Problem using pfuturbo for PCR

Alan Smith smitam01 at HOLMES.IPFW.EDU
Wed Jun 16 16:54:40 EST 1999


Hello

Over the past two weeks we have been trying to use pfuturbo DNA
polymerase to amplify a 2.3 Kb amplicon.  When using pfuturboDNA
polymerase we get an 800 bp amplicon, but when using taqDNA polymerase
we get the proper 2.3 KB amplicon.  My first hunch was that the pfuturbo
we were using was contaminated with another primer causing the problem
in amplification.  However, if there was contamination I would expect
two or more bands on my gels corresponding to one primer complementing
another (the contaminate) and another band corresponding to the primer
pair producing the proper amplicon, this was not the case.  The product
we get with pfuturbo is one very specific looking band on an agarose gel
at 800bp and when we use taq we get one specific band of the expected
size (2.3kb) and does prove to be the correct product when it is
digested with diagnostic restriction enzymes. We have just purchased
another batch of pfuturboDNA polymerase and we are having the same
problem agian .  So when I used the new pfuturbo plus new reagents
(dNTPs, water, etc) I get the same 800 bp band as seen with the old
pfuturbo I was using and this also means that there is no
contamination.  This leads me to believe that the pfuturbo for some
unknown  reason (to me) cannot amplify the correct product. Why can taq
produce the correct amplicon and pfuturbo cannot produce the correct
amplicon?  Has anyone ever experienced a problem like this before with
one polymerase giving the correct amplicon and another polymerase giving
an unexpected amplicon?   I seek advice in using pfuturbo DNA polymerase
to produce the proper 2.3 KB amplicon or any tricks of the PCR trade
that will help me through this oddity of a sistuation.  I have tried
adding DMSO and playing with the annealing temp (45-55 degrees) with no
luck.  Information is provided below on the reaction conditions used.

Primer 1 –> 51 mer with 20 homologous bases at 3' end.  The other 31 are
being added to the     template.  Tm= 56C for 20 homologous bases, and
Tm = 83 C for the whole 51 mer GC=52%
 Primer 2 –> 28 mer with 20 homologous bases at 3' end.  The other 8 are
being added (restriction site).  Tm=55C for 20 homologous bases, and Tm=
69C for whole 28 mer. GC=60.7%

Taq reaction–> 1uM of each primer, 2.5U taq, 0.200mM dNTPs, 2.0mM MgCL,
1X promega Taq Buffer, 25 ng plasmid DNA as the template. 100ul total
volume.
 94C 2 min
30x (94C 1 min, 50C 30 sec, 72C 1 min)
72C 7 min

PfuTurbo reaction–> 0.5 uM of each primer, 2.5U pfuturbo, 1X pfuturbo
stragene buffer, 0.200 mM dNTPs, 25 ng plasmid DNA as the template, 100
ul total volume
94C 2 min
30x (94C 1 min, 50C 30 sec, 72C 3 min)
72C 7 min
The polymerases are always added last in the reaction mixtures

Thank You for
Alan Smith





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