Evaluation of gel filtration resins for the removal of PCR-inhibitory substances from soils and sediments.
A variety of gel filtration resins (Sephadex G200 and G150; Sepharose 6B, 4B and 2B; Bio-Gel P100, P200; and Toyopearl HW 55, HW 65, and HW 75) were evaluated for their efficacy in removing PCR-inhibitory substances from feedlot soil DNA crude extracts using gravity-flow disposable columns. Sepharose resins demonstrated the best properties for DNA purification when compared to other gel filtration resins, and Sepharose 2B was the most efficient purification resin based upon flow rate and the elution of DNA and humic acids from the columns. A method for purifying large solution volumes of DNA extract economically was also developed using low-cost disposable Disposaflex columns. Crude DNA extracts of cattle feedlot soil and aquifer sediment impacted by animal and human wastes were easily purified using the Disposaflex column method regardless of whether a gentle chemical lysis or a bead mill homogenization DNA extraction method was employed.
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U.S. Meat Animal Research Center, USDA, ARS, P.O. Box 166, Clay Center, NE 68933, USA.
D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods*; D004269:DNA, Bacterial / Q000302:isolation & purification; D003911:Dextrans / Q000032:analysis; D005782:Gels; D019015:Geologic Sediments / Q000382:microbiology*; D006812:Humic Substances / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D012116:Resins, Plant / Q000032:analysis*; D012988:Soil Microbiology
D004269:DNA, Bacterial; D003911:Dextrans; D005782:Gels; D006812:Humic Substances; D012116:Resins, Plant; C025614:sephadex