1. Using a black pen, mark the filter (Amersham's Hybond-NTM,
82 mm dia.) to identify the plate.
2. Lay the filter on the agar surface to make a replica of
the colonies. Leave for 1-5 minutes.
3. Using a needle, pierce the filter and agar in an asymmetrical
pattern to mark the position of the filter on the plate.
4. Carefully lift the filter off the plate and place colony-side
up on a piece of Whatman 3MM paper. Return the agar plate to the 37o C
incubator to allow the colonies to grow.
5. To lyse the colonies and fix the DNA to the membrane, autoclave
the filter(s) @ 212o C for 1 minute on the liquid cycle. (Note: The
autoclave on the 6th floor of the Taub Building works the fastest, with the
entire cycle taking about 8 minutes.) Remember to reset the temperature
on the autoclave to 250o C after use.
6. The filter(s) may be prehybridized and hybridized according
to the standard Southern protocol.