Alk Phos treatment
robert at arbo.microbiol.uwa.oz.au
Sun Apr 26 21:38:02 EST 1992
In article <17001.9204220915 at subnode.susssys1.rdg.ac.uk> skspoidn at SUSSSYS1.RDG.AC.UK (Mike Poidinger) writes:
>In reply to:
>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' +
>----- End Included Message -----
>Yeah, all the time!
>We use calf intestinal phosphatase, and the problem of vector degradation
>occurs quite often. The way we minimise the effect is to
>a) Ensure that the CIP is pure, and not contaminated (when in doubt,
>buy a new batch)
>b) Use very little CIP in the reaction (about 0.1 to 0.01 units), even for
>a blunt end reaction
>Hope this helps.
>Dept of Microbiology
>University of Reading
and you can add to this not to overdo the time and when it says heat-kill you should kill
and extract (because just heat-killing might allow some of the vector degradation to
Dept of Microbiology
More information about the Methods