HELP! Exo III

Pamela Norton pnorton at lac.jci.tju.edu
Wed May 31 16:17:15 EST 1995


In article <3qhhom$k35 at bisance.citi2.fr>, cocea at bisance.citi2.fr (Cocea)
wrote:

> Dr I.M. Clark (imc1001 at cus.cam.ac.uk) wrote:
> : I have been attempting to make deletions from an 850bp fragment in pBLCAT3 
> : using Promega's Erase-a-Base.  Screening the final colonies yields almost 
> : entirely 'random' sized plasmids, and I lose my insert entirely.  This is 
> : despite the fact that the deleted plasmid prior to re-ligation appears to
> : be sequentially deleted across my time points.  Any tips, hints or just
> : comments would be appreciated.
> 
> : Ian.

> ....
> secondary structures, but who knows? Given the fact that many sequencing
> reactions gave blank lanes, I thing Exo III eats up the sequence for the
> annealing primer, and thus there was no initiation. What I thought to do next

> 
> Regards, Laurentiu COCEA (cocea at citi2.fr)

We have also been using the Erase-a-Base system with moderate success. Like
Lauentiu, we saw a number of blank sequencing lanes. However, pre-screening
by cutting with an enzyme that lies to either side of the insert reduced
the incidence of these (we didn't try to sequence things that had lost the
restriction site beyond the primer). We have concluded that the cutting was
incomplete with the site that leaves a 3' overhang, leaving that end
susceptible to ExoIII digestion. 

The original poster did not mention the temperature/time points he used.
850 bp is pretty small, if you have't tried lowering the temp and going for
really short times, you might want to give it a try. 

Hope some of this helps,

        Pam Norton

-- 
Pamela A. Norton, Ph.D.          Assistant Professor of Medicine
Thomas Jefferson University
Philadelphia, PA 19107           p_norton at lac.jci.tju.edu



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