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Zeocin and S. cerevisiae ...

[ISO-8859-1] Björn bjorn_johansson at bio.uminho.pt
Thu May 16 05:12:01 EST 2002


Hi,
I used zeocin alot for both laboratory and industrial strains of S.
cerevisiae with very good results. There are a few tricks:

Let the cells incubate for 3h in non-selective YPD before plating on
selective plate. Cayla, the manufacturer of zeocin (they licence it to
Invitrogen) recommends that you incubate the cells on ice one hour
prior to plating for lower background. I found this lowers the
background dramatically. Dont plate too many cells on each plate, this
increases the background.1

500µg /ml sounds like 10 times too much to me. I used 50µg/ml. Did you
adjust the pH to 7.5 as recommended ? Zeocin can be mutagenic, so
perhaps what you saw on 500µg /ml was some kind of mutants?

Finally, they stress the importance  of low salt when selecting for
zeocin. What about the sorbitol ? Is it compatible ? I dont know about
this one, I never used sorbitol for S. c. transformation (by LiAc meth
?)

Hope this can be of some help!

Cheers,
Björn Johansson
Centro de Ciencias do Ambiente-Departamento de Biologia
Universidade do Minho - Campus de Gualtar
4710-057 Braga
Portugal
http://www.uminho.pt/
tel +351-253604310
fax +351-253678980



Daniel Omar Espinoza <despinoza at rics.bwh.harvard.edu> wrote in message news:<abfl2h$dpt$1 at mercury.hgmp.mrc.ac.uk>...
> Hi,
> I would like to ask if anybody had good results using Zeocin in S.
> cerevisiae, and at what concentrations. I used synthetic liquid media
> and tried [Zeocin] from 0 to 500 ug/ml. The only concentration useful
> (were untransformed yeast didn't grow) seems to be 500ug/ml. Next, I
> transformed my strain with a Zeocin plasmid and plated it on synthetic
> medium + 500ug/ml Zeo. Now they don't seem to grow under these
> conditions (plates contain YNB + aa dropout mixture + 2% gluocose + 1M
> srobitol). From what I found, people use from 100 to 300ug/ml, but my
> strain grows at 300ug without any plasmid.
> 
> Any help/suggestions are welcome.
> Thank you very much.
> Sincerely,
> 
> Daniel Espinoza.
> 
> --
> Daniel O. Espinoza, Ph.D.
> Center for Neurologic Diseases
> Brigham and Women's Hospital
> Harvard Institutes of Medicine, HIM-616
> 77 Avenue Louis Pasteur
> Boston, MA 02115
> 
> Tel: 617-525-5746
> Fax: 617-525-5305

---





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