Mont ge dna gel extraction kit

The previously extracted viral DNAs were used to amplify the P32 gene, PRO30 gene and GPCR gene. The 20-µl total volume of the PCR reaction contained 2 µl of each purified genomic DNA, 10 µl of a 2X HotStart Taq Plus Master Mix (Qiagen, USA) containing 1.5 mM of MgCl2, 200 µM of each dNTP and 1 unit of HotStart Taq Plus DNA polymerase, 1 µl (10 µM) of each forward and reverse primer, 2 µl of MgCl2 (25 mM) and 4 µl of nuclease-free water. The thermal cycling parameters were 95ºC for 5 minutes for enzyme activation and initial denaturation, followed by 35 cycles of 94ºC for 30 seconds; the primers were annealing for 30 seconds (Table 1) and at 72ºC for 120 seconds, and a final extension step took place at 72ºC for 10 minutes. The amplified products were electrophoresed in 1.2% agarose gel stained with ethidium bromide and documented using the ultraviolet gel documentation system (Bio-Rad Laboratories, USA). The specific PCR products were extracted from the agarose gel, purified using the Montàge DNA gel extraction kit (Millipore, USA) and sequenced in an automated ABI 3730 DNA sequencer (Applied Biosystems, USA).