Bio-Rad Zeta-Probe Membranes - Manual (Page 10)

4.2 Formamide Protocol
Prehybridization
1. Seal the blotted Zeta-Probe membrane inside a heat-sealable
plastic bag. Prepare the following solution for prehybridization:
50% formamide
0.12 M Na
2
HPO
4
, pH 7.2
0.25 M NaCl
7% (w/v) SDS
1 mM EDTA
2. Cut one corner of the plastic bag and pipet the prehybridization
solution in, then reseal the bag.
3. Incubate at 43°C for 5 min.
Hybridization
1. Cut one corner of the bag, remove the prehybridization solution,
and replace it with the same buffer.
2. Add probe, then seal the open corner (taking care to exclude all
air bubbles). Mix the contents of the bag thoroughly. Incubate at
43°C for 4–24 hr with agitation.
Note: At no stage before washing should the membranes be
permitted to dry.
Washes
1. At the completion of hybridization, remove membranes from their
hybridization bags and place them in 2x SSC. Rinse briefly, then
wash them successively by vigorous agitation at room temper-
ature for 15 min in each of the following solutions:
2x SSC/0.1% SDS
0.5x SSC/0.1% SDS
0.1x SSC/0.1% SDS
15
2. Add the denatured probe and remove all bubbles before
resealing the bag. Hybridize for 4–24 hours at 65°C with
agitation.
3. Carefully remove the hybridization solution by cutting one
corner. Remove hybridized Zeta-Probe membrane plastic bag.
Note: At no stage before washing should the membranes be
permitted to dry.
Washes
1. Wash the membrane at 68°C, 2 times for 10 min each, in the
following:
1x SSC
0 . 1% (w/v) SDS
The first wash should be conducted at room temperature; the
second wash should be conducted in the hybridization oven.
2. Wash the membrane at 65°C, 2 times for 30–60 min each, in the
following:
0 . 1x SSC
0 . 1% (w/v) SDS
These washes should be conducted in the hybridization oven.
3. After washing, the blotted membranes are ready for autoradio-
graphy. 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.
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