Water used here and throughout method for all buffers is dou-
ble distilled water which is then autoclaved and kept in 2 L
bottles. Failure to autoclave often leads to DNA degradation
due to DNases that are often found in double distilled water
which is especially found if the water has been stored in direct
sunlight.
After 5 min of rehydration the rehydrated gel is about 0.5 mm
thick and quite tough, being resilient to folding without break-
ing and is treated from now on the same as you would a nylon
membrane.
The denaturing and neutralizing solutions can be reused many
times without loss of activity. Simply return the solutions back
to their bottles after use.
Prehybridization is made up from stock solutions of: 5% PVP
(0.4 mL), 5% ficoll (0.4 mL), 10 mg/mL herring testes DNA
(0.3 mL), 10% SDS (1 mL), 20× SSC (6 mL), and 1 mL of full
cream UHT milk. The herring testes DNA is sheared by repet-
itive pipetting and then added to the prehyb mix. The UHT
milk can be purchased from any supermarket. Prehybing can
be left from 24 to a maximum of 72 h.
When amplifying the DNA to be used as a probe it is a good
idea to amplify the desired gene from an organism that is dif-
ferent to the one being probed (e.g., if probing a gel of
DNA’s derived from E. coli strains use a Klebsiella pneu-
moniae strain containing the desired gene as a template for
the probe PCR as this will minimize any background hybrid-
ization problems).
The kit utilizes random primers, which are 9 bp single stranded
oligomers. These will bind randomly by chance to about every
200–300 bp of DNA. The minimum sized PCR product for
efficient labeling is therefore about 500 bp. However, in prac-
tice we find that PCR products between 800 and 1000 bp pro-
duce ideal probes.
The probe is added to the prehybridization solution that the
gel has been prehybridizing in. There is no necessity to change
this solution before adding the boiled probe.
Washing can be repeated several times to ensure low back-
grounds. The gel can even be left washing over a weekend
without any discernable reduction in probe signal. During
washing the gel can be removed from the hybridization tube
and background levels tested by holding a Geiger tube against
parts of the gel, which contain no sample (i.e., the small gel
section above the loading wells). In this way low backgrounds
can be assured before autoradiography.
We use trays containing developer and fixer rather than a machine
as the film can be left until all bands are visible before fixing.
