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:
> 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...
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:
363mM Tris pH 7.9
To make the 10x user stock add 909 ul 11x stock, 20 ul BSA (50ug/ul,
Promega), and 71 ul water.
11 ul BME
489 ul water
Works like a charm!
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