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College of Life Sciences Nutrition, Dietetics, and Food Science
Tessem Laboratory - Diabetes Research Group

DNA Extraction

DNA from tail biopsies:

  1. Remove 0.5mm of tail into microfuge tube (do not mince)
  2. Add 0.5 ml DNA digestion buffer with proteinase K added to 0.5 mg/ml final concentration
  3. Incubate overnight at 50-55°C with gentle shaking
  4. Quick spin tubes to get solution off inside of cap
  5. Add 0.7 ml neutralized phenol/chloroform/isoamyl alcohol (25:24:1)
  6. Mix fairly vigorously. (Do NOT vortex--We use a clinical rotator for 1 hour)
  7. Spin in microfuge at top speed 5 minutes and transfer 0.5 ml of the upper phase to a new microfuge tube.
  8. Add 1ml 100% ethanol at room temperature and invert untill DNA precipitate forms (approx. 1 min)
  9. Spin in microfuge at top speed 5 minutes and carefully remove and discard supernatant
  10. Add 0.5-1ml 70% ethanol (-20°C) and invert several times
  11. Spin in microfuge at top speed 5 minutes and carefully remove and discard supernatant
  12. Add 0.5-1ml 70% ethanol (-20°C) and invert several times
  13. Spin in microfuge at top speed 5 minutes and carefully remove and discard supernatant
  14. Air dry at room temp. (can leave anywhere to 1 hr - overnight)
  15. Add 100-200ul TE buffer and incubate at 65°C for 15 min to re-suspend DNA

DNA Digestion Buffer:

  • 50mM Tris-HCL pH 8.0
  • 100mM EDTA pH 8.0
  • 100mM NaCl
  • 1% SDS