Fill-in reaction

Stephen R. Lasky Stephen_Lasky at brown.edu
Mon Feb 19 08:35:10 EST 1996


Brian: Klenow will work fine for filling this in and there is not really
any need for using a heat stable polymerase, especially TAQ which can add
a non-template driven base onto the end.  Just add all four dNTPs, Klenow
reaction buffer, your DNA and the Klenow and incubate.  Several companies
make fill in kits, but if you don't need a kit to do this fill in.  Even
if you don't have any klenow laying around your lab, someone down the hall
should have some and some 10X buffer and dNTPs. 


SRLasky

> I need a good protocol to blunt-end a SalI site.  I was a molecular 
> biologist in a past-life, and I don't have much experience with the
> new-fangled polymerases these days.
> The SalI site is:
> 
>        5'...G^TCGAC..3'
>        3'...CAGCT^G..5'
> 
> I think the overhang is compatible with Klenow, Taq,vent, whatever.
> But.... I'm just not sure.  I need a sure-fire protocol.
> 
> To let you know how clueless I am, I now a structural biologist.. doing 
> molecular biology... life is truly cruel...
> 
> 
> Bryan Sutton
> University of Texas Southwestern Medical Center
> Dallas,TX 
> email: sutton at howie.swmed.edu

-- 
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Stephen R. Lasky Ph.D.   Brown U/Roger Williams Medical Center,  Providence, RI.   
Phone: 401-456-5672     Fax: 401-456-6569     e:mail: Stephen_Lasky at brown.edu
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America may be unique in being a country which has leapt from barbarism to decadence without touching civilization.  John O'Hara.
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