西瓜ISSR-PCR体系的正交优化研究

Optimization of ISSR-PCR System in Watermelon

采用正交试验设计的方法,建立了西瓜ISSR分析的优化反应体系,即20μL反应体系中含有1×buffer,dNTP200μmol/L、Primer1.0μmol/L、MgCl21.5mmol/L、Taq酶1U和模板DNA40ng。适宜的扩增程序为94℃5min,94℃30s,53℃45s,72℃90s,35个循环;72℃5min,4℃保存。

The optimal ISSR-PCR system in watermelon was established with orthogonal design. In a total volume of 20 μL ISSR-PCR system,it contains 1×buffer,200 μmol/L dNTP,1.0 μmol/L Primer,1.5 mmol/L MgCl2,1 U Taq DNA polymerase and 40 ng template DNA. The suitable PCR procedure is one cycle denaturing at 94 ℃ for 5 min;35 cycles each involved denaturing at 94 ℃ for 30 s,annealing at 53 ℃ for 45 s, extending at 72 ℃ for 90 s,one cycle extending at 72 ℃ for 5 min,and then remaining at 4 ℃?