利用正交设计建立洋桔梗RAPD最佳反应体系

Establishment of Eustoma grandiflorum Optimal RAPD Reaction System Using Orthogonal Design

利用正交设计法对影响洋桔梗RAPD反应的Mg2+、dNTPs、Taq DNA聚合酶、引物和模板等主要因素的作用进行了比较分析,建立了洋桔梗RAPD的最佳反应体系:即在25μL反应体积中,10×PCR buffer为2.5μL,Mg2+浓度为2.0 mmol/L,dNTPs浓度为0.4 mmol/L,引物浓度为0.2μmol/L,模板浓度为50 ng/μL,Taq DNA聚合酶用量为1.0 U;在此基础上,比较了不同退火温度对RAPD扩增产物的影响,退火温度经筛选选定为41℃;最后再将该反应体系应用于不同引物,结果表明该优化结果具有较好的稳定性和通用性。

The essential factors of the RAPD reaction system for Eustorna grandi.filorurn, such as： Mg^2＋ ,dNTPs, Taq DNA polymerase, primer and DNA template were optimized by using the method of orthogonal design. The optimal RAPD reaction system was set as follows： 10×PCR buffer 2. 5 μL, Mg^2＋ 2. 0 mmol/L, dNTPs 0. 4 mmol/L, random primers 0. 2 μmol/L, DNA template 50 ng/μL and Taq DNA polymerase 1.0 U, in a total 25 μL reaction volume. Then the effects of different annealing temperatures to the amplified products of RAPD were also investigated. The annealing temperature was selected as 41℃. Finally the optimized system was successfully applied to different random primers, indicating that the optimal system was stable and adapted.