白羊草ISSR-PCR反应体系的建立与优化

Establishment and Optimization of ISSR-PCR Reaction System for Bothriochloa ischaemum

采用正交试验与单因素、双因素设计结合的方法,对白羊草ISSR-PCR反应体系的Mg2+、dNTP、模板DNA、Taq DNA聚合酶及引物5种主要因素进行优化。确立了白羊草最佳反应体系及扩增程序:25μL体系中dNTP 0.2mmol/L、Taq酶1.0U、引物0.6μmol/L、Mg2+2.5mmol/L、DNA模板30ng、10×PCR Buffer 2.5μL;扩增程序:94℃预变性5min,94℃变性45s,50～60℃(退火温度随引物不同而定)退火60s,72℃延伸90s,共35个循环,72℃后延伸5min。

An orthogonal design combined with single factor experiment and two factors design were applied to optimize ISSR-PCR amplification system for Bothriochloa ischaemum.The five main reaction system factors on ISSR-PCR were optimized,including Mg2＋,dNTP,Taq DNA polymerase,DNA template and primer.The optimal PCR system for ISSR analysis was 0.2mmol/L dNTP,1U Taq DNA polymerase,0.6μmol/L primer,2.5mmol/L Mg2＋,30ng template DNA,10×PCR buffer 2.5μL and with total 25μL reaction solution,and the augmentation procedure were pre-denaturation at 94℃ for 5 minutes,denaturation at 94℃ for 45 s,annealing at 50℃~60℃ for 60 s,extension at 72℃for 90 s,reaction with 35 cycle,and extension at 72℃ for 5 minutes.