Recovery of bulk DNA from soil by a rapid, small-scale extraction method
- 259 Downloads
We describe an extraction method that yields up to 25 µg of DNA from 1 gram of soil. Samples are processed within 48 h. The purified DNA is 20–25 kb in size and can be digested by restriction enzymes. The soil is vortex-mixed with 6 mL mixing buffer in an Oakridge tube. Polyvinyl polypyrrolidone is added to adsorb the humic compounds that interfere with restriction enzyme activity. The entire soil homogenate is treated with lysozyme and Novozym, followed by sodium dodecyl sulfate to lyse the cells. The crude DNA extract is separated from the soil debris by low-speed centrifugation. Finally, the DNA is purified by CsCl gradient centrifugation and extracted with Geneclean.
KeywordsRestriction Enzyme Sodium Dodecyl Sulfate Extraction Method Lysozyme CsCl
Unable to display preview. Download preview PDF.
- 1.Fincham JRS (1989) Transformation in fungi. Microbiol Rev 53:148–168Google Scholar
- 2.Holben WE, Jansson JK, Chelm BK, Tiedje JM (1988) DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl Environ Microbiol 54:703–711Google Scholar
- 3.Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor, New York: Cold Spring Harbor LaboratoryGoogle Scholar
- 4.Ogram A, Sayler GS, and Barkay T (1987) The extraction and purification of microbial DNA from sediments. J Microbiol Methods 7:57–66Google Scholar
- 5.Ogram A, Sayler GS, Gustin D, Lewis RJ (1988) DNA adsorption to soils and sediments. Environ Sci Technol 22:982–984Google Scholar
- 6.Palmer JD (1986) Isolation and structural analysis of chloroplast DNA. Methods Enzymol 118:167–186Google Scholar
- 7.Somerville CC, Knight IT, Straube WL, Colwell RR (1989) Simple, rapid method for direct isolation of nucleic acids from aquatic environments. Appl Environ Microbiol 55:548–554Google Scholar
- 8.Steffan RJ, Goksøyr J, Bej AK, Atlas RM (1988) Recovery of DNA from soils and sediments. Appl Environ Microbiol 54:2908–2915Google Scholar
- 9.Torsvik VL (1980) Isolation of bacterial DNA from soil. Soil Biol Biochem 12:15–21Google Scholar
- 10.Torsvik V, Goksøyr J, Daae FL (1990) High diversity in DNA of soil bacteria. Appl Environ Microbiol 56:782–787Google Scholar
- 11.United States Department of Agriculture Soil Conservation Service (1975) Soil survey of Benton County area, Oregon, in cooperation with Oregon Agriculture Experiment Station. Washington DC: U.S. Government Printing OfficeGoogle Scholar