SAP degrades my vector

Daniel Mytelka mytelka at mendel.Berkeley.EDU
Thu Dec 15 17:09:40 EST 1994


In article <Pine.OSF.3.91a.941214223436.9395A-100000 at saul1.u.washington.edu>,
James Gray  <jp at u.washington.edu> wrote:
>
>	I have recently encountered problems using USB's Shrimp Alkaline 
>Phosphatase (SAP).  On two occasions dephosphorylating NotI digested 
>pBluescript I've noted the SAP enzyme to degrade the vector.  I'm using 
>overnight incubation at 37C to ensure a complete reaction.  


I find that SAP works quite well in my hands most of the time. As long
as your digest is complete, you can virtually eliminate vector
background.  Using it with an enzyme that's finicky and doesn't cut to
completion isn't particularly useful.

I've spoken to people at USB, and they say that the preps are always
contaminated with some amount of endonuclease. They specifically told
me not to not to exceed the stated enzyme concentrations or time
(1 hour). 

Dan Mytelka
mytelka at mendel.berkeley.edu
  




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