Variable PCR results - Ideas and Suggestions?

Bob Steinberg rsteinbe at etowah.ouhsc.edu
Fri Oct 31 18:11:13 EST 1997


Warren Lushia wrote:
> 

> What do you mean by PCR efficiencies?  More yield of the desired product
> per rxn?
Efficiencies are generally expressed as mean efficiency per cycle using
the formula: 
    Amplification factor = (1 +  efficiency)**(# cycles)--
efficiency can be estimated if you know the input sequence concentration
and run different #'s of cycles until you just get a quantifiable amount
of product.
    
> 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.
I would guess that there was something inhibitory in your dNTPs (other
than the dNTPs themselves)-- 50uM is only a couple of times the Kd of
Taq pol. for dNTPs, so you are working at less than maximum rates.

> 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? 
I agree that there are potentiall problems with excessive elongation
times, but we have found that a minute per kb is too short for good
efficiencies.



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