(by davidminde from gmail.com)
Mon Aug 10 12:33:34 EST 2009
... if your amplicon is "big" (e.g. 5-15 kb), you better choose phusion
polymerase (or the like) and it will just work ...(unless your template or
primers are degraded or you have problems with primer sequence specificity
which can be overcome by prolongation in many cases)
2009/8/10 malihe keramati <mlh_keramati from yahoo.com>
> hello all
> I am trying to pcr out a sequence with pfu. This
> > has worked before perfectly with taq, but now I get repeatedly emplty
> > I have tried to work with longer extension times as well
> > as annealing times,gradint pcr and increased enzyme, dnttp's ,
> > primers and mgso4 - but no success - also I digested the
> > genomic dna (bacterial ) and checked them for pcr . I keep getting
> > a gel with not the faintest track of a band.
> -Pfu and its buffer and dntp mix are functional because I check them by
> positive control and get a sharp band)).
> - I have checked these temperature :40,45,50,53,56,58,60.for pfu .
> taq pol pcr has sharp band in range of 52 to 62 C, lower temperature for
> taq has nonspecific band .
> the mg conc increased to 3mM and dNTP 0.25 mM of each and pfu conc is 1.25
> u/25 microlit .
> - the segment is about 1300 bp and extension time was set for 3 min in
> pfu reaction . However in all of experiences there is no pcr product, no
> band just primer dimmer,
> Would anyone of you have any idea on that?
> Best regards,
> melika .
> Methods mailing list
> Methods from net.bio.net
David Minde MSc (TUM)
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