Rumen Gram-positive bacteria Protocol for DNA extraction

       300ul aliquots of culture sample was centrifuged at 12 000g for 5 min. The supernatant was removed. The pellets were lyzed for 1h at 37 oC with 300uL of lysozyme lysis buffer (100 mM NaCl, 500 mM Tris [pH 8.0], lysozyme 10mg/ml). Then 200uL of SDS lysis buffer (100 mM NaCl, 500 mM Tris [pH 8.0], 10% [wt./vol.] SDS) and vortex. After incubation at 65 oC for 10 min, the mixture was centrifuged at 12 000g for 5 min. The supernatant was transferred into another microcentrifuge tube. Protein was removed by adding 500 μl of chloroform/isoamyl alcohol (24:1), vortexing for 5 s, incubating at 4 oC for 5 min, and centrifuging at 12,000g for 5 min. the upper solution was precipitated by adding a 0.5 vol. of 7.5 M ammonium acetate and a 1.0 vol. of isopropanol. After incubation at -20 oC for 15 min, DNA was pelleted at 12,000g for 10 min and washed three times with 75% ethanol. After being air-dried, pellets were dissolved in 100 μl of 10 mM Tris, pH 8.0.