齿裂菌属和皮下盘菌属ISSR-PCR最佳反应条件的研究

在利用ISSR技术分析齿裂菌属和皮下盘菌属遗传多样性的研究中,为获得条带清晰、重复性好的ISSR扩增结果,对影响ISSR-PCR的条件进行了筛选,确定了此类菌物ISSR-PCR反应的最适宜条件:在15μLPCR反应体系中,10倍Taq酶缓冲液1.5μL,DNA模板8ng/μL,MgCl22.5mmol/L,dNTP0.15mmol/L,引物浓度0.4μmol/L,Taq酶1.00U,ddH2O9.0μL。最佳退火温度因不同的引物而定,最佳循环次数为35次。

Optimization of ISSR-PCR Reaction Conditions for Coccomyces and Hypoderma

The factors which affect the ISSR analysis in the study of the genetic diversity of Coccomyces De Not.and Hypoderma De Not.,such as concentrations of template DNA,Taq DNA polymerase,Mg2+,primer and dNTP,annealing temperature,cycling times,etc.were filtrated.The optimal ISSR-PCR conditions in the experiments were as the following:10×Taq buffer 1.5 μL,template DNA 8 ng/μL,MgCl2 2.5 mmol/L,dNTP 0.15 mmol/L,primer concentration 0.4 mmol/L,Taq DNA polymerase 1.00 U,and ddH2O 9.0 μL in total 15 μL reaction volumes.Optimal annealing temperature is decided by different primers.Optimal circular time is thirty-five.The amplification system can give clear and reproducible banding patterns.