Abstract:

To optimize the inter simple sequence repeat(ISSR) reaction condition for Hopea chinensis genomic DNA, the concentrations of MgCl2, dNTPs, Taqpolymerase, primers and template DNA were studied with an orthogonal experimental design, and the optimal anneal temperature of primer and cycles were determined through gradient PCR. The optimal PCR system for ISSR analysis was 1×PCR buffer, 2 mmol/L MgCl2, 0.25 mmol/L dNTPs, 0.04 U/μL Taq polymerase, 0.2 μmol/L primer, 4 ng/μL template DNA in 25 μL reaction solution. And the augmentation procedure was pre-denaturation at 94℃ for 5 min, denaturation at 94℃ for 45 s, annealing at 53℃ for 45 s, extension at 72℃ for 1.5 min, reaction with 35 cycles, and extension at 72℃ for 7 min.