This is a cached page for the URL (http://wheat.pw.usda.gov/~lazo/methods/lazo/dnageneral.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
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.