Bio-Rad Zeta-Probe Membranes - Manual (Page 12)

2. Cut one corner of the bag, remove the prehybridization
solution, and replace it with the same buffer.
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 washes should the membranes be
permitted to dry.
Washes
1. Wash the membrane twice at 50 °C for 30 min in the following:
3x SSC
10x Denhardt’s
5% SDS 25 mM NaH
2
PO
4
, pH 7.5
2. Wash the membrane once at 50 °C for 30 min in the following:
1x SSC
1% SDS
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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Section 5
Hybridization Protocol for RNA Probes
Prehybridization
1. Seal the blotted Zeta-Probe membrane inside a heat-sealable
plastic bag. Prepare the following solution for prehybridization:
50% formamide
1.5x SSPE
1% SDS
0.5% BLOTTO
0.2 mg/ml carrier RNA
0.5 mg/ml carrier DNA
The carrier DNA must be denatured before adding it to the
hybridization solution by heating at 100°C for 5 min, followed by
rapid cooling on ice.
2. Cut one corner of the plastic bag and pipet prehybridization
solution in, then reseal the bag.
3. Incubate at 50°C for 0.5–24 hr.
Hybridization
1. Immediately before use, fragment and denature the probe and
carrier DNA as follows. Add to the precipitated RNA probe 0.1 ml
of yeast RNA (20 mg/ml), 0.5 ml of carrier DNA (10 mg/ml), and
0.6 ml of deionized formamide, mix thoroughly, and heat at 70°C
for 5 min.
2. Cut one corner of the bag, remove the prehybridization solution,
and replace it with hybridization buffer:
50% formamide
1.5x SSPE
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