Q: alkaline phosphatase and 3' overhangs

Dom Spinella dspinella at chugaibio.com
Mon May 5 13:05:34 EST 1997


Wolfgang Schechinger writes:
> Hi all!
> 
> Is alkaline phosphatase (currently, I want use calf intestine from 
> BM) suitable for dephosphorylating 3' overhangs as produced with 
> ApaI? 

I presume that it it is not the 3' overhang (which does not have a
phosphate) but the 5' recessed end that you are going after. Yes, you
can use CIP to de-phosphorylate 5' recessed ends.  However, it is a good
deal less efficient that de-phosphorylating 5' overhangs, so it may help
to use more enzyme (say 0.1-1 unit per pmol of ends).  Also, incubation
at 56 degrees rather than 37 is reported to help. Cheers. 
-- D.G. Spinella



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