极度濒危植物猪血木RAPD反应体系的优化

Optimal Choice for Reaction System in RAPD Analysis of Euryodendron Excelsum H.T.Chang

采用改进的CTAB法提取中国特有极度濒危植物猪血木(Euryodendron excelsum H.T.Chang)嫩叶总DNA,并对其进行RAPD分析.分别检测了模板DNA、引物、dNTP、Mg2+和Taq DNA聚合酶用量对反应结果的影响.筛选并建立了适合于猪血木RAPD扩增的反应体系:总体积25μL,其中10×PCR缓冲液(500 mmol/L KCl,100 mmol/L Tris-HCl,110%Triton X-100,20 mmol/L MgCl2)2.5μL,引物(1.5μmol/L)0.3μL,dNTPs(10 mmol/L)0.5μL,模板2μL(25 ng),Taq酶0.2μL(0.5 U).

The DNA templates of Euryodendron excelsum H. T. Chang extracted from young leaf were amplified by RAPD. Effects of the content of DNA temlates, primers, dNTP, Mg^2＋ and Taq DNA polymerase on experimental results were tested and the optimal reaction system of RAPD for Euryodendron excelsum H.T. Chang was determined as follows：25 ng DNA template,2.0 mmol/L Mg^2＋ ,0.018 μmol/L primer,250 μmol/L dNTP,0.5 u Taq DNA polymerase in total 25 μL reaction volume.