Obaid Yusuf Khan
obaidyusufkhan at yahoo.com
Sat Aug 19 05:19:30 EST 2000
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.
> 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...
> 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
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