SOUTHERN BLOT, con’t
Part II:
adapted from:
HYBRIDIZATION WITH DNA PROBE
Current Protocols in Molecular Biology
A note about volumes and vessels: Generally, we want to minimize volumes when using expensive reagents or when
trying to achieve a high concentration of some component (high concentrations make chance collisions more likely!),
and to use generous volumes for washing procedures. Wash volumes are not critical, but those containing some
specific reagent, such as the probe, or in the detection part, the SA-AP or BCIP /NBT are. Think about this before you
ask about volumes.
1. Wet the membrane with immobilized DNA in 2X SSC (make from 20X!)
2. Place membrane, DNA side up, in hybridization tube
3. Add about 1 ml APH (aqueous pre-hybridization) solution per 10 cm2 of membrane
4. Place tube in the oven, incubate 1 hr at 68 C
(aqueous prehyb does not contain formamide. When formamide is used in hybridization solution, the
hybridization temperature is typically 42 C.)
Very shortly before the end of this incubation:
5. Denature the probe DNA by heating for 10 min. at 100 C. Place on ice.
6. Add probe to a small volume (20-100uL per cm2 of membrane) of pre- warmed APH
The amount of probe to use depends in part on how well labeled it is. For radioactive probes we use 2 to 10
ng/ml, or sometimes a specific number of counts. For the colorimetric detection of biotinylated probe
(BluGene) recommends 100 ng/ml.
7. Pour the APH solution from the hybridization tube and replace with (pre-warmed) APH
solution containing denatured probe
8. Incubate overnight 68 C
APH
5 X SSC (make from 20X)
5 X Denhards (from 50X)
SDS, 0.5%, or 1%
Dextran Sulfate (5-10%) – optional
9.
50X Denhards
1 % (w/v) BSA Fraction V
1 % (w/v) Ficoll
1% (w/v) polyvinylpyrolidone
Washes: (specific concentrations of SSC and temperature depend on stringency desired;
volumes are not critical, but use enough to achieve good washing
In sequence do:
 2X SSC/0.1% SDS, 5-10 min. RT
 0.2X SSC/0.1% SDS, 2 x 5 min. RT
 0.16X SSC/0.1% SDS, 15 min. 65 C
 Briefly rinse in 2 X SSC
Proceed directly with detection or air dry.