针茅属植物RAPD条件优化

Optimal of RAPD amplification on Stipa L.

本试验以针茅属 ( Stipa L.)植物为材料 ,使用 CTAB法提取其基因组 DNA,通过对 RAPD( Random amplified polymorphic NDA,随机扩增的多态性 )条件优化分析 ,初步确定了针茅属植物 RAPD的最佳方案 ,即在 PCR扩增程序为 94℃预变性 3min,94℃变性 1 min,37℃复性 1 min,72℃延伸 1 .5 min,设 45个循环 ,最后 72℃保温 5 min时 ,2 5μL反应体系中包括 :0 .2 μL( 1 0 mmol/L) d NTPs,2 .0 μL( 1 0 mmol/L) Mg2 + ,0 .5 μL( 1 0 0 pmol/μL)引物 ,模板DNA80 ng,Taq酶 ( Sangon) 1 .0 U,2 .5 μL1 0×buffer缓冲液 ,其余部分用无菌三蒸水补平可以得到较好的扩增结果。

CTAB method was used to extract genome DNA from plant material of Stipa L.Through optimal analysis of RAPD,a PCR amplification program was determined.When the amplification was performed as,94℃ pre-denaturation for 3min,45cycles of 94℃ for 1min,37℃ for 1min,72℃ for 1.5min,and 5min 72℃ extension,the 25μL reaction mixture contained 80ng DNA sample,0.2μL(10mmol/L) of each dNTP,2.0μL(10mmol/L) Mg 2+ ,0.5μL(100pmol/μL) primer,2.5X taq polymerase buffer,and 1 unit Taq polymerase(Sangon).