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Isolation of DNA from Paraffin-Embedded Tissue

Method

Note: In general, the yield is pretty low and variable.

  1. Place 2 sections of tissue, 10 µm each, in a 1.5-ml tube.
  2. Spin at 12,000 rpm for 5 minutes to get sections to bottom of tube.
  3. Add 200 µl digestion buffer (50mM Tris, pH 8.2, 1mM EDTA, 0.5% Tween-20)
  4. Add 2 µl 20 mg/ml proteinase K, for a final concentration of 200 µg/ml.
  5. Incubate at 55°C for 3 hours O/N.
  6. Spin at 12,000 rpm for 10 minutes. A ring of paraffin will form on the top of the liquid. Pierce the ring and transfer the supernatant to a fresh tube.

    Make sure tubes are balanced to ensure that the paraffin ring forms well.
  7. Heat-inactivate the proteinase K by heating to 95°C for 5 minutes.
  8. Add 500 µl of 100% ethanol and 10 µl of 10M ammonium acetate. Incubate 30 minutes at -80°C.
  9. Centrifuge at 12,000 rpm for 10 minutes. Decant supernatant.
  10. Wash 2X with 70% ethanol, spinning at 12,000 rpm for 5 minutes each.
  11. Drain tube and invert to dry.
  12. Dissolve DNA in 50 µl TE buffer.
  13. Determine concentration by spectrophotometry.

© 2014 The University of Texas MD Anderson Cancer Center