Optimization of experiment conditions with ISSRPCRreaction system in Anemone obtusiloba

The genome DNA extracted by the improved method of CTAB from Anemone obtusiloba collected from Hezuo region in Gansu was used as mode to optimize the ISSR reaction conditions by orthogonal experiment and single factor experiment. This study showed that the optimal conditions of ISSR reaction for A. obtusiloba were 20 L reaction system, and it contained 10 Buffer 2 L, Mg2+ 2.5 mmolL-1, Taq enzyme 2 U, Genome DNA 2 mgL-1, dNTPs 0.3 mmolL-1 and ISSR primer 0.4 mmolL-1. The process of PCR were 94 ℃ for 5 min, 35 cycles (94 ℃ 30 s，51 ℃ 45 s，72 ℃ 90 s), and 72 ℃ for 7 min, and the consult based on primer UBC 807.