Variable PCR results - Ideas and Suggestions?

Warren Gallin wgallin at gpu.srv.ualberta.ca
Tue Oct 21 13:38:37 EST 1997


In Article <344cac3d.2019757 at news.telenet.net>, jonesd at telenet.net (Doug
Jones) wrote:
>I have been having a problem with an RT-PCR for murine IL-6.  After
>optimization, which went well, I have been unable to obtain consistent
>results, even in the same run.  
>
>The PCR is done in a mix using 1.5 mM MgCl, 50mM KCl, 10mM Tris, 0.1%
>Triton X-100, .1% NP-40, .1% Gelatin, 0.25 mM each dNTP's, 5 U Taq
>(Promega) and primers in a volume of 50 microliters.  The cycle is 1
>min at 95, 1 minute at 55, and 1.5 minutes at 72 degrees, for 35
>cycles in a Stratagene RoboCycler.  

Two things come to mind.  1) You have too much dNTP.  Anything over 200
micromolar inhibits the enzyme.  This is not simply due to complexing Mg, it
can not be rescued by adding more MgCl2.  2) I would try a range of Mg
concentrations.  The extra effort of making up three different Mg
concentration reactions is probably cheaper than floundering around with a
marginal result because you are not in the Mg optimum for the particular
primer pair that you are using.

Warren Gallin
Department of Biological Sciences
University of Alberta
Edmonton,  Alberta     T6G 2E9
Canada
wgallin at gpu.srv.ualberta.ca



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