Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 18)

Washes
1. At the completion of hybridization, remove the membranes from their hybridization bags into
2x SSC. Rinse briefly, then wash them sequentially with agitation for 15 minutes at room
temperature in the following solutions:
• 2x SSC/0.1% SDS
• 0.5x SSC/0.1% SDS
• 0.1x SSC/0.1% SDS
2. For DNA bound to nitrocellulose membranes, it may be necessary to include an RNase
treatment in the wash. Membranes are treated with 20 µg/ml RNase for 30 minutes at 37°C
in 2x SSC (Johnson et al. 1984).
3. After washing, the blotted membranes are ready for autoradiography. If no further cycles of
hybridization are to be done on the membrane, then the membrane can be dried. When
reprobing do not allow the membrane to dry between hybridizations. Expose moist
membranes between plastic wrap or enclosed in a sealable plastic bag. Do not allow a wet
membrane to come in contact with the film, because wet membranes will stick to the film, and
moisture on the film will cause black spots.
Note: To increase the rate of hybridization, include 10% dextran sulfate (final concentration)
in the hybridization solution (Maniatis et al. 1982). Prewarm hybridization solution to 50°C.
Denature the probe and carrier as above. Special care must be taken to ensure uniform
mixing of the denatured probe with the hybridization solution, since the solution is quite
viscous at 50°C.
7.4 Probe Stripping and Rehybridization
If reprobing is desired, do not allow the membrane to dry between hybridizations. The membrane
should be stripped as soon as possible after autoradiography.
1. Wash 2 times, 20 minutes each, in a large volume of 0.1x SSC/0.5% SDS at 95°C.
2. Check membrane for removal of autoradiaography patterns by overnight exposure.
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