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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:
>Hi:
>
>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.

Frosty



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