长寿老人体内分离的鼠李糖乳杆菌RAPD体系优化研究

Study on Lactobacillus rhamnosus RAPD System Optimization from Macrobian

采用CTAB改良法提取11株鼠李糖乳杆菌的基因组DNA,对其完整性进行了测定,并以此为模板进行RAPD反应。优化的RAPD反应体系为:模板50ng,Taq酶2U,dNTPs 3.25mmol/L,随机引物5mmol/L,10×PCR缓冲液2.5μl,Mg2+7.5mmol/L,反应总体积为25μl;PCR扩增参数:93℃2min,36℃1min,72℃2min1次循环;93℃1min,36℃1min,72℃2min,40次循环;93℃1min,36℃1min,72℃10min1次循环。

An improved DNA extraction protocol with CTAB was developed for Lactobacillus rhanmosus. DNA was extracted successfully from the samples of 11 strains and was suitable for RAPD analysis. The optimization of a 25 μl RAPD was determined as followed： template 50ng, 2 U TaqDNA polymerase, 3.25 mmol/L dNTPs for each, 5 mmol/L primer, 2.5μl 10× PCR buffer, and 7.5 mmol/L Mg^2＋. The reaction program was devised for one cycled at 93℃2 minutes, 36℃ 1 minutes, 72 ℃ 2 minutes, which is followed by 40 cycles, each I minutes at 93 ℃, 1 minute at 36℃, 2 minutes at 72 ℃, and a final extension at 93℃ 2 minutes, 36 ℃ 1 minutes, 72℃10 minutes.