[Yeast] RE: Slow galactose induction (Kerry Geiler)

Wang, Xin-Xiang via yeast%40net.bio.net (by WangXX from MedImmune.com)
Wed Sep 5 13:29:34 EST 2007


Hello Kerry,
 
I used to induce yeast for GFP expression, also using Gal promoter.  I noticed the trace amount of repressing medium (raffinose in your case or glucose in my case) was essential for GFP induction.  If I washed the trace amount of repressing medium away with induction medium, GFP was not induced in even a week!  But with the trace amount of glucose medium in, induction took only 15 hours (not test shorter time). 
 
Did you wash away the raffinose medium before induction or not? Good luck!
 
Xinxiang

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Sent: Wed 9/5/2007 1:02 PM
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Subject: Yeast Digest, Vol 28, Issue 2



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Today's Topics

   1. Slow galactose induction (Kerry Geiler)
   2. RE: Slow galactose induction (Z.J. Zhang)


----------------------------------------------------------------------

Message: 1
Date: Tue, 4 Sep 2007 13:54:57 -0400
From: "Kerry Geiler" <kgeiler from gmail.com>
Subject: [Yeast] Slow galactose induction
To: yeast from magpie.bio.indiana.edu
Message-ID:
        <3fa3f1ca0709041054o692505aek8f75bbd8b3d1d072 from mail.gmail.com>
Content-Type: text/plain; charset=ISO-8859-1

Hi,

I am looking for suggestions as to why my galactose induction process
takes 6 hours.  I am expressing CFP under a gal promoter in media with
no glucose and 2% galactose.  I am getting fluorescence, but it takes
6 hours to see it.  Any ideas why induction could be taking so long?

I grow the cells up in media containing no glucose (2%raffinose)
overnight.  Then I induce 150uL of this culture with 2% galactose and
measure fluorescence every 15 minutes.  I do not see fluorescence
distinguishable from background until about 6 hours later.

Thanks very much for any suggestions!  Please reply to kgeiler from gmail.com.
Thanks,
Kerry



------------------------------

Message: 2
Date: Tue, 4 Sep 2007 15:27:43 -0600
From: "Z.J. Zhang" <ZZhang from uwyo.edu>
Subject: RE: [Yeast] Slow galactose induction
To: "Kerry Geiler" <kgeiler from gmail.com>, <yeast from magpie.bio.indiana.edu>
Message-ID:
        <C9C1AF307F12AF4087DEF87CB0E6A4AD01A3881F from TELEGRAPH5.uwyo.edu>
Content-Type: text/plain;       charset="US-ASCII"

Kerry:

One possibility is the slow maturation of your CFP protein, rather than
slow induction.

Depending on the version of the CFP, and where the CFP is tagged, it
could take hour(s) to mature.

Zhaojie

Zhaojie Zhang, Ph.D.
Director, Microscopy Core Facility
Department of Zoology and Physiology
University of Wyoming
Laramie, WY 82071
TEL: 307-766-3038
FAX: 307-766-5625
zzhang from uwyo.edu
http://www.uwyo.edu/microscopy





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