Alk Phos treatment

Robert Coelen 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...
>
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>
>
>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.
>
>Mike Poidinger
>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
continue

Robert Coelen
Dept of Microbiology



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