摘要
以酒酒球菌SD-2a为试验材料,研究了酒酒球菌基因组DNA的提取方法,对影响RAPD-PCR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RAPD反应体系。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RAPD反应的需要;建立的RAPD-PCR反应体系为:25μL反应体积、1.0UTaq酶、160μmol/LdNTP、0.4μmol/L随机引物、3.0mmol/LMg2+、10ng的DNA模板用量;PCR反应程序为:94℃变性300s,1个循环;94℃变性60s,36℃退火60s,72℃延伸90s,45个循环;72℃延伸300s。
In this study, Oenococcus oeni was used as experimental materials. The method of genomic DNA extraction was studied in Oenococcus oeni and the primary conditions of RAPD-PCR were optimized. Finally, RAPD reaction system suitable for our lab was established. The results showed that high-grade genomic DNA which could meet the requirements of RAPD reaction was obtained by the modified methods. The established RAPD-PCR reaction system was as follows： 1.0 UTaq polymerase, 160 μmol/L dNTP, 0.4 μmol/L random primer, 3.0 mmol/L Mg^2＋, and 10 ng DNA template, and 25 μL reaction volume. The reaction program of PCR was devised as follows： 5 min degeneration at 94 ℃ （one cycle）, then lmin degeneration at 94 ℃, 1 min annealing at 36℃, and 1.5 min extension at 72 ℃（45 cycles）, and then 5 min final extension at 72 ℃.
出处
《酿酒科技》
2009年第4期46-50,53,共6页
Liquor-Making Science & Technology
关键词
微生物
酒酒球菌
DNA提取
RAPD
优化
microbe
Oenococcus oeni
DNA extraction
random amplified polymorphic DNA （RAPD）
optimization
