Clone gene by PCR

Rafael Maldonado rafael at howard.genetics.utah.edu
Wed Nov 1 15:36:56 EST 1995


On 1 Nov 1995, Walter Ogston wrote:

> dtyyu at ucla.edu (David Tak Yan Yu) writes:
> > 
> > I have been able to clone eukaryotic genes by RT-PCR for expression.  Can I 
> > clone a bacterial gene using the bacterial DNA as template?
> > 
> The only thing you have to watch out for, as far as I know, is
> possible prokaryotic DNA sequences contaminating the Taq
> polymerase.  If you put lots of the target DNA into the
> amplification, keep to low cycle numbers, 

Don't bother on that. I have never got any contamination with my Taq 
polymerase from Perkin-Elmer (no relation etc.). It is easier with DNA 
since you don't have to worry about RT step, and the complexity of the molecule 
is lower, because of the smaller number of genes of prokariotes.

Some discussion had been going around about some DNA contaminant in the 
Taq polymerase preps. But if you are using matching oligos (not 
degenerated), and your gene is really in the bacteria, the proportion of 
any contaminant DNA will be ridicolous if you compare with your target 
gene. You will amplify almost 100% the desired gene.

> and do your
> no-template controls it should be OK.  

Yes, do always the controls. Walter is right!!!

	Rafa

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