茶藨子总DNA提取及ITS-PCR体系的优化

Extraction of Genomic DNA from Ribes and Establishment of ITS-PCR Reaction Systems

利用Tiangen DP305试剂盒对茶藨子(Ribes L.)总DNA进行提取,应用单因子试验分析了DNA模板、dNTPs、引物和Taq酶对ITS-PCR扩增结果的影响,并建立了茶藨子ITS-PCR扩增反应的优化体系,最优反应体系为:25μL体系中,10×PCR buffer 1μL、Mg2+0.4 mmol/L、引物浓度0.5μmol/L、dNTP浓度为1 mmol/L、Taq酶的用量1.0 U、DNA模板用量为50 ng。优化后的反应体系可作为茶藨子属植物ITS-PCR的基本反应体系,为进一步开展茶藨子属的分类和系统发育研究奠定基础。

Single factor optimization was carried out in order to study the effects of concentrations of template DNA,dNTPs,primer and Taq polymerase activity on ITS-PCR amplification.The total DNA was extracted from ribes by Tiangen DP305.The optimal conditions for ITS-PCR on ribes were obtained as 10×PCR buffer 1 μL,Mg2＋ concentration 0.4 mmol/L,primer 0.5 μmol/L,dNTP 1 mmol/L,Taq DNA polymerase 1.0 U,and total DNA 50 ng in a 25 μL reaction system.The optimized reaction system can be used as the ITS-PCR basic reaction system for ribes plants.