Isolation of Genomic DNA—DNA Extraction Procedure

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 –20°C.
4. Add 0.5 mL 250 mM Tris (pH 8.0), 10 mg/mL lysozyme to frozen suspension, and let it 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, and 1 mg/mL proteinase K. Place in a 50°C water bath for 60 min.
6. Extract with 6 mL Tris-equilibrated phenol and centrifuge at 10000 xg for 15 minutes. Transfer top layer to new tube (avoid interface). Redo this step if necessary.
7. Add 0.1 vol 3M Na acetate (mix gently), then add 2 vol 95% ethanol (mix by inverting).
8. 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 4°C.
9. Extract with equal volume chloroform (mix by inverting) and centrifuge at 10000 xg for 5 min. Transfer the top layer to a new tube.
10. Add 0.1 vol 3M Na acetate (mix gently), then add 2 vol 95% ethanol (mix by inverting).
11. Spool out DNA and dissolve in 2 mL 50 mM Tris (pH 7.5), 1 mM EDTA.
12. Check the purity of the DNA by electrophoresis and spectrophotometric analysis.