瓠瓜RAPD-PCR反应体系的优化

Optimization of RAPD-PCR system of bottle gourd

采用正交试验设计,研究瓠瓜RAPD-PCR反应体系中5个反应组分的不同浓度对PCR扩增结果的影响,以期建立瓠瓜RAPD-PCR的最优反应体系。结果表明,在25μL的最优反应体系中各组分适合浓度为:1×PCR buffer,2.5mmol/L MgCl2,100μmol/L dNTP,0.2μmol/L引物,1U Taq DNA聚合酶,30ng模板DNA。通过梯度PCR测验,37℃为最适的退火温度。

The effects of the 5 factors and their different levels of RAPD-PCR system on the PCR amplification of bottle gourd were studied through an orthogonal experiment in order to establish an optimal RAPD-PCR system of bottle gourd. The results showed that it was appropriate for a 25 μL reaction system to contain 1 × PCR buffer, 2.5 mmol/L MgC12,100 μmol/L dNTP, 0.2 μmol/L primer, 1 U Taq DNA polymerase and 30 ng template DNA. The gradient PCR test indicated that the optimal annealing temperature was 37 ℃.