犊牛盲肠微生物总DNA的提取方法

Extraction of Calves Cecum Microbial Total DNA

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.