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R.E.W. Hancock Laboratory: Methods Chromosomal DNA Isolation

METHOD:
  1. Grow cells in 2-5 ml broth to late log phase.
  2. Pellet 1-2 ml cells in microfuge.
  3. Resuspend cells in 400 µl TES (50 mM Tris-HCl, 10 mM NaCl, 10 mM EDTA, pH7.5).
  4. Add: 17 µl 30% (w/v) sarkosyl (final concentration of 1%) and 5 µl 10 mg/ml proteinase K (final concentration 100 µg/ml).
  5. Incubate at 37ºC for 30-60 minutes, or until the solution clears.
  6. Add 400 µl 4 M NH4OAc and mix well.
  7. Extract 2X with 1:1 phenol:chloroform.
  8. Extract 1X with chloroform.
  9. Precipitate with an equal volume of isopropanol at RT for 10 minutes.
  10. Resuspend pellet in 400 µl 0.1 M NaOAc pH 6.0. Pellet may not resuspend completely. Reprecipitate DNA with 800 µl EtOH for 15 minutes at RT.
  11. Centrifuge at 11K for 15 minutes to pellet DNA.
  12. Rinse pellet in 1 ml 70% EtOH for 5 minutes.
  13. Dry pellet by placing open tube in 37C incubator or on bench top for 15-20 minutes.
  14. Resuspend DNA in 50 ul TE (10 mM Tris pH 8.0, 0.1 mM EDTA) overnight at 4C. May add 1 µl RNase to help DNA dissolve. Yields 25-50 ug of DNA; use 1-5 µl per digest.

TES:

5.0 ml 1 M Tris pH 7.5

0.2 ml 5 M NaCl

2.0 ml 0.5 M EDTA

93 ml sterile MQ water