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Protocols: ES Cell Culture - Gene Targeting - Protocols

Colony Hybridization

	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.