Hi MWt PCR artefact

John Thompson jrt at home.com
Fri Jul 28 13:28:48 EST 2000


Mark Dowton <Mark_Dowton at uow.edu.au> wrote:

>A student in our lab is reproducibly seeing a high M.Wt band (it only
>enters the gel a mm or so after an hours electrophoresis in 1% agarose)
>after PCR of genomic DNA.  We think we've ruled out bacterial
>contamination (-ive Taq control does not show the band - this also rules
>out the possibility that it's genomic DNA), or any reagent artefact, as
>fresh reagents also show the problem, and we can amplify a house-keeping
>gene (28S rDNA) without getting the high M.Wt band.  I would guess that
>it's a bona fide PCR product, but it is far too big (>20 kbases)  to be
>produced in the 2.5 min extension we're giving this (we do have a
>Taq/PFU polymerase mix though). It only appears with ca. 3 mm Mg++ in
>the PCR reaction, much less is produced with 1 mm.  Any ideas?

A few years back we switched to a tricine-based buffer system for PCR.
Our PCRs maxed out at 20-25 cycles where we'd been using 30-40 cycles
in tris buffered systems.  Before we realized that fewer cycles were
needed, we saw high mw smears up the gel which I've dubbed overcycling
artifacts. Never tried to figure out their exact origin; just happy
that fewer cycles solved the problem. So pull a few tubes 5-15 cycles
early and take a look.

John Thompson
Merck Research Laboratories






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