粗毛淫羊藿ISSR-PCR体系的正交优化

Optimization of ISSR-PCR system in Epimedium acuminatum Franch.

采用正交试验设计方法,建立了粗毛淫羊藿ISSR分析的优化反应体系:即20μL反应体系中含有10×buffer、2.0 mmol.L-1Mg2+、0.10 mmo.lL-1dNTP、1.5U Taq酶、40 ng模板DNA和0.6μmol.L-1引物;PCR反应程序为:95℃5 min;94℃30 s,52℃45 s,72℃2 min,共35个循环;72℃10 min,4℃保存。

The optimal ISSR - PCR system in Epimedium acuminatum was established using orthogonal design in the current case. A total volume of 20μL ISSR -PCR system, containing 10 × buffer, 2.0 mmol. L^-1 Mg^2＋ ,0.10 mmol. L^-1 Dntp, 1.5 U Taq polymerase, 40 ng template DNA and 0.6 μmol. L^- 1 primer was proved to better compared with tested cocktails. Further, and the suitable PCR procedure is initiated with one cycle of denaturation at 95℃ for 5 min, followed by 35cycles of denaturing at 94℃ for 30 s, annealing at 52℃ for 45 s, and extending at 72℃ for 2 min, then finally extended at 72℃ for 10 min. The amplified mixture is deposited at 4℃.