[Objective] The aim was to obtain the optimum RAPD-PCR reaction system for Glycyrrhiza uralensis. [ Method] Orthogonal design was adopted to screen the suitable concentration of major factors (dNTPs, primer, Taq polymerase, DNA template) in PCR reaction system. [ Result] The optimal protocol was accomplished by orthogonal design in 25 μl reaction volume containing 10 × PCR buffer solution(include MgCl2) 2.5 μl, 10 mmol/L dNTPs 2.5 μl, 50 ng DNA template 2.0 μl, 10 μmol/L primer 2. 0 μl, 5 U/μl Taq polymerase 0.4 μl; the optimum annealing temperature was 34 ℃. [ Conclusion] Orthogonal design was an effective method for the optimization of RAPD-PCR reaction system for C. Uralensis.%[目的]建立一套适合甘草分子学研究的RAPD-PCR反应体系.[方法]以甘草种质为试材,采用正交试验法设计,对影响RAPD-PCR扩增的主要因素dNTPs、引物、Taq酶和DNA模板进行优化筛选.[结果]总体积25μl的甘草RAPD-PCR最佳反应体系为:10 ×PCR缓冲液(含MgCl2 )2.5 μl,10 mmol/L dNTPs 2.5 μl,50 ng DNA 2.0μl,10 μmol/L引物2.0μl,5 U/μl Taq酶0.4μl.对引物的退火温度进行了梯度筛选,34℃时扩增效果较好.[结论]进行甘草RAPD-PCR反应体系的正交优化非常有效.
展开▼
