Protocol for DNA isolation from paraffin slices

(Randy Johnson lab and David Looney lab 2005)

  1. Take care and work in the fume hood
  2. Add 1 ml. of xylene to paraffin tissue section in the eppendorf tube
  3. vortex well.
  4. Spin 5 min at 10K g, and aspirate supernatant.
  5. Wash 2X with 1 ml. of ethanol, centrifuging for 5 min at 10, 000 g each time and aspirating supernatant each time.
  6. Centrifuge briefly again and aspirate remaining ethanol.
  7. Added 180 ul Qiagen buffer ATL and 20 ul 18 mg/ml Proteinase K. (Sigma)
  8. Incubate at 55oC, Overnight , vortexing occasionally over the course of several hours.
  9. Add 20 ul of 20 mg/ml RNAseA, and let the tube sit for 1 min. at room temperature.
  10. Add 200 ul Qiagen buffer AL.
  11. Vortex well, and incubated for 10 min in a 70oC heating block.
  12. Add 200 ul of 100% ethanol, and vortex well.
  13. Load onto Qiamp columns in collection tubes.
  14. Centrifuge 1 min at 10,000 g, and discard flow through volume.
  15. Place columns in new collection tubes.
  16. Wash with 500 ul of buffer AW1.
  17. Briefly centrifuge for1 min. at 10,000 g. Aspirate through volume.
  18. Add 500 ul of buffer AW2. Centrifuge for 3 minat10,000 g and discard the wash.
  19. Place columns in new collection tubes.
  20. Add 200 ul of preheated (70oC) buffer AE.
  21. Let sit 1 min. at room temperature.
  22. Centrifuge 1 min. at 10,000 g.
  23. Transfer eluant to Eppendorf tubes.
  24. Added 1 ul of 10 mg/ml glycogen, at 1/10 volume in 3M NaOAc (pH 5.2) and 2.5 volumes of ethanol. Let it sit at 20oC freezer overnight.
  25. Centrifuge 10 min at maximum speed in microfuge.
  26. Remove ethanol.
  27. Wash with 500 ul of 70% ethanol.
  28. Centrifuge for 5 min., and removed ethanol.
  29. Centrifuge briefly, remove remaining ethanol.
  30. Dissolve in ----ul H2O. Measure O.D. 260 of 3.5 ul in 66.5 ul H2O.
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