Reagents
Denaturation solution-- standard recipes
Transfer solution-- standard recipes
Hybridization solution
Reagent Final [ ] Stock [] Stock volume
SSPE 1.5x 20 x 7.5 ml
SDS 7% 20% 35 ml
SSS DNA* 100 µg/ml 10 mg/ml 1 ml
PEG 8000** 10% w/v 50% w/v 20 ml
dH20 55 M 55 M 36.5 ml
100 ml
Procedure
1) Blot and bake the filter per usual protocols.
2) Prewash the filter for a few minutes in 1 x SSC, 0.1% SDS.
3) Prepare the hybridization solution as above less the carrier DNA. About 0.1
ml/cm2 of filter area is sufficient.
4) SSS DNA is kept separate from the rest of the solution and is boiled at
the same time as the probe for 2-5 minutes just prior to the hybridization.
5) Prehybridization is not necessary. Add denatured probe (<10 x 106 Cerenkov
cpm/ml) and carrier to the hybridization solution; add hybridization solution
to the filter in a plastic bag and heat seal.
6) Hybridize for 12-24 hours at 65 C.
7) Wash beginning with the below protocol; all SSC wash solutions contain 0.1% SDS.
[SSC] Time Temperature
2 x SSC 5-10 min 20 C
2 x SSC 20 min 65 C
0.5 x SSC 20 min 65 C
0.2 x SSC 30 min 65 C