Pcr cfx96

Analysis using real-time PCR was carried out with instrument of PCR CFX96 (Bio-Rad, USA) using total volume of 10 µL, which consisted of consisting of 5 µL SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA), 0.5 µL forward primer of pig 5 μM, 0.5 µL reverse primer of pig 5 μM, 0.5 µL forward primer of chicken 5 μM, 0.5 µL reverse primer of chicken 5 μM, 0.5 µL mixed DNA pork-chicken (145.65 ng/µL), and 2.5 µL nuclease free water. The thermocycler of PCR was programmed as follows: pre-denaturation at 95°C for 30 s, followed by 40 cycles of denaturation at 95°C for 15 s, annealing temperature was optimized at 50-60 o C for 30 s based on Tm of primer, and elongation at 72°C for 10 s.

Real-time PCR analysis was performed using PCR CFX96 (Biorad, USA) with total volume of 20 µL consisting of 1× THUNDERBIRD® Probe qPCR Mix (Toyobo Co, USA), 1 µL forward primer and 1 µL reverse primer with final concentration of 500 nM, 1 µL DNA template (50 µg/mL), and nuclease-free water until 20 µL. The amplification program was as follows: pre-denaturation at 95°Cfor 3 min, followed by 40 cycles of denaturation at 95°C for 15 s, annealing temperature at 50-62°C for 15 sec, and extension at 72°C for 45 sec. Each sample, positive control, and template control was run in triplicates.