Abstract:

The conditions of SRAP-PCR reaction were optimized using the method of single-factor experiment respectively. The optimal ISSR-PCR amplification was established as follows: Each 20-μL PCR mixture consisted of 1.0 U Taq DNA polymerase (TaKaRa Biotechnology), 6X PCR buffer, 0.6 mM dNTP, 0.35 μM primer, 1.5 mM Mg2+, and 25-200 ng template DNA. Thermal cycling (Biometra T1 Thermocycle) started with 5 min at 94°C for initial denaturing, and 5 cycles of 30 s at 94°C, 30 s at 35°C, and 45 s at 72°C, followed by 40 cycles of 30 s at 94°C, 30 s at 4°C and 45 s at 72°C. The last cycle was followed by a 7-min extension at 72°C. Amplified products were analyzed on 2% (w/v) gels and visualized with the Vilber Gel Documentation System.