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[Yeast] no FRET signal!

Nicolas Brandes nbrandes at umich.edu
Tue Feb 28 16:44:15 EST 2006


Hi,
I have a problem:

I have a CFP-YFP FRET construct, which already worked in C.elgans and 
COS7 cells. Now I cloned this construct into the p414GALL vector (SpeI, 
ClaI) and transformed this plasmid into EG103 cells (MATalpha leu2-3,11 
his 3delta1 trp1-289a, ura 3-52). I selected my transformants with 
SC-TRP plates which worked fine. But now I have a problem with the 
expression, because I can't see any GFP under the microscope and I 
don't get any FRET signal in the fluorometer.

Where is the problem? To do these experiments I grow my cells into 
minimal media with raffinose (glucose represses the promoter) and -TRP. 
After they reach an OD 1 I use whole cells samples to measure FRET at 
433nm. But I get no signal, only a large background signal because of 
my buffer.

For the microscope I streak out cells onto SC-TRP plates, let them grow 
  and stamp them then onto a minimal media plate with raffinose, 
galactose for the induction (glucose represses the promoter) and -TRP. 
But I get no GFP in the microscope.

Any ideas???

Nico


-- 
Nicolas Brandes <nbrandes at umich.edu>
University of Michigan
Department of MCDB
Room 4041 (Jakob lab)
830 N.University
Ann Arbor, MI 48109-1048




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