Need Help Cloning in Charomid (Cosmid) Vectors

Peter T. Boag BOAGP at QUCDN.QUEENSU.CA
Mon Jul 25 09:22:53 EST 1994


We are trying to clone some minisatellites from birds. The method of
choice has been to size fractionate genomic DNA into the 6-16kb
range, and clone in Charomid (type of cosmid) vectors, constucted
with stuffer fragments so that they preferentially accept inserts in
the  10kb size range (we are using the 9-36 Charomid specifically).
We have grown up the charomid and can linearize it ok, and
distinguish between uncut and linearized vector on a low % gel with
large size markers. But we have been unable to get the vector to
ligate, to itself or to insert, ie. vector ligated in the absence of
insert apparently does not recircularize. The same reaction
conditions lead to ligation of the genomic DNA alone, and to to
ligation of more traditional vectors alone also. But not the
charomids.

Has anyone had experience with ligation of cosmids that might have
some suggestions? And more specifically, does anyone know what a
self-ligated vector of this type should look like on an ethidium
stained gel? Will it run at the same place as the original vector,
or is it nicked enough so it runs more like the linear form? Or
somewhere in between? Thanks.
***************************************************************

Peter T. Boag			TEL: (613) 545-6160
Department of Biology		FAX: (613) 545-6617
Queen's University		EMAIL: BOAGP at QUCDN.QUEENSU.CA
Kingston, Ontario
Canada  K7L3N6

***************************************************************



More information about the Methods mailing list