Variable PCR results - Ideas and Suggestions?

Warren Lushia walush0 at ukcc.uky.edu
Thu Oct 30 18:05:11 EST 1997


Bob Steinberg wrote:
> 
> 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
> > Canada
> > 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--

What do you mean by PCR efficiencies?  More yield of the desired product 
per rxn?  I had a similar problem with inconsistent results, for many 
months, and the trick that solved everything (by that point I thought I 
had tried everything) was to lower the dNTP's to 50uM.  Interestingly, 
when the PCR would work, the yield of the desired product was huge, but 
when it wouldn't work, there was nothing (using the higher dNTP conc.)  
Now it is extremely consistent, although not quite as much product as I 
know is possible (from my previous experiences.)  BTW, this was for 
Taq.....with a different enzyme (Deep Vent) I found the higher dNTP 
conc. worked better.  I don't have an explanation, but I'm sure that 
dNTP conc. was in fact the major contributor to inconsistencies.

The original poster does not state how big the desired product is and 
what template is being used for the amplification?  These could have 
important implications...most of the tips seem pretty sound though (WAY 
too much Taq in the mixture, and the cycling parameters could use some 
adjustment)....



> 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.

I would have to agree with Cetus on this.....the only thing I have found 
increasing extension time significantly beyond what is required (you are 
suggesting ~8-fold increase) is I have to wait longer for the tubes to 
come out of the machine.  Seems it could potentially cause problems as 
well (non-specific amplification products).  Have you seen this anywhere 
else, or is this from personal experience?  Just wondering .... seems 
everyone has their own way to do PCR (Pure Chance Reaction)...

Warren Lushia
University of Kentucky



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