4 DNA EXTRACTION PROCEDURE - GENERAL 1) Grow cells overnight in 500 ml broth medium. 2) Pellet cells by centrifugation, and resuspend in 5 ml 50 mM Tris (pH 8.0), 50 mM EDTA. 3) Freeze cell suspension at -20C 4) Add 0.5 ml 250 mM Tris (pH 8.0), 10 mg/ml lysozyme to frozen suspension, and let thaw at room temperature. When thawed, place on ice for 45 min. 5) Add 1 ml 0.5% SDS, 50 mM Tris (pH 7.5), 0.4 M EDTA, 1 mg/ml proteinase K. Place in 50C water bath for 60 min. 6) Extract with 6 ml Tris-equilibrated phenol and centrifuge at 10,000X g for 15 min. Transfer top layer to new tube (avoid interface). Re-do this step if necessary. 7) Add 0.1 vol 3M Na acetate (mix gently), then add 2 vol 95% ethanol (mix by inverting). Spool out DNA and transfer to 5 ml 50 mM Tris (pH 7.5), 1 mM EDTA, 200 g/ml RNase. Dissolve overnight by rocking at 4C. 8) Extract with equal volume chloroform (mix by inverting) and centrifuge at 10,000X g for 5 min. Transfer top layer to a new tube. 9) Add 0.1 vol 3M Na acetate (mix gently), then add 2 vol 95% ethanol (mix by inverting). 10) Spool out DNA and dissolve in 2 ml 50 mM Tris (pH 7.5), 1 mM EDTA. 11) Check purity of DNA by electrophoresis and spectrophotometric analysis.
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