Alkaline phosphatase problem...

Richard Sucgang rs54 at cunixf.cc.columbia.edu
Wed Apr 22 00:37:01 EST 1992


In article <1992Apr21.161143.9 at wums.wustl.edu> wetsel_r at wums.wustl.edu writes:
>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...
>

Well, we have had some experience with some contaminating activity
that can chew in overhangs during dephosphorylation. In general,
blunt ends are not so affected. The solution, as recommended by
tech support from NEB, is to use a buffer of 50 mM Tris (pH 8.5) and
1 mM EDTA, and 1/100 U of CIP per picomole of overhanged ends;
1 U per pmole blunt ends. Incubate at 50 deg for 1 hr, then 
at 65 deg for 30 min.

Worked for our sma cut vector...
-rich
Richard Sucgang : Dept. of Anatomy and Cell Biology
Columbia University (sucgang at cuhhca.hhmi.columbia.edu; 
de slime god         rs54 at cunixf.cc.columbia.edu)



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