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采用正交设计方法,对影响铜鱼(Coreius heterodon(Bleeker))ISSR-PCR扩增结果的5个因素,如Mg2+、dNTP、引物、TaqDNA聚合酶和模板DNA的浓度,在4个水平上进行了比较优化,建立了适合铜鱼的最佳ISSR-PCR反应体系:在25μL反应体系中含有1.5 mmol/L Mg2+,0.16 mmol/L dNTP,0.2μmol/L引物,1UTaqDNA聚合酶和140 ng模板DNA.检验结果表明,采用该反应体系对铜鱼进行ISSR-PCR扩增可获得多态性高,稳定性和重复性好的电泳条带.
Orthogonal design was used to determine the concentration of five factors affecting the amplification results of ISSR-PCR of Coreius heterodon (Bleeker), such as Mg2 +, dNTPs, primers, Taq DNA polymerase and template DNA at four levels The optimal ISSR-PCR reaction system for copper fish was established, including 1.5 mmol / L Mg2 +, 0.16 mmol / L dNTP, 0.2 μmol / L primer, 1 Ua DNA polymerase and 140 ng template DNA in 25 μL reaction system The test results show that using this reaction system for ISSR-PCR amplification of copperfish, we can obtain electrophoresis bands with high polymorphism, good stability and repeatability.