1. Start with mouse tail biopsies in Falcon 2059 tubes. Add 2 ml SET buffer (10 mM Tris, pH 7.5, 300 mM NaCl, 5 mM EDTA, 1 % SDS) and 30-50 µl 10 mg/ml proteinase K (100 mg proteinase K in 10 ml water). Incubate at 45-55 °C with vigorous shaking until tissue is digested and only hair and small bones are left. Digestion should be complete in 2-3 hr.

2. Spin in clinical centrifuge 3,000 RPM (1,900 x g) 7 min at room temperature. Make sure supernatant is clear. If not, revortex and respin.

3. Transfer 400 µl supernatant to a 1.5 ml microfuge tube.

4. Add 800 µl EtOH. Mix by inverting rack.

5. Spin 2 min in microfuge.

6. Remove supernatant and resuspend pellet in 12 µl 2 M NaCl, 0.1 M NaOH (do not dry pellet).

7. DNA is ready for denaturation and dotting. DNA preps from non-albino mice will contain some pigment material; this will not affect the results of the blots.

DOT BLOTS

1. Start with tail DNA that has been resuspended in 2 M NaCl, 0.1 M NaOH. Heat samples to 100 °C for 3-5 min. Use Fisher 05-406-16 microfuge tubes to avoid caps popping during boiling. Quick spin to bring down condensation and mix before dotting.

2. Dot 5 µl onto gridded nitrocellulose with parafilm under nitrocellulose (or use dot blot apparatus). Dot on every other crosshair. Rinse pipet tip between samples 3 times with water and 2 times with 95% ethanol (a little residual ethanol in pipet tip helps spread dot).

3. Let filter air dry, rinse in 2X SSCP (20X SSCP stock is 0.2 M sodium phosphate, pH 7.0, containing 3 M sodium chloride and 0.3 M sodium citrate), and bake at 80 °C for 1 hr.

4. Wet filter by floating on 2X SSCP and prehybridize in 10 ml of 50% formamide, 5X SSCP, 5X Denhardt's solution (50X stock is 10 mg/ml each Ficoll, polyvinylpyrrolidone, and BSA), 0.1% SDS, and 100 µg/ml denatured salmon sperm DNA at 42 °C for 2 hr.

5. Hybridize filter overnight at 42 °C with [32P]-labeled DNA probe. We ususally label 25 ng of probe with >6,000 Ci/mmole [32P]dCTP and use the whole labeling reaction in the hybridization. Denatured probe can be added directly to the prehybridization solution.

6. Wash filter 2 x 15 min in 2X SSC + 0.1% SDS and 3 x 30 min in 0.1X SSC + 0.1% SDS at 65 °C.

7. Air dry filter briefly, wrap in plastic wrap, and expose to X-ray film at -70 °C.

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