Fill-in reaction

Gary K. gtk10583 at glaxo.com
Fri Feb 23 16:15:49 EST 1996


In article <4g3mji$ko6 at swsu65.swmed.edu>, "R. Bryan Sutton"
<sutton at howie.swmed.edu> wrote:

> 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,

Resuspend your DNA in the following:

   2 ul 10x buffer (see below)
   0.4 ul BME (see below)
   2 ul 20mM dNTP's
   0.5 ul T4 DNA polymerase
   Water to 20 ul total volume

Incubate 10 min. at 37C

10x buffer is made from 11x stock:
   11x stock:
      363mM Tris pH 7.9
      726mM KoAc
      110mM Mg(oAc)2
   To make the 10x user stock add 909 ul 11x stock, 20 ul BSA (50ug/ul,
Promega), and 71 ul water.

BME stock:
   11 ul BME
   489 ul water

Works like a charm!

Gary



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