTTTTCTCTATATCTATTAGCTGAGGTAATC-3′). For all reactions, GAPDH was used as an internal control. Analyses were completed as illustrated earlier [22 (link)]. The Invitrogen Platinum II Hot-Start Green PCR Master Mix (2X) (Thermo Fisher Scientific, Waltham, MA, USA) was used to perform PCR. Briefly, the MMTV-like virus gene was amplified for an initial denaturation at 94°C for 2 min, followed by 40 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s. The samples were finally incubated for 10 min at 72°C for a final extension. HPV and EBV genes were amplified as described previously [22 (link)]. The PCR product was resolved using 1.5% agarose gel electrophoresis. For each experiment performed, respective positive and negative controls were used; positive control for HPV included normal oral epithelial cell line expressing E6/E7, for EBV diluted DNA plasmids for EBV nuclear antigen 1 and LMP1, while for MMTV-like virus, we used MDA-MB-231 as the positive control [35 (link)]. Nuclease-free water instead of DNA was used for the negative control [15 (link)].