Isolation of DNA from Paraffin-Embedded Tissue
Method
Note: In general, the yield is pretty low and variable.
- Place 2 sections of tissue, 10 µm each, in a 1.5-ml tube.
- Spin at 12,000 rpm for 5 minutes to get sections to bottom of tube.
- Add 200 µl digestion buffer (50mM Tris, pH 8.2, 1mM EDTA, 0.5% Tween-20)
- Add 2 µl 20 mg/ml proteinase K, for a final concentration of 200 µg/ml.
- Incubate at 55°C for 3 hours O/N.
- 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. - Heat-inactivate the proteinase K by heating to 95°C for 5 minutes.
- Add 500 µl of 100% ethanol and 10 µl of 10M ammonium acetate. Incubate 30 minutes at -80°C.
- Centrifuge at 12,000 rpm for 10 minutes. Decant supernatant.
- Wash 2X with 70% ethanol, spinning at 12,000 rpm for 5 minutes each.
- Drain tube and invert to dry.
- Dissolve DNA in 50 µl TE buffer.
- Determine concentration by spectrophotometry.