Variable PCR results - Ideas and Suggestions?
rsteinbe at etowah.ouhsc.edu
Wed Oct 22 13:47:10 EST 1997
Warren Gallin wrote:
> 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
> wgallin at gpu.srv.ualberta.ca
I don't know where the idea that high dNTPs are inhibitory comes from--
the original PCR protocols called for 0.5 mM dNTPs and worked fine-- in
our hands, the higher dNTPs generally gave higher PCR efficiencies-- the
reason for decreasing dNTPs to 0.2 mM or lower is that the fidelity of
Taq polymerase is higher at lower dNTP concentrations--
As far as Doug's basic problem, I was struck by the fact that replicates
gave different results-- this sounds like sample well variability within
the thermal cycler, either because the wells are not uniform or because
the tubes are not making equal contact with the wells.
I would also note that Doug is taking a big hit on his Taq by denaturing
for 1 min at 95oC-- for DNAs with normal GC content, it is sufficient to
bring the tubes to 92oC for efficient denaturation-- using a machine
that regulates based on tube temperature, we denature 1 second at 92oC--
for a machine regulating on block temperature, you need to know
something about the relationship between block and tube temperature--
with our "MJ Research" machine, we use 10 seconds at 96oC, which we
believe brings the sample just up to about 92-93oC. Also, the annealing
time is excessive (10 to 30 seconds is more than enough), and it might
help (depending on product size) to increase elongation time-- although
Cetus claims a kb or so per min, we have found that efficiencies are
better if you allow about 1 min per 120-150 nucleotides.
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