Hi MWt PCR artefact

Anders Gorm Pedersen gorm at cbs.dtu.dk
Fri Jul 14 02:36:34 EST 2000

Mark Dowton 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?

>From the extension time I think you're right - it's probably not as
large as it appears. Could it be something in the primer stock?
(Something sticky or denaturing or something?).

Puzzled - 


 Anders Gorm Pedersen, cand.scient., Ph.D.  (gorm at cbs.dtu.dk)

 Center for Biological Sequence Analysis
 Technical University of Denmark
 Bldg 208, DK-2800 Lyngby, Denmark

 phone: (+45) 45 25 24 84
 fax:   (+45) 45 93 15 85

 Web: http://www.cbs.dtu.dk/gorm/

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