Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments （lysozyme, sodium dodecyl sulfate （SDS）, sonication, mechanical mill and the...Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments （lysozyme, sodium dodecyl sulfate （SDS）, sonication, mechanical mill and thermal shock） on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg （total DNA）/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis （denatured gradient gel electrophoresis） to be possible.展开更多
基金This work was supported by the China Postdoctoral Science Foundation(No.20060390060).
文摘Purification of the total DNA extracted from activated sludge samples was studied. The effects of extraction buffers and lysis treatments （lysozyme, sodium dodecyl sulfate （SDS）, sonication, mechanical mill and thermal shock） on yield and purity of the total DNA extracted from activated sludge were investigated. It was found that SDS and mechanical mill were the most effective ways for cell lysis, and both gave the highest DNA yields, while by SDS and thermal shock, the purest DNA extract could be obtained. The combination of SDS with other lysis treatment, such as sonication and thermal shock, could apparently increase the DNA yields but also result in severe shearing. For the purification of the crude DNA extract, polyvinyl polypyrrolidone was used for the removal of humic contaminants. Cetyltrimethyl ammonium bromide, potassium acetate and phenol/chloroform were used to remove proteins and polysaccharides from crude DNA. Crude DNA was further purified by isopropanol precipitation. Thus, a suitable protocol was proposed for DNA extraction, yielding about 49.9 mg （total DNA）/g volatile suspended solids, and the DNA extracts were successfully used in PCR amplifications for 16S rDNA and 16S rDNA V3 region. The PCR products of 16S rDNA V3 region allowed the DGGE analysis （denatured gradient gel electrophoresis） to be possible.
