正交设计优化大白菜ISSR-PCR反应体系

以大白菜为试材,采用新型植物基因组DNA提取试剂盒提取大白菜基因组DNA,采用正交实验设计方法,对dNTPs、Mg2+、Taq DNA聚合酶、引物、及模板DNA五因素四水平进行优化,筛选并建立了适合大白菜的ISSR-PCR反应体系。结果表明:25μL的反应体系中含有1.5mmol/L Mg2+、200μmol/L dNTP、0.5UTaq DNA聚合酶、0.7μmol/L引物、30ng模板DNA。在此基础上探讨了最佳循环次数,应用该优化反应体系,用3个不同循环数对资源DNA进行ISSR-PCR扩增,结果显示优化的反应体系适宜的循环次数是30。

Optimization of an ISSR-PCR Reaction System of Chinese Cabbage by Orthogonal Design

Chinese cabbage genomic DNA,extracted from its fresh young leaves by DNA extraction kit was applied to optimize ISSR-PCR amplification system based on orthogonal design.Five factors were considered such as dNTP,Mg2+,Taq DNA polymerase,primer and template DNA at four levels.A suitable ISSR-PCR reaction system was established.The results showed that 25 μL reaction system containing 1.5 mmol/L Mg2+,200 μmol/L dNTP,0.5 U Taq DNA polymerase,0.7 μmol/L primer and 30 ng DNA template.The number of cycles was also investigated.Three different cycle numbers of were used to test the optimized reaction system.The results indicated that the appropriate cycle numbers of the optimized reaction was 30.