Home Overview People Projects Protocols Sample collection for planktonic DNA DNA extraction from sediments Standard DGGE procedures Leucine incorporation rate, field procedures Methane production and oxidation rates Data/Posters/Reports Photos & Maps Publications Links	DNA extraction from sediments Michele Bahr, April 2000 Equipment: Bead beater (BioSpec 311BX) Zirconium beads (mix 106, 425-600, 710-1180 um, Sigma, bake in conical flask w/ metal spatula) -20ºC freezer, -80ºC freezer Refrigerated microcentrifuge 2 ml sterile microcentrifuge tubes with o-ring (Biospec #10832 or Fisher #05-669-9) 60ºC water bath Solutions: DNAse, RNAse-free water 10 x Extraction buffer pH 8.0 (500 mM NaAcetate, 10 mM EDTA) 1 x buffer pH 8.0 (50 uM NaAcetate, 1 mM EDTA) 20% SDS (N-Lauroylsarcosine, e.g., Sigma #L9150 or L-7414) Phenol, equilibrated, pH 8.0 (Sigma #P4557) Phernol: chloroform:isoamyl alcohol mixture at 25:24:1, equilibrated, pH 8.0 (Sigma #P2069) Chloroform:isoamyl alcohol mixture at 25:24:1, pH 8.0 (e.g., Sigma # CO549) 7.5 M NH4Acetate 100% isopropanol at room temperature 80% ethanol Resuspension buffers TE buffer, 10mM TRIS and 1 mM EDTA, pH 8.5 10mM TRIS buffer, pH 8.5 Procedure: Prepare 2 microcentrifuge tubes/sample. In each tube weigh 0.5 g zirconium bead mix, then add 0.2 g sample (100-300 mg) 50 ul 10x buffer 35 ul 20% SDS 500 ul phenol Vortex tubes c. 20 sec. Bead-beating Bead beat at max setting (5000 rpm) for 30 seconds (to 3 min.) Incubate in 60o water bath for 10 min. Bead beat at max setting (5000 rpm) for 30 seconds Centrifuge 10K x g to pellet beads and separate phases Transfer aqueous phase to a clean microfuge tube Re-extract sample with 200 ml 1 x buffer and add to aqueous from step e. Keep tubes on ice until final suspension in TE Phenol-Chloroform extraction Work in a fume hood and extract combined supernatants with an equal volume of phenol, vortex vigorously c. 20 sec., centrifuge 13K rpm for 5 min at 4o Transfer super to a clean tube and extract with an equal volume of phenol:chloroform:isoamyl alcohol 2x Transfer super and extract with an equal volume of chloroform:isoamyl alcohol 2x Combine the two tubes from each sample Precipitate Add 0.5 volume NaAcetate Add 0.7 volume isopropanol, vortex Hold at -20o overnight Centrifuge 13K rpm for 30 minutes Discard supernatant Wash pellet with 1 ml 80% ETOH, spin 13K x g for 10 minutes Dry in speed-vac Suspend pellet in 150 ml TE, store at -80o Dilute working aliquot 1:10 in 10 mM Tris (e.g., 50 ul sample in 450 ul Tris) prior to PCR amplification   From Lin, C. and D.A. Stahl. 1995. Taxon specific probes for the cellulytic genus Fibrobacter reveal abundant and novel equine-associated populations. Appl. Environ. Microbiol. 61:1348-1351
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