DNA EXTRACTION PROCEDURE - GENERAL

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).
  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 4C.
  9. Extract with equal volume chloroform (mix by inverting) and centrifuge at 10,000X g for 5 min. Transfer 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 purity of DNA by electrophoresis and spectrophotometric analysis.