Methods Digest, Vol 51, Issue 9

[Virash Gupta]

Check with normal taq for integrity of template DNA

On 8/10/09, methods-request from oat.bio.indiana.edu <
methods-request from oat.bio.indiana.edu> wrote:
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> Today's Topics:
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>   1. pfu pcr  (malihe keramati)
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> ----------------------------------------------------------------------
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> Message: 1
> Date: Mon, 10 Aug 2009 07:27:24 -0700 (PDT)
> From: malihe keramati <mlh_keramati from yahoo.com>
> Subject: pfu pcr
> To: "methods from iubio.bio.indiana.edu" <methods from magpie.bio.indiana.edu>
> Message-ID: <870519.19574.qm from web112519.mail.gq1.yahoo.com>
> Content-Type: text/plain; charset=us-ascii
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>
> 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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> End of Methods Digest, Vol 51, Issue 9
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-- 
Dr V K Gupta
Sr Microbiologist (Molecular Biology)
Insect Molecular Biology Lab
Department of Entomology
Punjab Agricultural University
Ludhiana (Pb)-141004- India
M: 09815963210