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no FRET signal!

no FRET signal! - Yeast Forum

no FRET signal! - Discuss research on yeast the model organism. Post questions and topics on the molecular biology and genetics of Saccharomyces cerevisiae and other yeast species.


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Old 02-28-2006, 09:44 PM
Nicolas Brandes
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Default no FRET signal!



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 <[Only registered users see links. ]>
University of Michigan
Department of MCDB
Room 4041 (Jakob lab)
830 N.University
Ann Arbor, MI 48109-1048


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