Hi MWt PCR artefact

Mark Dowton Mark_Dowton at uow.edu.au
Thu Jul 13 19:15:27 EST 2000


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?

--
Mark Dowton
Australian Flora and Fauna Research Centre
Dept Biology
Wollongong University
Wollongong  NSW  2522
AUSTRALIA
Phone: 61-2-42125897
Fax: 61-2-42214135
Email: mdowton at uow.edu.au

And

Dept Applied and Molecular Ecology
Waite Campus, Adelaide University
PMB #1
Glen Osmond  SA  5064
AUSTRALIA







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