User Manual

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

(Bio-Rad Laboratories 1987) (Jerome and Jaehning 1986)

50% formamide 50% formamide

1.5x sodium, sodium phosphate, EDTA (SSPE) 0.1% SDS

1% SDS 5x SSPE

0.5% non fat 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 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 DNA Bound to

Zeta-Probe Membrane 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 DNA Bound to

Zeta-Probe Membrane 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: After beginning hybridization the membranes should not be permitted to dry.