pfu pcr

David-Paul Minde via methods%40net.bio.net (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)
best wishes,
David

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
> lanes.
> >
> >  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  .
>
>
>
>
>
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-- 
David Minde MSc (TUM)

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