Cloning vector with kanamycin instead of ampicillin resistance

Klaus Salger salger at zi.biologie.uni-muenchen.de
Thu Aug 8 21:30:26 EST 1996


Harry Witchel wrote:
> 
> Yo dudes and dude-ettes of molecular surfing --
>    I am looking for a general cloning vector (along the lines of pUC18
> or pGEM3z) which has the gene for kanamycin resistance instead of
> ampicillin resistance.  If anyone can send me an aliquot or give me
> the name of a supplier I would be stoked.
>    The reason I want it is that when I clone from
> one plasmid to another, there is always some left over vector from the
> first plasmid, and by having the second vector have a novel resistance
> (eg kanamycin), there will be no carry over of uncut original plasmid,
> nor will there be any original vector being recloned.
>    Many thanks,
>    Harry.Witchel at Bristol.AC.Uk
> Harry Witchel
> Dept. Physiology
> Medical School
> Bristol  BS8 1TD
> England

Hi Harry,

using another resistance on the target vector is one way to reduce
background but you may want to consider another alternative.

Cutting the source vector with ScaI or another enzyme that would destroy
its amp resistance cassette will also lead to decreased background.

I did it several times, not even preparing fragments - just putting the
digested DNAs together. Always gave at least something like 50% correct
clones (if I remember correctly...).

Of course, one has to choose an enzyme which won't cut inside the
insert...


Hope this helps
  Klaus


-- 
Klaus Salger		phone : +49 (0)89 5902 	-502
Zoologisches Institut	FAX   :			-450
AG Wetterauer		e-mail: salger at zi.biologie.uni-muenchen.de
Luisenstr. 14
80333 Muenchen
Germany

BioLinks: http://www.zi.biologie.uni-muenchen.de/~salger/salger.html



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