Pre-hyb/hyb for Radioactive Probes
互联网
1172
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.
