山药基因组DNA的提取和RAPD反应条件的优化

Isolation of Genomic DNA and Optimization of RAPD Conditions of Dioscorea opposita Thunb.

研究了山药基因组DNA的提取方法;对Mg2+浓度、dNTPs浓度、引物浓度和Taq酶用量进行了优化,建立了最佳的RAPD反应体系:在25μL反应体系中Mg2+浓度为2.0 mmol.L-1,dNTPs浓度为0.25 mmol.L-1,引物浓度为20 pmol.L-1,Taq酶1.5 U,模板DNA为200 ng.扩增程序为:95℃预变性7 min;94℃变性30 s,36℃退火45 s,72℃延伸2 min,共35个循环;72℃终延伸10 min.

This article studied the method of genomic DNA isolation of Dioscorea opposita Thunb. and the optimization of Mg^2＋ , dNTPs, primer and Taq DNA polymerase concentration. An optimal PCR system for RAPD analysis was selected as follows： 2.0 mM Mg^2＋ , 0.25 mM dNTPs, 0.2 μM primer, 1.5 U Taq DNA polymerase and 200 ng template DNA in 25 μ1 reaction volume. The PCR program for RAPD analysis was determined： preheat denatured at 95 ℃ for 7 mini denatured at 94℃ for 30 s, primer annealing at 36 ℃ for 45 s, extension at 72 ℃ for 2 min, 35 cycles in total; at last extension 72 ℃ for 10 min.