| PubMed I Cell Nature Science Scientific American I HHMI Nobelprize I Weather |
0.5 M Sodium Phosphate pH 7.2
1 mM EDTA pH 8.0
7% (w/v) SDS
1% Bovine Serum Albumin (Use electrophoresis grade BSA)
Pre-hybridize the membrane for about 1 hour in the hybridization solution at 65°C. If you need to probe more than one membrane or the membrane is too big, I recommend you place a nylon hybridization mesh between the membrane folds. The mesh provides space for the hybridization solution to move easily.
After an hour, replace the prehybridization solution with fresh hybridization solution at 65°C.
Denature the probe at 95°C or at 65°C in 50 percent formamide for 5 min. Place the denatured probe right away on ice.
Add the probe into hybridization buffer directly. Make sure the concentrated probe does not come to direct contact with the membrane.
Hybridize 16 hours at 65°C.
A proper wash is necessary to remove the unspecific binding of the probe.
Briefly wash the membrane twice with 2X SSPE (or SSC), 0.1% SDS at room temperature. Then, wash the membrane twice with pre-warmed 0.1X SSPE (or SSC), 0.1% SDS for 15 minutes at 65°C. Check the signal with Geiger counter to see how hot the membrane is.
Expose the membrane to X-ray or Phosphorimager screen.
Note: Never allow the membrane to dry completely anytime during hybridization or washing, if you want to strip the probe from membrane. If you let the membrane dry, the probe will be permanently cross-linked to the membrane, and become impossible to strip it.
Good luck!
Posted by Erbay Yigit