马铃薯SSR-PCR反应体系的建立和优化

摘要

应用L16(45)正交设计对影响马铃薯SSR-PCR的主要参数进行优化,建立适于马铃薯SSR-PCR反应的最佳体系.结果表明:各因素不同水平浓度对PCR反应结果均有显著影响.马铃薯SSR-PCR优化反应体系为:10×PCR Buffer 1.0μl、模板DNA约30 ng、dNTP 200 mol/L、SSR引物66 ng、Taq DNA聚合酶0.25 U、Mg2+ 2.25 mmol/L,加ddH2O至终体积10.0 μl.PCR扩增程序如下:94℃预变性5 min;94 ℃变性1 min,53℃复性1 min,72℃延伸1.5 min,共35个循环;72℃延伸10 min,然后4℃保存.%Used L16 (45) orthogonal designs for optimizing the main factors of the SSR-PCR reaction system of potato and establishing the optimum SSR reaction system finally. The result showed that there were significant effects on the results of SSR-PCR at different concentration of each factor,and the optimized SSR-PCR reaction system was: 10 × PCR Buffer 1.0μl,DNA template 30 ng, dNTP 200μmol/L,SSR primer 66 ng,Taq DNA polymerase 0.25 U, Mg2+ 2.25 mmol/L. , adding ddH2O to terminal volume 10.0μμl. The PCR amplification procedure was: predenaturation for 5 min at 94 ℃, followed by 35 cycles of denaturation for 1 min at 94 ℃, anneal for 1 min at 53 ℃ ,extension for 1.5 min at 72 ℃ ,the amplification was completed after extension for 10 min at 72 ℃, then stored at 4 ℃.