10 Hybridization Methods (Southern and Northern Blotting)
145
• Heating block (95°C)
• X-ray cassette with intensifying screen
• X-ray film (e.g. Kodakor Dupont)
Procedure
1. Wet the RNA filter in Sx SSPE.
2. Transfer the wet RNA filter into a plastic bag. Seal the bag at four sides.
Cut off one corner.
3. Preheat prehybridization solution to 42°C. Add 0.1 ml/cm 2 to the bag
through the open corner. Try to avoid bubbles by careful pipetting.
Squeeze out all air bubbles from the bag. Seal the bag completely.
4. Incubate the bag in a shaking water bath or between two glass plates in an
oven for 2-4 hours at 42°C. Make sure that there are no air-bubbles on the
filter surface.
5. Denature the probe for 10 minutes at 95°C. Immediately chill on ice for 5
minutes.
6. Cut off a corner of the hybridization bag. Empty the prehybridization
buffer into a 15 or 50 ml Falcon tube. Add probe at approximately
10-20 ng/ml (random primed labeling). In general, 10 6 -10 7 cpm/ml
should suffice. Mix gently. Add the hybridization solution to the filter
by using a disposable plastic pipet.
Note: If it is necessary to improve sensitivity (see above), prepare hybridization solution with dextran sulfate ( 10% w/v) freshly, preheat to 42°C, then
add denatured probe. Mix gently, and add the solution to the filter. Do not
add radioactive probe directly into the bag.
7. Squeeze out all air bubbles through the open corner of the bag. Use a
paper towel to remove traces of hybridization buffer that come out.
Seal the bag carefully. A void leakage. If necessary, seal the bag in a second
bag to prevent contamination.
Note: W ear gloves when working with radioactive probe. W ork according to
your laboratory's safety rules. Dispose of radioactive waste properly.
8. lncubate for 12-16 hours at 42°C in a shaking waterbath or between two
glass plates in an oven.
Hybridization
145
• Heating block (95°C)
• X-ray cassette with intensifying screen
• X-ray film (e.g. Kodakor Dupont)
Procedure
1. Wet the RNA filter in Sx SSPE.
2. Transfer the wet RNA filter into a plastic bag. Seal the bag at four sides.
Cut off one corner.
3. Preheat prehybridization solution to 42°C. Add 0.1 ml/cm 2 to the bag
through the open corner. Try to avoid bubbles by careful pipetting.
Squeeze out all air bubbles from the bag. Seal the bag completely.
4. Incubate the bag in a shaking water bath or between two glass plates in an
oven for 2-4 hours at 42°C. Make sure that there are no air-bubbles on the
filter surface.
5. Denature the probe for 10 minutes at 95°C. Immediately chill on ice for 5
minutes.
6. Cut off a corner of the hybridization bag. Empty the prehybridization
buffer into a 15 or 50 ml Falcon tube. Add probe at approximately
10-20 ng/ml (random primed labeling). In general, 10 6 -10 7 cpm/ml
should suffice. Mix gently. Add the hybridization solution to the filter
by using a disposable plastic pipet.
Note: If it is necessary to improve sensitivity (see above), prepare hybridization solution with dextran sulfate ( 10% w/v) freshly, preheat to 42°C, then
add denatured probe. Mix gently, and add the solution to the filter. Do not
add radioactive probe directly into the bag.
7. Squeeze out all air bubbles through the open corner of the bag. Use a
paper towel to remove traces of hybridization buffer that come out.
Seal the bag carefully. A void leakage. If necessary, seal the bag in a second
bag to prevent contamination.
Note: W ear gloves when working with radioactive probe. W ork according to
your laboratory's safety rules. Dispose of radioactive waste properly.
8. lncubate for 12-16 hours at 42°C in a shaking waterbath or between two
glass plates in an oven.
Hybridization