褐黄孢链霉菌基因组DNA的提取及RAPD体系的优化

Genomic DNA Extraction and Optimization of RAPD Amplifying Conditions of Streptomyces Gilvosporeus

以改进的SDS法提取褐黄孢链霉菌基因组DNA,对其进行随机扩增多态性(RAPD)研究。通过单因素和正交试验相结合的方法,建立了适于褐黄孢链霉菌RAPD分析的PCR反应体系:包括Taq聚合酶、Mg^(2+)、随机引物、dNTPs和模板浓度。在20μl体系中,模板DNA浓度60~150mg,Taq聚合酶为1.0~1.5U,引物浓度0.3~0.4mmol/L,dNTPs浓度200~250μmol/L,Mg^(2+)浓度2.5~3.0mmol/L。在此反应体系下可扩增出条带数目多且清晰稳定的电泳图谱。

An improved SDS method was used to extract genomic DNA from Streptomyces gilvosporeus.The RAPD conditions were optimized by single factor experiment and orthogonal experiment,including Taq DNA polymerase,Mg^(2+),primer,dNTPs and template DNA.The results were as follows;DNA 60~150 ng,Taq DNA polymerase 1.0 ~ 1.5U,primer concentration 0.3~0.4 mmol/L,dNTPs concentration 200~250μmol/L,Mg^(2+) concentration 2.5~3.0 mmol/L in 20μl PCR system.Under above optimal conditions,the abundant,stable and clear strips could be obtained.