摘要
以改进的CTAB-溶菌酶-蛋白酶K裂解法抽提土壤可培养真菌总DNA,直接进行随机扩增多态DNA(RAPD)分析。分别测试了镁离子浓度,4×dNTP浓度,模板DNA量,引物用量,Taq酶用量和牛血清白蛋白浓度对反应结果的影响,通过各因子的组合研究,确定了土壤可培养真菌遗传多样性分析的稳定的RAPD反应体系:15μlPCR反应体积,1×Taq酶配套缓冲液(10mmolL-1Tris·HClpH9.0,50mmolL-1KCl,0.1%TritonX-100),1.5mmolL-1MgCl2,2UTaq酶(上海华美公司),20ng模板DNA,15pmol引物(上海Sangon公司);dATP、dCTP、dGTP、dTTP各0.2mmolL-1;1mgmL-1牛血清白蛋白。
The total soil cultured fungal DNA was extracted by the improved CTAB-lysosome-protease K method and was analyzed by random amplified polymorphic DNA markers. The effect of concentration of Mg^(2+), Dntp, DNA templates, primer, Taq polymerase and bovine serum albumin on experimental results were tested and the optimal reaction system of RAPD for soil cultured fungi was determined as follows: 1×Taq polymerase corresponding buffer (10mmol L^(-1) Tris·HCl pH9.0,50mmol L^(-1) KCl, 0.1%Triton X-100), 1.5mmol L^(-1) MgCl_2, 1.5U Taq DNA polymerase, 20ng template DNA, 20pmol primer, 0.2mmol L^(-1) dATP, dCTP, dGTP, dTTP for each in total 15μl reaction volume.
出处
《土壤通报》
CAS
CSCD
北大核心
2004年第3期295-298,共4页
Chinese Journal of Soil Science
基金
台州学院校立项目资助(2001-14)
