cip & geneclean

michael coyne michael.coyne at channel1.com
Sun Aug 9 22:38:40 EST 1992


Hello fellow molbio.metters:

I have a question to which I have been unable to get a clear answer.  I
do not expect a cut-and-dried answer, but perhaps a consensus would be
helpful.

We have been attempting to clone a fragment of DNA into a high
copy-number vector (a derivative of the pUC plasmids).  We have had some
problems with incomplete digestion of the vector causing high
background, thus making identification of recombinant clones difficult
(using lacZ blue/white identification).  We have tried recovering the
linearized plasmid by excising the fragment from agarose gels after
electrophoresis, then CIP'ing the vector to minimize self-ligation, and
ligating a mixture of the vector and our fragment.  We have so far been
unsuccesful at recovering a clone.

This seems a pretty straight-forward experiment, and we are puzzled as
to why it has been so far unsucessful.  While discussing the
possibilties of why this hasn't worked (including the possibility of
gene dosage effects due to the high copy number of the vector) one of us
asked whether it would be better to CIP the vector digest *before*
running it out on a gel, rather than after the ends have been
dephosphorylated.

It may sound trivial, but is there any benefit to this?  In other words,
is there a difference between "vector digestion - gel electrophoresis -
recovery of fragment - CIP treatment" and "vector digestion - CIP
treatment - gel electrophoresis - fragment recovery"?

Thanks for any comments...

Mike Coyne
michael.coyne at channel1.com

---
 ~ SLMR 2.1a ~ If I knew what I was doing, would it be called research?
--
Channel 1 (R)   Cambridge, MA



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