strange cloning result

David L. Haviland dhavilan at imm2.imm.uth.tmc.edu
Wed Mar 29 19:27:16 EST 2000


Nicolas Lemee wrote:
> 
> I was told it has a non negligible tendency to overchew. Don't ask me for ref.
> or what not, never looked into it :) I just don't use it whenever possible, and
> usually it is.

Nicolas:

I wouldn't say non-negotiable but with concentration and time you can
minimize it.  I've cloned quite a bit with CIAP and have had problems on
occasion.  SAP has circumvented all of the woes.  I've used CIAP
anywhere from the prescribed 0.01U/pmole of ends all the way to 1U/pmole
of ends.  Most of the troublesome vectors were prepared with the higher
concentration of CIAP.  I had the best results with the CIAP sold from
Sigma that comes in what I recall is a NH4Cl stock where the CIAP is in
precipitated form.  We'd mix the tube, take a aliquot, spin it,
resuspend/redisolve the CIAP it in 1X CIAP buffer, and dilute
accordingly.  Some in the lab didn't like this form of CIAP as it took
too much time to prepare (read: lazy).   The premade suspensions from
other companies that are akin to restriction enzymes never worked
consistently for us.

As far as detailing the woes of CIAP, Hiranya couldn't have summarized
it any better (kudos!).

Many moons ago, we were given a tube of SAP from our Roche rep, then
BMB.  We never looked back.  We've been buying ever since.
 
Hope this helps,
David
===========
David L. Haviland, Ph.D., Asst. Prof. Immunology 
University of Texas - Houston, H.S.C.
Institute of Molecular Medicine, R907
2121 W. Holcombe Blvd.,  Houston, TX  77030 
713.500.2413-Voice//713.500.2424-FAX
-----------------
If everything seems to be going so well, you have obviously 
overlooked something.
==========
---




More information about the Methods mailing list