Hybridization of filters can be performed in either rotisserie
bottles or heat-seal bags. In our lab both work well.
We routinely hybridize up to seven filters in one bag and
up to five filters in a large (7.5cm diameter) bottle (with
nylon mesh between filters). The filters have been successfully
hybridized using a number of different published "Southern"
DNA hybridization protocols incorporating a variety of buffer
systems. It has been our experience that all of these
established protocols will work with the high density filters.
In our lab we routinely follow the following procedure:
Wet filters in minimum volume of "Church Buffer"
in large petri dish. Keep track of volume of buffer
absorbed into the filters as this will contribute to your
overall volume. If using bottles, place nylon mesh
between filters as you wet them.
Place filters in bottle or bag, add approx. 25ml additional
"Church Buffer" and seal.
Pre-hybridize one hour at 65°.
Add labeled probe(s) so that the total cpm of each probe
is 1X106 to 11X107. We routinely
label our probes by random prime labeling. PCR incorporation
also works well for smaller (>100-200bp) probes.
Hybridize overnight (16-18 hours) at 65o C.
Wash filters (in bag or bottle) with Wash I solution
at 65o C. Repeat wash about 4 to 6 times
until most of the non-bound probe is removed. At
this point the filters can continue to be washed in the
bag or bottle or placed in a large tray in a 65o
C water bath for more efficient washing.
Wash filters with wash II solution repeatedly at 65o
C until the level of cpm (monitored with a Geiger counter)
removed remains constant (no longer decreases).
Rinse filters in water and wrap individually in plastic
wrap. Take care to remove air bubbles between filter
and wrap by pressing with a wipe to force out air.
Place two filters in a large cassette (35x43cm), there
will be a small overlap of filters but this normally is
not a problem in interpreting data. We place two
filters per cassette to maximize film use.
Expose for 2 to 72 hours at -80o C.
This depends on the intensity of signal from filters.
We have found it best to develop one piece of film after
4-5 hours exposure to determine the optimum exposure time
required for all of the filters.
Interpret the position of the positive clones following
the protocol supplied with the filters using the transparent
overlay as an orientation guide.
Store the used filters in a sealed hybridization or zip-lock
bag in "Church Buffer" to keep them wet.
Allow the radioactivity to decay out for 2-4 half lives
before using again. We do not recommend stripping
the filters as smearing of the DNA may result and the
overall life of the filters may also be diminished.