Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples

@article{citeulike:2889915, abstract = {We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.}, author = {Miller, D. N. and Bryant, J. E. and Madsen, E. L. and Ghiorse, W. C. }, citeulike-article-id = {2889915}, comment = {phenol chloroform extraction best (add SDS?) bead beat for 30 - 120s best try Sephadex G-200 columns or Sepharose 4B (if I can actually get a decent yield first. }, journal = {Appl. Environ. Microbiol.}, keywords = {dnaextraction, methods, soil}, month = {November}, number = {11}, pages = {4715--4724}, posted-at = {2008-06-12 20:10:08}, priority = {0}, title = {Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples}, url = {http://aem.asm.org/cgi/content/abstract/65/11/4715}, volume = {65}, year = {1999} }

TY - JOUR ID - citeulike:2889915 L3 - citeulike-article-id:2889915 TI - Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples JF - Appl. Environ. Microbiol. VL - 65 IS - 11 SP - 4715 EP - 4724 N2 - We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification. KW - dnaextraction KW - methods KW - soil N1 - phenol chloroform extraction best (add SDS?) bead beat for 30 - 120s best try Sephadex G-200 columns or Sepharose 4B (if I can actually get a decent yield first. AU - Miller, DN AU - Bryant, JE AU - Madsen, EL AU - Ghiorse, WC PY - 1999/11/01/ UR - http://aem.asm.org/cgi/content/abstract/65/11/4715 ER -