荷斯坦奶牛RAPD反应条件的优化

以中国荷斯坦奶牛为实验材料,研究了Mg2+浓度、dNTPs浓度、引物浓度、模板DNA浓度、TaqDNA聚合酶,以及退火温度对RAPD反应的影响,建立一套适合中国荷斯坦牛的最佳RAPD反应体系。反应总体积为20μL,Mg2+浓度为2.5mmol/L,TaqDNA聚合酶浓度为1U,引物浓度为2μmol/L,dNTPs浓度为400μmol/L,模板DNA浓度为100ng。PCR反应程序:94℃预变性2min→40循环(94℃变性1min→36℃退火1min→72℃延伸1min)→72℃延伸5min。

Study on RAPD Reaction Conditions for Dairy Cattle

In this paper, the effects of concentration of Mg2+, dNTPs, random primers, template DNA,TaqDNA polymerase and anneal temperature on RAPD were analyzed with Holstein cattle. An optimalized RAPD system with total volume 20μL including Mg2+ at 2.5 mmol/L, TaqDNA polymerase at 1 U, random primers at 2μmol/L, dNTP at 400μmol/L, template DNA at 100ng.The reaction program with 2 minutes at 94℃,which is followed by 40 cycles ,each with 1 minutes at 94℃,1 minutes at 36℃,1 minutes at 72℃,and a final extension at 72℃ for 5 minutes.