Sybr green 1 master

Real-time PCR amplification was performed using Bio-Rad SYBR Green I Master according to the manufacturer’s instructions; 500 nM primers and a variable amount of DNA standard were used in a 20 μl final reaction volume. Amplification was performed in a programmable thermal cycler (Bio-Rad 480, USA) under the following conditions: after 10 min of denaturation at 95 ^oC, 40 amplification cycles (95°C for 5 s; 60°C for 5 s; 72°C for 20 s) were performed with a single fluorescence reading taken at the end of each cycle. For the long amplicon, the elongation was at 72°C for 90 s. Each reaction combination was performed with three replicates, and all the runs were completed with a subsequent melt curve analysis. The PCR product was resolved on ethidium-bromide-stained 1% agarose gel to confirm the specificity of bands and absence of primer dimers.