Bio-Rad Bio-Dot® and Bio-Dot SF Microfiltration Apparatus - Manual (Page 17)

2. After washing, the blotted membrane is ready for autoradiography. 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. Make the autoradiographic
exposure with the moist membrane wrapped in plastic wrap or enclosed in a sealable plastic
bag. Do not allow the wet membrane to come in contact with the film, because wet
membranes will stick to the film, and any moisture on the film will cause black spots.
7.3 Hybridization Protocols for RNA Probes
The following protocols are for RNA probes to DNA blots. Casey and Davidson (1977) contains
protocols for RNA-RNA hybridizations.
Prehybridization
1. Place the blotted membrane inside a heat-sealable plastic bag. Seal three sides, leaving the
top side open.
2. Pipet in the prehybridization solution:
For DNA Bound to For DNA Bound to
Zeta-Probe Membrane Nitrocellulose
50% formamide 50% formamide
1.5x sodium, sodium phosphate, EDTA (SSPE) 0.1% SDS
1% SDS 5x SSPE
0.5% nonfat dry milk 5x Denhardt’s solution
200 µg/ml carrier RNA 200 µg/ml denatured salmon sperm DNA
500 µg/ml denatured salmon sperm DNA
The carrier DNA must be denatured before adding it to the prehybridization solution by
heating at 100°C for 5 minutes, followed by rapid cooling in ice.
3. Seal the bag and incubate.
DNA Bound to Zeta-Probe Membrane DNA Bound to Nitrocellulose
30 minutes at 50°C 4 hours at 42°C
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 minutes.
2. Cut one corner of the bag, remove the prehybridization solution, and replace it with
hybridization solution.
DNA Bound to Zeta-Probe Membrane DNA Bound to nitrocellulose
50% formamide 50% formamide
1.5x SSPE 1x Denhardt’s solution
1% SDS 0.1% SDS
0.5% nonfat dry milk 100 µg/ml denatured salmon sperm DNA
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 hours.
Note: Once hybridization has begun, do not allow the membrane to dry.
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