Any problems with Pfu Turbo
svetlov at oncology.wisc.edu
Thu Sep 10 10:13:41 EST 1998
In article <6t8arn$q0l at sun0.urz.uni-heidelberg.de>, January Weiner
<nospam_jweiner1 at ix.urz.uni-heidelberg.de> wrote:
> We switched to Pfu - Turbo from Stratagene and seem to have many problems
> with it. Under the specified conditions (te=1min/kbp, template 0,5 ug
> genomic bacterial DNA) sometimes there is no product at all, even though it
> ought to be quite small (~3.5 kb). The only non-standard thing is that one
> of the oligonucleotides has a coupple of nucleotides more bearing a
> restriction site.
> What are your experiences with Pfu Turbo?
The possible reason for this behavior is that Pfu itself appears to be more
sensitive to the polymerization inhibitors present in the oligos or the
template. In a number of parallel trials we were able to amplify sequences
from the yeast or coli genomic DNA with Pwo whereas yield in Pfu-driven
reaction it was barely detectable. Pwo alone (w/o Taq) amplified fragments
up to 5 kb in length while Pfu or Pfu/Taq mixes did not do well in
amplification of 1 kb + fragments. Try to purify better your template and
oligos or try other polymerase source.
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