一种高效提取桑树根围土壤微生物总DNA的方法

为了建立桑树根围土壤微生物群落结构的分子生态学试验技术体系,采取不同的细胞裂解方法及DNA沉淀方法直接提取桑树根围土壤微生物总DNA,并以采用试剂盒提取的桑树根围土壤微生物总DNA为参照,筛选出一种能有效提取高纯度、多样性土壤微生物总DNA的方法:以十六烷基三甲基溴化铵(CTAB)、溶菌酶、蛋白酶K、十二烷基磺酸钠(SDS)为裂解液,经反复冻融裂解细胞,在DNA提取液中添加聚乙烯吡咯烷酮K30(PVP K30)、牛血清蛋白(BSA)、CaCl2去除腐殖酸,并结合聚乙二醇8000(PEG8000)沉淀和纯化DNA。该方法简便、快速,获得的桑树根围土壤微生物基因组DNA分子长度在23 kb以上,土壤鲜样的微生物DNA产率为11.2μg/g,且纯度较高,D(260 nm)/D(230 nm)=1.42,D(260 nm)/D(280 nm)=1.37,可直接用于PCR扩增及变性梯度凝胶电泳(DGGE)分析。

A Highly Effective Method for Total DNA Extraction from Rhizospheric Soil Microorganisms of Mulberry Tree

In order to establish an experimental protocol for molecular ecology on microbial population structure in mulberry rhizospheric soil,various methods of cell lysis and DNA sedimentation were used to extract total DNA from mulberry rhizospheric soil microorganisms directly which were then compared with that extracted with commercial DNA extraction kit.As a result,a highly effective protocol for extracting high purity total DNA from diversified soil microorganisms was established: using cetyltrimethylammonium bromide(CTAB),lysozyme,proteinase K,sodium dodecyl sulfonate(SDS) as lysate,cells were lysed by repeated freezing-thawing;and then polyvinyl pyrrolidone K30(PVP K30),bull serum albumin(BSA) and CaCl2 were added to the DNA extract for the removal of humic acids;finally,DNA was precipitated and purified with polyethylene glycol 8000(PEG8000).This method is easy and quick.The obtained genomic DNAs from mulberry rhizospheric soil microorganisms are longer than 23 kb,with high purity DNA of soil microorganisms 11.2 μg/g,D(260 nm)/D(230 nm)=1.42,and D(260 nm)/D(280 nm)=1.37,which can be used for PCR amplification and denatured gradient gel electrophoresis analysis(DGGE) directly.