太湖流域土壤微生物基因组总DNA分离纯化及其质粒文库的初步构建

EXTRACTION AND PURIFICATION OF SOIL MICROBIAL GENOMIC DNA FROM TAIHU AREA AND PRIMARY CONSTRUCTION OF PLASMID LIBRARIES

采用研磨 /冻融和SDS/蛋白酶K热处理以及CTAB处理等理化方法 ,直接从太湖流域土壤样品中抽提得到微生物总基因组DNA .所得粗DNA用透析袋法以及NucleaotrapsuspensionDNA纯化试剂盒进行了纯化 .纯化过的DNA能够满足常用限制性内切酶酶切、细菌 16SrDNA通用引物和随机引物进行PCR扩增的要求 .将所得纯品DNA经过EcoRI部分酶切之后与酶切并脱磷酸的pSK-空载体连接 ,再以大肠杆菌JM10 9为宿主细胞初步构建了土壤微生物基因组文库 .图 6参

A method has been developed for extracting genomic DNA from samples of Taihu area. In this method, the environmental samples were treated first by grinding and freezing/thawing and subsequently by SDS/proteinase K and CTAB-based DNA extraction. The crude extraction from these samples was purified by dialysis bag and Nucleaotrap respectively. Both purification methods resulted in complete removal of the brown color from crude DNA solution. The purified DNA could be digested well by 4 kinds of restriction enzymes and was pure enough for amplification of 16S rDNA gene and other random primers. Mixed genomic DNA libraries for the environmental samples were constructed by inserting part restriction digesting fragments (3-8 kb) into plasmid pSK- and then transforming competent E. coli JM109 with the resultant plamids. Fig 6, Ref 7