10 Hybridization Methods (Southern and Northern Blotting)
135
radiation for different times (e.g. 1, 2, 3, 4, 5 minutes). Hybridize the fllter to
the appropriate probe, autoradiograph, and determine the optimal signal
strength.
20. The filter is now ready for hybridization or can be stored at 4°C. Nitrocellulose filters should be storedunder vacuum. Nylonfilters should
be sealed in plastic bags.
Subprotocol 3
Hybridization of DNA Blots
Many different protocols have been described for the hybridization ofDNA
blots. In general, they fall into two categories: hybridization at 42°C in the
presence of formamide, and hybridization at 65°C without formamide. The
speed of nucleic acid reassociation is higher at 65°C, so that 16 hours hybridization are sufficient to detect single-copy genes in genomic DNA blots.
In the following, a standard protocol for hybridization at 65°C is given. Normally, it is used with radioactively-labeled probes. With modifications,
however, it may also work when non-radioactively-labeled probes are
used. Refer to the manufacturer's instructions if you use non-radioactive
label.
Materials
• DNA probe labeled to high specific activity (approximately 1 x 10 9 cpm/
f..lg) with e.g. random primed labeling (for a protocol see Chap. 9)
• DNA filter
• 20x SSPE (3.6 M NaCl, 0.2 M NaH2P04, 0.02 M EDTA, adjust the pH to 7.5
with 10 N NaOH [approximately 6 ml/1])
• 100x Denhardt's solution (2 g/100 ml bovine serum albumin, 2 g/100 ml
Pieoll 400 [e.g. Pharmacia], 2 g/100 ml polyvinylpyrrolidine [PVP])
• Denatured, fragmented salmon sperm DNA (10 mglml). After complete
dissolutionpass 15 times through a 17-gauge hypodermic needle. Alternatively, sonicate at 70 W for 2 minutes on ice, then denature by boiling
for 10 minutes. Chili quickly on ice.
• 10% SDS in water
135
radiation for different times (e.g. 1, 2, 3, 4, 5 minutes). Hybridize the fllter to
the appropriate probe, autoradiograph, and determine the optimal signal
strength.
20. The filter is now ready for hybridization or can be stored at 4°C. Nitrocellulose filters should be storedunder vacuum. Nylonfilters should
be sealed in plastic bags.
Subprotocol 3
Hybridization of DNA Blots
Many different protocols have been described for the hybridization ofDNA
blots. In general, they fall into two categories: hybridization at 42°C in the
presence of formamide, and hybridization at 65°C without formamide. The
speed of nucleic acid reassociation is higher at 65°C, so that 16 hours hybridization are sufficient to detect single-copy genes in genomic DNA blots.
In the following, a standard protocol for hybridization at 65°C is given. Normally, it is used with radioactively-labeled probes. With modifications,
however, it may also work when non-radioactively-labeled probes are
used. Refer to the manufacturer's instructions if you use non-radioactive
label.
Materials
• DNA probe labeled to high specific activity (approximately 1 x 10 9 cpm/
f..lg) with e.g. random primed labeling (for a protocol see Chap. 9)
• DNA filter
• 20x SSPE (3.6 M NaCl, 0.2 M NaH2P04, 0.02 M EDTA, adjust the pH to 7.5
with 10 N NaOH [approximately 6 ml/1])
• 100x Denhardt's solution (2 g/100 ml bovine serum albumin, 2 g/100 ml
Pieoll 400 [e.g. Pharmacia], 2 g/100 ml polyvinylpyrrolidine [PVP])
• Denatured, fragmented salmon sperm DNA (10 mglml). After complete
dissolutionpass 15 times through a 17-gauge hypodermic needle. Alternatively, sonicate at 70 W for 2 minutes on ice, then denature by boiling
for 10 minutes. Chili quickly on ice.
• 10% SDS in water