Reagents
Denaturation solution-- standard recipes
Transfer solution-- standard recipes
Hybridization solution
Reagent Final [] Stock [] Stock volume
NaPO4, pH 7.2 0.5 M 1 M 50 ml
SDS 7% 25% 28 ml
EDTA 1 mM 500 mM 0.2 ml
BSA 1% 5% 20 ml
dH20 1.8 ml
100 ml
Procedure
1) Blot the filter per usual protocols.
2) Prewet the filter for a few minutes in 1 x SSC, 0.1% SDS.
4) Prehybridization at 50 to 65 C for 2 to 12 hours: After getting rid of dripping
liquid, put the filters into bags or the containers. Add 5 to 10 ml hybridization
buffer for each filter.
5) Denature the probe (DsDNA) by boiling in water for 5 minutes and put immediately into icewater.
6) Hybridization at 50 to 65 C for 12 to 36 hours: Add 1 to 10 x 106 Cerenkov
cpm/ml of denatureed probe to the pre-hybridization solutions containing the
filters.
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 50 to 65 C
0.5 x SSC 20 min 50 to 65 C
0.2 x SSC 30 min 50 to 65 C
0.1 x SSC 30 min 50 to 65 C
You can repeat any washing step if necessary, you can also stop washing at any point if your background is low enough (100 cpm by survey meter).