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KAI-OLAF NETZER
• 20x SSC
• 20o/o (w/v) dextran sulfatein water (optional)
• Ultrapure water
• Prehybridization buffer. To make 10 ml, add
20x SSPE
100x Denhardt's solution
Salmon sperm DNA
Water
10% SDS
2.5 ml
0.5 ml
0.1 ml
6.8 ml
0.1 ml
(5.9 ml for nylon membranes)
{1.0 ml for nylon membranes)
The solution should be prepared fresh from stock solutions before use.
Add SDS as last component. Denhardt's and salmon sperm DNA stock
solutions should be stored frozen at -20°C.
• Hybridization buffer (onlyrequiredifdextran sulfate is tobe used; otherwise, prehybridization buffer can be used; see note below). To make 10
ml, add add
20x SSPE
2.5 ml
100x Denhardt's solution
0.5 ml
Salmon sperm DNA
0.1 ml
20% dextran sulfate
5.0 ml
(optional, see below)
Water
1.8 ml
(0.9 ml for nylon membranes)
10% SDS
0.1 ml
(1.0 ml for nylon membranes)
Note: Dextran sulfate may be added to increase sensitivity. However, increased background problems sometimes occur with dextran sulfate. Hybridization solution should be prepared fresh from stock solutions before
use. Add SDS as last component.
• W ash buffer 1 (2x SSC, 0.1 o/o SDS)
• Wash buffer 2 (0.25x SSC, 0.1 o/o SDS)
• Hybridization bag
• Heat sealer (industrial standard is preferable over household plastic bag
sealer)