Simple and rapid method for direct extraction of microbial DNA from soil for PCR. Cullen, D., W. & Hirsch, P., R. Soil Biology & Biochemistry, 30(8-9):983-993, 1998.
abstract   bibtex   
A simple and rapid procedure for direct extraction of DNA from soils was developed to yield DNA of a high purity and quality suitable for amplification using the polymerase chain reaction (PCR). Go-extracted humic material from soil was a major contaminant of DNA and methods were devised to overcome this problem. Oligonucleotide PCR primers were designed to detect and monitor a genetically-modified (GM) Rhizobium leguminosarum by, vicine strain RSM2004 (marked with Tn5) which had become established in Rothamsted field soils. The key steps of the procedure were alkaline-SDS buffer assisted lysis of indigenous soil bacteria in a bead-beater and the purification of extracted DNA by separate PVPP and Sephadex G-75 spin-column chromatography. The mean yield from Rothamsted soil was 25 +/- 1.7 mu g crude DNA g(-1) wet soil (i.e. 20 mu g g(-1) dry soil), sheared to fragment sizes of about 22-25 kb. The recovered DNA was easier to purify and of a higher quality, as verified by PCR amplification of a 442 bp target sequence of Tn5, than DNA extracted by a hot-SDS lysis method. The detection limit was demonstrated to be one culturable cell of RSM2004 (i.e. a single copy of Tn5) 10 mg(-1) soil against a background of 10(7) diverse non-target bacteria. (C) 1998 Elsevier Science Ltd. All rights reserved.
@article{
 title = {Simple and rapid method for direct extraction of microbial DNA from soil for PCR},
 type = {article},
 year = {1998},
 pages = {983-993},
 volume = {30},
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 abstract = {A simple and rapid procedure for direct extraction of DNA from soils was developed to yield DNA of a high purity and quality suitable for amplification using the polymerase chain reaction (PCR). Go-extracted humic material from soil was a major contaminant of DNA and methods were devised to overcome this problem. Oligonucleotide PCR primers were designed to detect and monitor a genetically-modified (GM) Rhizobium leguminosarum by, vicine strain RSM2004 (marked with Tn5) which had become established in Rothamsted field soils. The key steps of the procedure were alkaline-SDS buffer assisted lysis of indigenous soil bacteria in a bead-beater and the purification of extracted DNA by separate PVPP and Sephadex G-75 spin-column chromatography. The mean yield from Rothamsted soil was 25 +/- 1.7 mu g crude DNA g(-1) wet soil (i.e. 20 mu g g(-1) dry soil), sheared to fragment sizes of about 22-25 kb. The recovered DNA was easier to purify and of a higher quality, as verified by PCR amplification of a 442 bp target sequence of Tn5, than DNA extracted by a hot-SDS lysis method. The detection limit was demonstrated to be one culturable cell of RSM2004 (i.e. a single copy of Tn5) 10 mg(-1) soil against a background of 10(7) diverse non-target bacteria. (C) 1998 Elsevier Science Ltd. All rights reserved.},
 bibtype = {article},
 author = {Cullen, D W and Hirsch, P R},
 journal = {Soil Biology & Biochemistry},
 number = {8-9}
}
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