墨兰ISSR-PCR反应体系建立及优化

Establishment of ISSR-PCR Reaction System of Cymbidium Sinense and its Optimization

为建立墨兰的ISSR-PCR反应体系,采用正交试验分析了5个因素(Tag酶浓度、Mg2+浓度、模板DNA浓度、引物浓度、dNTP浓度)在4个水平上对ISSR-PCR反应的影响。结果表明:Tag酶浓度为1.6U/25μL、Mg2+浓度为2.4mmol/L、dNTP浓度0.96mmol/L、Primer浓度为0.64μmol/L、DNA浓度3.2ng/25μL是墨兰ISSR-PCR最优反应体系。

In order to establish theISSR -PCR reaction systemofISSR -PCR reactionsystem ,this article use-sorthogonalexperiment to analyze the influences of fivefactors including Tagenzyme concentration ,Mg2＋ con-centration ,DNA templateconcentration ,primer concentration ,and dNTP concentrationatfourlevelsof the or-thogonal teston theCymbidiumISSR -PCRreaction system .The results show thatwhen Tag enzyme concen-trationis 1 .6U/25μl ,M g2＋ concentration is 2 .4mmol/L ,dN T Pconcentration is 0 .96 mmol/L ,concentration of primeris 0 .64μmol/L ,DNAconcentrationis 80ng/25μl ,theISSR-PCRreaction system is optimal .