犊牛盲肠微生物总DNA的提取方法  

Extraction of Calves Cecum Microbial Total DNA

作　　者：于萍[1] 王加启[1] 卜登攀[1] 邓露芳[1] 刘开朗[1] 魏宏阳[1] 周凌云[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所动物营养学国家重点实验室,北京100193

出　　处：《乳业科学与技术》2009年第1期14-16,共3页JOURNAL OF DAIRY SCIENCE AND TECHNOLOGY

基　　金：国际科技合作重点项目(2006DFB32160);"十一五"支撑计划"奶牛高效饲养关键技术研究与开发"(2006BAD12B03)。

摘　　要：采用反复冻融法、玻璃珠法和超声波+反复珠磨的方法提取犊牛盲肠的微生物总DNA,并对以上三种方法进行比较,从中确定最佳的方法,以实现普通实验条件下成功提取符合PCR扩增要求的DNA。经紫外分光光度分析表明,超声波+反复珠磨的方法所得的DNA的A_(260)/A_(280)的比值为1.81,0.8%琼脂糖凝胶电泳结果显示,所提DNA片段分子量大于20 kb,适于酶解和PCR扩增要求。以提取的DNA样品为模板,利用细菌通用引物,对其16S rDNA进行PCR扩增,获得了1.7 kb大小特异性很好的预期条带。这是研究犊牛盲肠微生物的关键一步。In order to extract DNA from calves cecum, three methods（repeated freeze-thaw, bead beating, uhrasionic cell-break method combined with repeated bead beating） were used and compared, results showed that the ultrasionic cell-break method combined with repeated bead beating was the most efficient one. The high-quality total DNA from a wide variety of bacterial specie was isolated and determined by a spectrophotometer （A260/A280=1.81） and 0.8% agarose gel electrophoresis. The purified DNA with the molecule bayond 20 kb was suitable for PCR. The 16S rDNA was amplified from these DNA samples through a set of baceria-universial primers. The molecule of PCR products was about 1.7 kb. It is critical for investigating microbial ecology in gastrointestinal tract of calves.

关 键 词：犊牛盲肠微生物 细菌总DNA PCR扩增 超声波细胞破碎 反复珠磨法 

分 类 号：S852.6[农业科学—基础兽医学]