Large PCR products

Randy P. Rasmussen Rasmussen at Bioscience.utah.edu
Thu Feb 3 14:01:39 EST 1994


In article <2imq1h$nou at mserv1.dl.ac.uk>, Jose_Costoya
<Jose-Costoya at SEINS.usc.es> wrote:

>
>    I would like to get some protocol for amplification of large 
> PCR products (> 1500 bp). 
>  
I can pass along two references.  The first is "In vitro Amplification of
DNA fragments > 10 kb" by Kainz et al in Analytical Biochemistry 202,46-49
(1992).  They recommend Tub polymerase, claiming that it is more processive
than Taq or Vent.  I have no first hand experience with Tub.

The second reference is "Effect of Heat Denaturation of Target DNA on the
PCR amplification" by Gustafson et al in Gene 123, 241-244 (1993).  In this
paper they use Taq to make DNA in the 2.5 kb range.  They claim that it is
critical to keep the amount of time spent at the denaturation temperature
to an absolute minimum.  When they boil their DNA before amplification for
1 minute they get good yields of large products, when they boil the DNA for
3 minutes they get almost no product.  Smaller products do not show this
effect.  They also use an Idaho Technology rapid air thermo-cycler to keep
the time at denaturation during the amplification at a minimum.

We use this second protocol and the air-cycler in our lab and we amplify
1000 to 4000 bp products all the time. 



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