白桦SRAP反应体系的建立与优化

Construction and Optimization of SRAP Reaction System for Betula platyphylla

以白桦(Betula platyphlla Suk)基因组DNA为模板,利用趋势面设计对白桦SRAP-PCR反应体系的5个因素(Taq酶、Mg2+、模板DNA、dNTP、引物P)在3个水平上进行优化试验,筛选出各反应因素的最佳水平,建立了白桦SRAP-PCR反应的最佳体系。该反应体系为20μL:0.21g/LDNA,1.5μL;25mmol/LMg2+,1.4μL;5U/μLTaq酶,0.25μL;2.5mmol/L,2μL;10μm/L引物,0.35μL。PCR反应程序为:94℃预变性5min,94℃变性1min,35℃复性1min,72℃延伸1min,5个循环;94℃变性1min,50℃复性1min,72℃延伸1min,30个循环,72℃延伸7min。

Using Betula platyphylla genome DNA as template,response surface methodology was used to optimize SRAP-PCR amplification system for B.platyphylla in terms of five factors （Taq DNA polymerase,Mg^2＋,DNA template,dNTP and primer） in three levels.An optimal SRAP-PCR system for B.platyphylla was obtained as DNA template （0.21 g/L） 1.5 μL,Mg^2＋（25 mm/L） 1.4 μL,Taq DNA polymerase（5 U/μL） 0.25 μL,dNTP（2.5 mm/L） 2 μL,and primer（10 μm/L） 0.35 μL for 20 μL of reaction system.The optimal protocol was an initial denaturation at 94 degrees C for 5 min fo-llowed by 5 cycles of denaturation at 94 degrees C for 1 min,35 degrees C for 1 min and 72 degrees C for 1 min,then 30 cycles of denaturation at 94 degrees C for 1 min,35 degrees C for 1 min and 72 degrees C for 1 min,and final extension at 72 degrees C for 5 min.