dephosphorylation by SAP

Ian J. Mehr ijmehr at merle.acns.nwu.edu
Wed Jul 17 13:24:34 EST 1996


In article <Pine.SUN.3.91.960711132751.17038B-100000 at verdi>, hroychow at NMSU.EDU 
says...
>
>
>Hello All,
>        I had been using SAP lately. I use it the same way as I >would CIP
[snip]

Hiranya,

I use two-year-old SAP with some frequency, obtaining quite good results.  I 
simply add a unit or two directly to my restriction enzyme reaction, let it 
incubate at 37oC for about an hour, and kill it at 65oC for about 20min.  Not 
very precise, but it works like a charm.  To address your background problems, 
I take the clean (i.e., no salt, as it inhibits ligase), digested, SAPed DNA, 
and ligate it to itself in a dilute mix for a short period, such as an hour.  
I then run this ligation on an agarose gel, and gel isolate the linear, 
un-ligated form of the vector.  By doing so I reduce vector background in 
cloning to practically zero! 

Hope this helps,

Ian Mehr
Northwestern University Medical School
Dept. Microbiology and Immunology
ijm at nwu.edu




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