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Pre-hyb/hyb for Radioactive Probes

Stuff you need:

50X Denhardt's reagent:
1% Ficoll
1% Polyvinylpyrrolidone
1% Bovine Serum Albumin

Prehyb/hyb solution:
5X SSC
50% formamide
100 µg/ml SS DNA (heat denatured at 95 deg C)
5X Denhardt's
0.2% SDS

Blot Wash 1:
2X SSC
0.1% SDS

Blot Wash 2:
0.1X SSC
0.1% SDS

Stripping Solution
5 mM Tris-HCl, pH 8.0
2 mM EDTA, pH 8.0
0.1X Denhardt's

1. After transfer, crosslink and hybridize for 1 hour in hybridization buffer at 42 deg C.

2. Boil probe in 500µL hybridization buffer. Cool on ice.

3. Add probe to blot and incubate overnight at 42 deg C.

4. Remove blot from bottle and do the following washes in a pyrex dish:

5 min RT Blot Wash 1
15 min 55 deg C Blot Wash 2

Check radioactivity associated with corner of blot. If still hot: 15 min 55 deg C Blot Wash 2.

5. Wrap blot in plastic wrap and put down to film. Cassette at -70 deg C.

6. To strip, add stripping solution and incubate at 65 deg C for 2 hours, or until radioactivity is removed.

7. Store blot between two sheets of Whatman at 4 deg C.

Prepared By:
Michael McMurray
(206) 667-6660
Last Modified: Aug 29, 2001