Problem using pfuturbo for PCR

Dr. Duncan Clark Duncan at nospam.demon.co.uk
Thu Jun 17 03:18:14 EST 1999


In article <37681FC3.86DD16B5 at holmes.ipfw.edu>, Alan Smith
<smitam01 at HOLMES.IPFW.EDU> writes
>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%
>

I wonder if the 3'-5' exonuclease activity of the PfuTurbo is shortening
your primers (in the time it takes to prepare the PCR mix and get it to
94C) such that they then anneal elsewhere on your target. Try adding the
enzyme to the PCR mix in tubes at 94C on the PCR machine i.e. a manual
hotstart.

Duncan
-- 
The problem with being on the cutting edge is that you occasionally get 
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk



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