麻疯树ISSR-PCR反应体系的建立和优化

Optimization of ISSR-PCR reaction system for Jatropha curcas

以2年生的麻疯树嫩叶提取的DNA作为模板,固定ISSR-PCR扩增程序,对影响PCR扩增的模板DNA、dNTP、引物、TaqDNA聚合酶以及Mg2+浓度等5因素(不同水平)进行单因素设计优化,得到适合于麻疯树ISSR-PCR扩增的体系为:25μL反应体系中,含模板DNA300 ng、dNTP 0.15 mmol/L、引物0.20μmol/L、TaqDNA聚合酶1.0 U及Mg2+3.0 mmol/L。

Through using new leaf from two years old of Jatropha curcas as templat DNA,fixing ISSR-PCR amplification procedure,optimizing one design factor which influence five fators,such as template DNA,dNTP,TaqDNA polymerase,primer and Mg^2＋ concentration（different levels）,we could obtain PCR amplification reaction system which was fit for Jatropha curcas： PCR was performed in a 25 μL reaction system mixture with 1 U Taq polymerase,0.15 mmol/L of each dNTP,3 mmol/L Mg^2＋,300 ng DNA,and 0.2 mmol/L random primer.