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Environmental DNA Extraction






Source:      5/7/99

To each one ml sediment sample, one ml of extraction buffer (40 mM Tris [pH 8.0], 0.1 mg of polyA, 1 mM CaCl, 1 mg of proteinase K and 0.25% SDS) was added and incubated at 50°C for 30 minutes.  SDS was added to a final concentration of 1 % along with 2 g 0.1 mm zirconia/silica beads (Biospec Products) and the samples were shaken at high speed for 3 minutes on a Mini-Beadbeater-1 (Biospec Products).  The samples were centrifuged for 3 minutes at low speed to remove solid material and the beads from the aqueous phase.  The supernatant was extracted twice with equal volumes of extraction buffer-saturated phenol/chloroform and once with chloroform.  The extract was separated into two phases by centrifugation. The upper, aqueous layers were pooled and the DNA precipitated by addition of 1/10 vol 3 M NaOAc (pH 5.2) and 1 vol isopropanol followed by a 30 minute centrifugation at 11,000 g. After a 70% ethanol wash, the DNA was resuspended in TE (10 mM Tris, 1 mM EDTA).  The DNA was further purified by Wizard DNA clean-up system (Promega), according to the manufacturer's protocol.  High molecular weight DNA was isolated on a 1% low-melting-point agarose gel (SeaPlaque, GTG, FMC BioProducts) with 2% polyvinylpyrrolidone.



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