DNA EXTRACTION PROCEDURE - GENERAL

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Grow cells overnight in 500 ml broth medium.

Pellet cells by centrifugation, and resuspend in 5 ml 50 mM Tris (pH 8.0), 50 mM EDTA.

Freeze cell suspension at -20C

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.

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.

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.

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.

Extract with equal volume chloroform (mix by inverting) and centrifuge at 10,000X g for 5 min. Transfer top layer to a new tube.

Add 0.1 vol 3M Na acetate (mix gently), then add 2 vol 95 ethanol (mix by inverting).

Spool out DNA and dissolve in 2 ml 50 mM Tris (pH 7.5), 1 mM EDTA.

Check purity of DNA by electrophoresis and spectrophotometric analysis.

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