Bacillus subtilis were isolated by streak platemethod. It was identified that was Bacillus subtilis by using the 16S rDNA gene. Bacillus strains were used as experimental materials. The method of genomic DNA extraction was studied in Bacillus and the conditions of ERIC-PCR. Finally, ERIC-PCR reaction system was suitable for our lab was established. The results showed that high-grade genomic DNA which could meet the requirements of PCR reaction was obtained by the modified methods. The established ERIC-PCR reaction system was as follows:10 ×Buffer( Mg2+ ) 2.5μL, 20pmol/μL primer El lμL, 20pmol/μL primer E2 lμL, DNA template 2μL, 2.5mmol/L dNTPs 1.6μL,Taq polymerase 0.9μL, ddH2O 16μL,25μL reaction volume. The reaction program of PCR was devised as follows:4min degeneration at 94℃ (one cycle), then 30s degeneration at 94℃,40s annealing at 58℃, and 1 min extension at 72℃(30 cycles),and then 4min final extension at 72℃.%利用稀释平板法从米醋沉淀中分离获得细菌,通过对其进行16S Rdna分子鉴定,表明该细菌为枯草芽孢杆菌 (Bacillus subtilis).以该菌为实验材料,研究了细菌基因组DNA的提取方法,并将ERIC-PCR法首次应用于醋液菌种.对此反应体系的主要因素进行了优化,最终建立了适合于本实验室的ERIC-PCR体系.结果表明,采用改良的传统细菌基因组DNA提取方法,所提取的DNA质量较高,能够满足ERIC-PCR反应的需要;建立的反应体系为:25μL反应体积10×扩增缓冲液(含Mg2+)2.5μL,20pmol/L ERIC-PCR引物E1 1.0μL,20pmol/Μl ERIC-PCR引物E21.0μL,DNA模板21×L,2.5mmol/L dNTPs混合液1.61μL,taq聚合酶0.9μL,双蒸水补齐;PCR反应程序为:94℃变性4min,1个循环;94℃变性30s,58℃退火40s,72℃延伸1min,30个循环;72℃C延伸4min.
展开▼
