Bio-Rad Zeta-Probe Membranes - Manual (Page 13)

Section 6
Probe Stripping and Rehybridization
If reprobing is desired, do not allow the Zeta-Probe membrane to dry
between hybridizations.
The Zeta-Probe membrane should be stripped as soon as possible
after autoradiography.
Wash the membrane twice for 20 min each in a large volume of 0.1x
SSC/0.5% SDS at 95°C.
2 Check membrane by overnight exposure.
21 20
1% SDS
0.5% BLOTTO
3. Add probe, then seal the open corner (taking care to exclude all
air bubbles). Mix the contents of the bag thoroughly. Incubate at
50°C for 4–24 hr.
Note: At no stage before washing should the membranes be
permitted to dry.
Washes
1. At the completion of hybridization, remove the membranes from
their hybridization bags and place them in 2x SSC. Rinse briefly,
then wash them successively by vigorous agitation for 15 min 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. After washing, the blotted membranes are ready for autoradiog-
raphy. If no further cycles of hybridization are to be done on the
membrane, 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 Zeta-Probe membrane will stick to the
film.
Note: To increase the rate of hybridization, include 10% dextran
sulfate (final concentration) in the hybridization solution. Prewarm
the hybridization solution to 50°C. Denature the probe and carrier
as above. Special care must be taken to insure uniform mixing of
the denatured probe with the hybridization solution, since the
solution is quite viscous at 50°C.
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