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
