体外培养瘤胃微生物总DNA的提取与纯化

摘要

主要目的是寻找一种能最大限度获取所有类型的瘤胃微生物大片段DNA的提取方法,为后期分子生物学技术研究瘤胃微生物奠定基础。采用液氮冻融＋溶菌酶＋CTAB和SDS相结合的方法处理瘤胃样品并充分破壁,再采用酚/氯仿/异戊醇抽提、异丙醇沉淀获得DNA粗提物,经过试剂盒纯化后得到瘤胃微生物DNA样品。经特异性引物PCR检测,本方法所提取的DNA包含细菌、古细菌、真菌及原虫四类微生物信息。因此,采用该方法提取的DNA可用于后期实时定量PCR或DGGE等研究。%This study aimed to find an utmost extraction method of total DNA from rumen microbes in vitro fermentation for the post research on the rumen microbial structure and diversity.The method of liquid nitrogen Freeze-thaw,lysozyme＋CTAB and SDS were combined together in order to break microbal cells for releasing DNA as much as possible.Then phenol/chloroform/Isoamyl alcohol extraction and isopropanol precipitation were used to get the total DNA extracts.The purified DNA was obtained by the DNA purification kit,and identified by PCR experiments.Total DNA extracts included all four kinds of microorgism as bacterium,Archaebacteria,fungi and protozoa.Therefor,DNA extracted by this method can be used in the quantitative-PCR,DGGE and other researchs.