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CIPPers for breakfast

Frosty Loechel pulm012 at uabdpo.dpo.uab.edu
Fri Jul 22 12:37:56 EST 1994

In Article <CtCIzM.Fw1 at demon.co.uk>, Anthony Tomlinson
<anthony at pplros.demon.co.uk> wrote:
>I am trying to clone a ClaI fragment in to a ClaI site of a
>pBR322-derived vector, and am treating the vector with NEB's CIP at 10U
>of CIP to 5yg of 10Kb fragment, in 20yl of 1x Boehringer H buffer, for 2
>h at 37!C, followed by gel purification (Prepagene). It's not working. 
>Work with control fragments and vectors suggests that the ends are
>damaged, either in the ClaI digest (doubtful) or in the CIP step.  Does
>anyone know if CIP (especially NEB's) damages 5' overhangs?  I have
>previously CIPped blunt ends with no trouble.

You always have to be careful not to use too much CIP, or the ends do in
fact get damaged.  I've had good luck using Boehringer Mannheim CIP; I
haven't tried NEB's.  I used to make a point of using CIP buffer, but I've
found that it generally doesn't make any difference what buffer you use. 
Nowadays I just throw in the CIP for the last half hour of the restriction
digest, then extract with Strataclean to stop the reaction.  I frequently
have to remind myself that with CIP, more is *not* better.


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