水杉RAPD扩增条件的优化

Effect on Experimental Results of Content of Compositions in RAPD Experiment of Metusequoia glyptostroboides

以改进SDS法抽提濒危植物水杉嫩叶总DNA,进行随机扩增多态DNA(RAPD)分析,分别测试了镁离子,dNTP,模板DNA含量,引物和DNA聚合酶量对反应结果的影响,通过各因子的组合研究,可知水杉RAPD分析较适宜的扩增条件是:15μlPCR反应体积,1×Taq酶配套缓冲液(10mmol/LTris HCl pH 9,0.50mmol/L KCl,0.1％Triton X-100),1.5mmol/L MgCl_2,0.5U Taq酶(上海华美公司),5ng模板DNA,20pmol引物(上海Sangon公司);2μg/μlBSA;dATP、dCTP、dGTP、dTTP各0.2mmol/L。

The DNA templates of Metasequoia glyptostroboides extracted from young leaf were amplified in RAPD. The effectof content of Mg^(2+), dNTP, DNA templates, primers and DNA polymerase on experimental results were tested and the optimalreaction system of RAPD for Metasequoia glyptostroboides was dryermined as follows: 1×Taq polymerase corresponding buffer(10mmol/L Tris·HCl pH9.0,50mmol/L KCl,0.1% Triton X - 100), 1.5mmol/L MgCl_2 ,0.5U Taq DNA polymerase, 5ngtemplate DNA,20pmol primer,2μg/μl BSA,0.2mmol/L dATP,dCTP,dGTP,dTTP for each in total 15μl reaction volume.