米醋中蜡状芽孢杆菌DNA提取及其ERIC-PCR体系建立

Extraction of Bacillus cereus DNA in Vinegar and the Establishment of ERIC-PCR System

以蜡状芽孢杆菌(Bacillus cereus)为试验材料,比较细菌基因组DNA的提取方法。将ERIC-PCR应用于醋液分离菌的研究,对此反应体系的主要因素进行优化,最终建立了适合于此菌种的ERIC-PCR体系。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。反应体系:25μL反应体积10×扩增缓冲液(含Mg2+)2.5μL,20pmol/μL ERIC-PCR引物E11μL,20pmol/μL ERIC-PCR引物E21μL,DNA模板2μL,2.5mmol/LdNTPs混合液2.0μL,taq聚合酶1.1μL,双蒸水补齐;PCR反应程序为:94℃变性3min,1个循环;94℃变性30s,55℃退火40s,72℃延伸1min,30个循环;72℃延伸4min。

Bacillus cereus was 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 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＋） 2.5 μL,20 pmol/μL primer E1 1 μL,20 pmol/μL primer E2 1 μL,DNA template 2 μL,2.5 mmol/L dNTPs 2 μL,Taq polymerase 1.1 uL,ddH2O 15.4 μL,25 μL reaction volume. The reaction program of PCR was devised as follows： 3 min degeneration at 94 ℃（one cycle） ,then 30 s degeneration at 94 ℃,40 s annealing at 55 ℃,and l min extension at 72 ℃（30cycles） ,and then 4min final extension at 72 ℃.