Reamplification of PCRs = Maximum errors?

Bill Burnett w.j.burnett at ncl.ac.uk
Mon Nov 2 13:46:33 EST 1998



David Hills wrote:

> From the latest Qiagen news booklet I see that to obtain a mutation  that
> represents >10% of a PCR product you need to start with <5 copies of a
> template and have a base misincorporated in the first cycle. SO then once
> I have generated a product (eg. extremely difficult PCR on tough template
> etc etc using touchdown with Amplitaq Au and it was my last aliquot of
> template also...) then can I routinely go back to a dilution of that
> amplification and reamplify with confidence knowing I'll never be inthe
> <5 templates ball park again?
> Dave
>
> Dr D Hills
> Roslin Institute

  Hmmm.  This doesn't mean >90% of copies in your PCR are faithful though,
does it? It just means no single error is represented by more than 10% of
copies...  Or am I wrong? (I hope so...)  Anyone know what % perfect copies I
should have after 30 cycles with taq?  I'd like to know if the "diversity" I
see among inserts in a shotgun cloning experiment reflects reality or just
taq errors...

Thanks...

Bill.
------------------------------------------
Dr. B. Burnett, University of Newcastle.  w.j.burnett at ncl.ac.uk






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