Mark_Dowton at uow.edu.au
Sun Aug 20 18:43:16 EST 2000
You could also try adding DNA before the Pwo. If you add Pwo first, the only
thing for it to chew on are the primers, but with DNA in there first, the
primer-chewing activity of Pwo will likely be quenched.
Obaid Yusuf Khan wrote:
> Try a mix of Pwo and Taq in a ratio of 1:5. Usually, the proofreading
> polymerases start chewing up your primers from the 3' end and proper
> annealing does not take place. This happens with primers of lengths greater
> than 15 bp. The 20% Pwo in the polymerase mix is supposed to do the
> proofreading for you while the main job is done by the good old Taq. You can
> try different ratios of the two enzymes and use the on that haas the highest
> Pwo ratio and gives you the best clean band.
> Good luck!
> > Hi all,
> > I have tried to use PWO (proofreading) DNA polymerase for PCR cloning.
> > With 'normal' Taq polymerase I get a nice one band PCR product. When I
> > use PWO with similar PCR conditions I get a smear on my agarose gel. I
> > have tried increasing the Mg-concentration, increasing the annealing
> > temp., changing both the enzyme and template concentration, the number
> > of cycles and elongation time, with and without DMSO. So probably most
> > common problems have been eliminated. Has anyone any other ideas???? I
> > am out of any...
> > Claudia
> > --
> > K N Harvey
> > Dept of Cell Biology & Genetics
> > PO Box 1738
> > 3000DR Rotterdam
> > The Netherlands
> > Tel: + 31 10 4087282
> > Fax: + 31 10 4087282
> > mailto harvey at ch1.fgg.eur.nl
> Do You Yahoo!?
> Get your free @yahoo.com address at http://mail.yahoo.com
Australian Flora and Fauna Research Centre
Wollongong NSW 2522
Email: mdowton at uow.edu.au
Dept Applied and Molecular Ecology
Waite Campus, Adelaide University
Glen Osmond SA 5064
More information about the Methods