HYBRIDIZATION PROCEDURE

Hybridization

Hybridization buffer [for 4 probes]

ddH20	25.0 mL
20X SSPE	12.0 mL
100X Denhardt's solution	2.0 mL
20% SDS	1.0 mL
Salmon sperm DNA [10 mg/mL; boil for 5 minutes and set on ice 5 minutes before adding]	0.2 mL
dextran sulfate	2.0 g
TOTAL	40.2 mL

1. Replace prehybridization buffer in each hybridization bottle with 10 mL of hybridization buffer.
2. Add probe to hybridization bottle. NOTE: do not allow the probe to come in direct contact with the membrane when first adding the probe. Make sure the probe goes directly into the hybridization solution in the bottle.
3. Allow probes to hybridize for 18–24 hours at 65°C.