title: An improved method for extracting bacteria from soil for high molecular weight DNA recovery and BAC library construction.
abstract: Separation of bacterial cells from soil is a key step in the construction of metagenomic BAC libraries with large DNA inserts. Our results showed that when combined with sodium pyro-phosphate and homogenization for soil dispersion, sucrose density gradient centrifugation (SDGC) was more effective at separating bacteria from soil than was low speed centrifugation (LSC). More than 70% of the cells, along with some soil colloids, were recovered with one round of centrifugation. A solution of 0.8% NaCl was used to resuspend these cell and soil pellets for purification with nycodenz density gradient centrifugation (NDGC). After purification, more than 30% of the bacterial cells in the primary soil were extracted. This procedure effectively removed soil contamination and yielded sufficient cells for high molecular weight (HMW) DNA isolation. Ribosomal intergenic spacer analysis (RISA) showed that the microbial community structure of the extracted cells was similar to that of the primary soil, suggesting that this extraction procedure did not significantly change the the soil bacteria community structure. HMW DNA was isolated from bacterial cells extracted from red soil for metagenomic BAC library construction. This library contained DNA inserts of more than 200 Mb with an average size of 75 kb.
authors.affiliation: Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of Agriculture, College of Life Sciences of Nanjing Agricultural University, Nanjing, Jiangsu, 210095, PR China.
authors.forename: Juan
authors.initials: J
authors.lastname: Liu
authors.forename: Jingquan
authors.initials: J
authors.lastname: Li
authors.forename: Li
authors.initials: L
authors.lastname: Feng
authors.forename: Hui
authors.initials: H
authors.lastname: Cao
authors.forename: Zhongli
authors.initials: Z
authors.lastname: Cui
mesh_terms: D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D015373:Bacterial Typing Techniques; D044822:Biodiversity; D002499:Centrifugation, Density Gradient / Q000379:methods; D022202:Chromosomes, Artificial, Bacterial; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D015723:Gene Library; D005827:Genetics, Microbial / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D058889:Molecular Typing; D008970:Molecular Weight; D012988:Soil Microbiology
chemical_list: D004269:DNA, Bacterial; D021903:DNA, Ribosomal Spacer