Alkaline phosphatase problems

Gopalakrishna Dasika DGK at ZEUS.VETSCI.WISC.EDU
Thu Apr 23 15:39:02 EST 1992


Hi!

> In reply to:
> Message-Id: <9204220016.AA20570 at genbank.bio.net>
> To: methods-and-reagents at net.bio.genbank
> From: wetsel_r at edu.wustl.wums
> Subject: Alkaline phosphatase problem...
> Date: 21 Apr 92 22:11:42 GMT
> Hi There:
>
> A unique problem has surfaced in our lab and I was wondering if anyone else has
> had a similar experience.  When using alkaline phosphatase to remove 5'
> phosphates from cloning vectors, has anyone ever had the problem of having the
> alkaline phosphatase treatment digest one's vector as well?  Various reagents
> have been changed, water, Tris, ZnCl2, the enzyme, all to no avail.  Does
> anyone have any suggestions or comments?
>
> Thanks in advance...
>
> ===========================================================================
> +  David L. Haviland, Ph.D.        Internet:"haviland at kids.wustl.edu"   +
> +  Washington Univ. School of Med.   A.K.A : The Compiler                 +
> +  Dept. of Peds./Pulm. Box 8116     ICBM-Net : Just hit St. Louis        +
> +  400 S. Kingshighway               &-6  <- User is Brain Dead...        +
> +  St. Louis, MO  63110              FAX: 314-454-2476                    +
> +  (314) 454-6076                                                         +
> +  "...just because you know how to win an election doesn't mean you      +
> +      know how to run the country..."  - Robbie Sinclair, from the       +
> +                  TV show  'Dinosaurs'                 +
> ===========================================================================
>
In the past we used to get CIP from Boehringer Mannheim and never had
this problem. Recently we got it from Promega and I am having trouble
with it.( I am not saying that one is better than the other ).I am
trying to figure out the optimum concentration and time required for
the dephosphorylation. I use the buffer supplied by Promega. I always
inactivate the enzyme by heating to 65 degrees C in the presence of
EDTA for 1 hr followed by phenol:Chloroform extraction.

Gopal.




**********************************************************************
Gopalakrishna Dasika                                1655, Linden Dr.
Dept.of Animal Health & Biomedical Sciences         Madison WI 53706
University of Wisconsin-Madison                     PH:(608)262-2317
**********************************************************************









More information about the Methods mailing list