Optimization of ISSR-PCR Reaction System for Paphiopedilum

The genomic DNA of Paphiopedilum was extracted with improved CTAB method,consequently,the effects of Mg2+,dNTP,and primer concentrations,as well as the usage of Taq DNA polymerase on ISSR-PCR amplification were optimized using an orthogonal experimental design.The optimal anneal temperature of primer were determined by gradient PCR.The 25 μL ISSR-PCR cocktail contained 2.5 uL 10×PCR buffer,1.5 mmol/L Mg2+,0.15 mmol/L dNTP,0.6 umol/L primer,1.0 U Taq polymerase and 40 ng template DNA gave the best amplification result.PCR procedures were described as follows: 94℃ for 5 min,followed by 40 cycles of denaturing at 94℃ for 30s,annealing at 56.2℃ for 45s,and extending at 72℃ for 1 min,finally an extension at 72℃ for 7 min.