1. Rinse blot with 2X SSC and air dry
2. Place blot into hybridization tube with ~100 ml 0.5% SDS, 0.1 SSC. Prehybridize for 1 hour at 65C.
3. Discard prehybridization solution and add hybridization buffer; use approximately 7-10 ml per large hybridization tube.


 

25 ml 1.0 M NaHPO4

200 µl 0.5 M EDTA

50 ml formamide

7 g SDS

H2O to 100 ml


 
4. Prehybridize for ~5 - 10 minutes at 37-42ºC.
5. Add denatured and labeled probe.
6. Hybridize 16 - 24 hours at 37-42ºC.
7. Discard hybridization solution to radioactive waste. Wash the membrane briefly in 2X SSC, 0.1% SDS at 23ºC (room temperature).
8. Wash the membrane in ~ 100 ml 0.1X SSC , 0.1% SDS for 15-30 minutes at 37-65ºC.
9. Repeat step 8.
10. Blot the membrane onto filter paper, wrap in plastic and expose to film.

Wash the blot twice in 100 ml 0.1X SSC, 0.1% SDS at 95ºC for 30 minutes.

Wash the blot as in step 1 but at 23ºC.