[Yeast] tagging for overexpression (Cedric)

Cinzia Pagliuca via yeast%40net.bio.net (by cinthiait from yahoo.com)
Wed Jun 11 02:34:08 EST 2008


Hallo Cedric,

Usually I replace the stop codon with a restriction enzyme site and then insert the tag. You can find under "primer design" in the website, the software you wish and download. 

Ciao,
Cinzia


--- On Tue, 6/10/08, yeast-request from oat.bio.indiana.edu <yeast-request from oat.bio.indiana.edu> wrote:

> From: yeast-request from oat.bio.indiana.edu <yeast-request from oat.bio.indiana.edu>
> Subject: Yeast Digest, Vol 37, Issue 4
> To: yeast from magpie.bio.indiana.edu
> Date: Tuesday, June 10, 2008, 7:04 PM
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> Today's Topics:
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>    1. plasmid with secreted alkaline phosphatase (Ga?l
> Yvert)
>    2. tagging for overexpression (Cedric)
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> 
> ----------------------------------------------------------------------
> 
> Message: 1
> Date: Tue, 10 Jun 2008 13:40:53 +0200
> From: Ga?l Yvert <Gael.Yvert from ens-lyon.fr>
> Subject: [Yeast] plasmid with secreted alkaline phosphatase
> To: yeast from magpie.bio.indiana.edu
> Message-ID: <484E6845.9030808 from ens-lyon.fr>
> Content-Type: text/plain; charset=ISO-8859-1; format=flowed
> 
> Dear All,
> 
> We are setting up a reporter system requiring secreted
> enzymatic 
> activity in cerevisiae. The plasmid YEpFLAG-1 BAP from
> Sigma would be 
> perfect but it's no longer in their catalog and my
> contact at Sigma said 
> the product is discontinued. Would anybody having this
> plasmid be so 
> kind as to send a few microliters?
> 
> Many thanks for any help you can provide.
> Gael.
> 
> ----------------------------------
> Gaël YVERT
> LBMC CNRS UMR5239
> Ecole Normale Supérieure de Lyon
> 46 Allée d'Italie
> 69364 LYON cedex 07, FRANCE
> 
> Ph:   +33 4 72 72 87 17
> Fax: +33 4 72 72 80 80
> 
> http://www.ens-lyon.fr/LBMC/gisv/
> -----------------------------------
> 
> 
> 
> 
> ------------------------------
> 
> Message: 2
> Date: Tue, 10 Jun 2008 16:48:59 +0200
> From: Cedric <cedric.desmet from gmail.com>
> Subject: [Yeast] tagging for overexpression
> To: yeast from magpie.bio.indiana.edu
> Message-ID:
> 	<827989df0806100748o7a7c3ee9h55c67d3859d7b4a2 from mail.gmail.com>
> Content-Type: text/plain; charset="iso-8859-1"
> 
> Hello everyone,
> 
> I would like to tag a gene C-terminally with a His6HA3 tag.
> How do I decide
> where to insert this tag (directly before the stop codon?)
> and how do I
> design the primers for this? Is there a website or software
> available?
> 
> Cheers,
> Cedric De Smet
> Utrecht University
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