摘要
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.
出处
《东北林业大学学报》
CAS
CSCD
北大核心
2010年第9期1-3,共3页
Journal of Northeast Forestry University
基金
科技基础性工作专项课题(2007FY110400-3)