Biochemical Techniques and Instrumentation

PreHybridization Reaction

1. Place nitrocellulose blot in a seal-a-meal bag and seal 0.5 cm from each edge on 3 sides leaving the top open.
2. Prepare 25 ml of hybrization cocktail as follows:
   Fluka Formamide 12.5 ml
   20X SSC 6 ml
   1 M Tris pH 7.5 0.2 ml
   50X Denhardt's Solution 2.5 ml
   10% SDS 0.5 ml
   50% Dextran Sulfate (Oncor) 2.5 ml
   Water to 25 ml
3. Boil 0.25 ml of salmon sperm DNA (10 mg/ml in TE) for 7 min in an eppendorf tube.
4. Quick freeze both the salmon sperm DNA on dry ice.
5. Thaw the salmon sperm DNA at 68°C quickly.
6. Add the salmon sperm DNA to the premixed and warmed hybridization buffer and mix well.
7. Add the hybridization cocktail to the Southern blot in the seal-a-meal bag. Avoid bubbles in the hybridization bag by rolling them out with a 10 ml pipette on top of a glass plate. Make sure that the blot is uniformly wet.
8. Pre-hyb at 42^oC for 2 hrs.