藜ISSR-PCR反应体系的优化

Optimization of ISSR-PCR system for chenopodium album L

采用TaKaRa的PCR反应系统,以从藜(Chenopodium album L)幼叶中提取纯化后的总DNA为模板,进行UBC 859号引物的ISSR-PCR扩增实验,分别测试了镁离子、模板DNA和Taq DNA聚合酶含量对反应结果的影响,通过各因子的浓度梯度组合实验,确定了在25 mL体积的PCR反应中:酶配套缓冲液2.5μL,dNTPs0.2μmmol/L,引物0.2 mmol/L,Mg2+1.5 mmol/L,模板含量为40 ng,Taq DNA聚合酶为1.5 U是藜最佳的ISSR-PCR反应体系。

In this study,the factors which affect the ISSR-PCR reaction were detected by TaKaRa PCR reaction system of Chenopodium album L.The purified template DNA was extracted from fresh young leaves,the effect of content of Mg2＋,templates DNA and Taq DNA polymerase were tested.According to the results of amplifications in gradient it was determined that the optimization ISSR-PCR reaction system in rolumes of 25 mL contained 2.5 μL to XPCR buffer,0.2 mmol/L mixture of dNTPs,0.2 mmol/L primer,1.% mmol/L Mg2＋,40 ng of DNA and 1.5 unit Taq DNA Polymerase.