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KAI-OLAF NETZER
• 100x Denhardt's solution (2 g/100 ml bovine serum albumine, 2 g/100 ml
Pieoll 4000 [e.g. Pharmacia], 2 g/100 ml polyvinylpyrrolidine [PVP])
• Denatured, fragmented salmon sperm DNA (10 mg/ml). After complete
dissolutionpass 10 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
• 20x SSC
• Dextran sulfate
• Ultrapure water
• Prehybridization buffer. To make 10 ml, add
20x SSPE
2.5 ml
lOOx Denhardt's
0.5 ml
Salmon sperm DNA
0.1 ml
Formamide
5.0 ml
Water
0.9 ml
10% SDS
1.0 ml
The solution should be prepared freshly from stock solutions before use.
Add SDS as last component.
• Hybridization buffer is identical to prehybridization buffer (see above).
10% (w/v) dextran sulfate may be added to the hybridization solution if
sensitivity is a problem. However, increased background problems
sometimes occur with dextran sulfate. If adding dextran sulfate, reduce
the amount of water appropriately. Heat the buffer to 42°C to dissolve
dextrane sulfate. Mix gently.
• Wash buffer 1 (2x SSC, 0.1% SDS)
• Wash buffer 2 (0.25x SSC, 0.1% SDS)
• Hybridization bag
• Heat sealer (industrial standard is preferable over hausehold plastic bag
sealer)
• Water bath (42°C) or hybridization oven (42°C)