Protocol for DNA isolation from paraffin slices
(Randy Johnson lab and David Looney lab 2005)
- Take care and work in the fume hood
- Add 1 ml. of xylene to paraffin tissue section in the eppendorf tube
- vortex well.
- Spin 5 min at 10K g, and aspirate supernatant.
- Wash 2X with 1 ml. of ethanol, centrifuging for 5 min at 10, 000 g each time and aspirating supernatant each time.
- Centrifuge briefly again and aspirate remaining ethanol.
- Added 180 ul Qiagen buffer ATL and 20 ul 18 mg/ml Proteinase K. (Sigma)
- Incubate at 55oC, Overnight , vortexing occasionally over the course of several hours.
- Add 20 ul of 20 mg/ml RNAseA, and let the tube sit for 1 min. at room temperature.
- Add 200 ul Qiagen buffer AL.
- Vortex well, and incubated for 10 min in a 70oC heating block.
- Add 200 ul of 100% ethanol, and vortex well.
- Load onto Qiamp columns in collection tubes.
- Centrifuge 1 min at 10,000 g, and discard flow through volume.
- Place columns in new collection tubes.
- Wash with 500 ul of buffer AW1.
- Briefly centrifuge for1 min. at 10,000 g. Aspirate through volume.
- Add 500 ul of buffer AW2. Centrifuge for 3 minat10,000 g and discard the wash.
- Place columns in new collection tubes.
- Add 200 ul of preheated (70oC) buffer AE.
- Let sit 1 min. at room temperature.
- Centrifuge 1 min. at 10,000 g.
- Transfer eluant to Eppendorf tubes.
- 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.
- Centrifuge 10 min at maximum speed in microfuge.
- Remove ethanol.
- Wash with 500 ul of 70% ethanol.
- Centrifuge for 5 min., and removed ethanol.
- Centrifuge briefly, remove remaining ethanol.
- Dissolve in ----ul H2O. Measure O.D. 260 of 3.5 ul in 66.5 ul H2O.
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