Which should be CIPed, 7K one or 3K one?

Bernard P. Murray, PhD bpmurray*STUFFER* at socrates.ucsf.edu
Tue Aug 25 17:19:27 EST 1998


In article <199808251554.JAA169320 at nestor.NMSU.Edu>, hroychow at NMSU.EDU
(Hiranya Roychowdhury) wrote:

> At 08:59 PM 8/24/98 -0700, Yang Sheng wrote:
> >Hi, all!
> >
> > I am to link a 3K fragment with a 7k one. They were cut out by ScaI from
> two plasmids 
> >derived from pBluscript SK(+). I considered that one should treated with
> CIP to prevent it from 
> >self-circlized. So, which should be CIPed?
> >
> >Thank you for your advice.
> 
> 
> Most simply put, the reason for dephosphorylating is to prevent
> intramolecular or concatenated recircularization of the vector molecule. Now
> it is up to you to decide, since you have not supplied us with any other
> info about your 3k and 7k fragments.  
> 
> Dr. Hiranya Sankar Roychowdhury

I think part of the reason that the answer is not an absolute one
is that under some circumstances it is more advantageous to
deP the insert; eg. when ligating a very small insert to a vector
where dephosphorylating the insert would prevent concatamerisation
of the insert.  However, since intramolecular ligation is usually
very efficient this is a rare problem.  I would consider it very
unlikely that a tandem insertion of 3 kb (let alone 7 kb) fragments
would occur so dephosphorylating the vector would be more
appropriate in the example above.
     Bernard
-- 
Bernard P. Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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