Trouble with PCR amplification of vector inserts

Robert M Bellin rbellin at iastate.edu
Mon Nov 28 20:14:57 EST 1994


To all,

I'm trying to PCR amplify vector inserts directly from bacterial
colonies using the protocol by Gussow and Clackson in "Nucleic Acids
Research", Vol. 17, p. 4000 (1989), using primers for the cloning site.
The problem is that in some cases my only PCR product seems to be the
cloning site sequence without the insert.  The ligation doesn't seem to
be the problem, and I've tried picking colonies from fresh plates to try
to minimize insert loss, but most attempts still result in no insert
amplification.  Any ideas would be greatly appreciated.

Thanks in advance.

Rob Bellin
-- 
Rob Bellin
rbellin at iastate.edu




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