Source: Martin Scott (7/93)
Reagents
Hybridization solution
Reagent Final [] Stock [] Stock volume
SSC 6x 20 x 300 ml
Denhardt's 2.5 x 50x 50 ml
SDS 0.1% 10% 10 ml
SSS DNA* 100 µg/ml 10 mg/ml 10 ml
EDTA 2.5 mM 500 mM 5 ml
NaPyrophosphate 1 mM 125 mM 8 ml
dH20 55 M 55 M 617 ml
1000 ml
Procedure
1) After blotting and baking of the filter, prewash it in 0.1x SSC,
0.5% SDS for one hour at 68 C.
2) Prepare the hybridization solution as above, about 0.1 ml/cm2 of filter
area; SSS DNA is kept separate from the rest of the solution and is boiled for
2-5 minutes prior to addition just before incubation with the filter.
3) Place the filter and solution in a plastic bag and heat seal.
3) Prehybridize at hyb temperature for at least 1 hours (lower limit not known and may be zero). Hyb temperature should be at least 2 C lower than the melting temperature in degrees C computed by the following formula:
Tm = 2(A + T) + 4(G + C)
Note also that 55-65 C is satisfactory for computed Tm of 70-80 C.
4) Add the probe to be used (1.0-2.0 x 106 Cerenkov cpm/ml final volume) to 1-2 ml of hybridization solution. For plasmid DNA's, hybridization times of 2 hours may be satisfactory.
5) Wash twice at room temperature for 10-15 minutes in 6 X SSC, then once for 5 minutes at the hyb temperature or 55-65 C maximum. Air dry and expose to film.