冷暖型小麦基因差异的最佳RAPD反应体系研究

为了建立小麦RAPD反应的最佳体系,保证反应结果的稳定性和可靠性,寻找小麦冷暖型基因差异,降低试验成本,在相同PCR扩增程序(94℃预变性5 min,94℃变性45 s,38℃退火1 min,72℃延伸1.5 min,40个循环,72℃延伸10 min,4℃保存)下,对冷暖型小麦基因组DNA的RAPD扩增体系各参数进行比较筛选,创建其最佳反应体系为:25μl反应体系中,模板DNA 20 ng,10×PCR缓冲液2.5μl,dNTPs 0.2 mmol/L,Taq酶1 U,Mg2+浓度为2.0 mmol/L,ddH2O 12μl,随机引物10 ng。

Study on RAPD Reaction System for Gene Difference of the Cold and Warm Type of Wheat

In this research,with the same PCR program:94?℃/5 min→94?℃/45?s,38?℃/1 min,72?℃/1.5?min,45 cycle→72?℃/10 min→4?℃ forever,the effects of the compositions of RAPD-PCR reaction system on the gene difference of the cold and warm type of wheat were studied.An optimalzed RAPD system was set up: total volume was 25 μl,including DNA 20 ng template DNA,10×PCR buffer at 2.5?μl,dNTPs at 0.2 mmol/L,Taq DNA polymerase at 1?U,Mg~(2+) at 2.0 mmol/L,ddH_2O 12?μl and 10 ng random primers.