10 Hybridization Methods (Southern and Northern Blotting)
137
• Water bath (65°C) or oven (65°C)
• Heating block (95°C)
• X-ray cassette with intensifying screen
• X-ray film (e.g. Kodak X-OMAT AR or Dupont Cronex)
Procerlure
1. Wet the DNA filter in Sx SSPE.
2. Transfer the wet DNA filter into a plastic bag. Seal the bag at four sides
and cut off one corner.
3. Add prehybridization solution (0.1 ml/cm 2 l 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 65°C. Make sure that there are no air-bubbles on the
fllter 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 to the prehybridization
buffer or to freshly prepared hybridization buffer (if the use of dextran
sulfate is intended) at approximately 10-20 ng/ml (random primed labeling) or 50-100 ng/ml (nick translation). 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 the use of dextran sulfate is necessary to improve sensitivity (see
above), prepare fresh hybridization solution, preheat to 65°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 blot off 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.
Hybridization
137
• Water bath (65°C) or oven (65°C)
• Heating block (95°C)
• X-ray cassette with intensifying screen
• X-ray film (e.g. Kodak X-OMAT AR or Dupont Cronex)
Procerlure
1. Wet the DNA filter in Sx SSPE.
2. Transfer the wet DNA filter into a plastic bag. Seal the bag at four sides
and cut off one corner.
3. Add prehybridization solution (0.1 ml/cm 2 l 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 65°C. Make sure that there are no air-bubbles on the
fllter 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 to the prehybridization
buffer or to freshly prepared hybridization buffer (if the use of dextran
sulfate is intended) at approximately 10-20 ng/ml (random primed labeling) or 50-100 ng/ml (nick translation). 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 the use of dextran sulfate is necessary to improve sensitivity (see
above), prepare fresh hybridization solution, preheat to 65°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 blot off 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.
Hybridization