Christa Baumstark-Khan wrote:
>> Dear GFPlers
> I try to transfect CHO cells with the Clontech pGFP-N1 vector using a
> liposome transfection assay. So far we succeded in the experiment as
> under selection conditions (G418, 1.5 microgram per ml medium) we got
> realy nice colonies, surviving the procedure. So far, so good, but non
> (realy none!!!) of the colonies gives any shine of green fluorescence.
> We harvested such colonies and cells, which have not been transfected
> in order to measure fluorescence output in a spectrofluorimeter.
> Again, ther was not a glimpse of fluorescence. It was our 3rd
> transfection with the same result. Something is wrong, but what is it?
> Has anyone an idea?
> Thanks
> Christa
>> Dr. Christa Baumstark-Khan
>> DLR, Institut fuer Luft- und Raumfahrtmedizin
> Linder Hoehe
> 51147 Koeln
>> Tel.: (49) 02203-601-3145
> Fax: (49) 02203-61970
> e-mail christa.baumstark-khan at dlr.deHi Christa,
my first question is, are you using the native GFP from Clontech? We
have tried to use GFP-N2 and get absolutely no fluorescence whatsoever
using our confocal set ups. We get much better results with one or other
of the GFP mutants which are currently availabe (eg S65T). Many of these
mutants have a much higher fluorescence than the native protein and are
also much more stable. There's lots of info about them in the archives
to this newsgroup. Clontech now do a mutant GFP - eGFP (enhanced GFP)in
the same formats as GFP-N1-3 and GFP-C1-3. But whether they are any
better, I don't know. We gave up using Clontech GFPs as we could not cut
the sequence out of the plasmid in an attempt to replace it with a
mutant sequence.
Hope this helps,
--
***********************************************
Dr Andrew Doherty
Department of Anatomy
School of Medical Sciences
University Walk
Bristol
UK
BS8 1TD
e-mail Doherty at bsa.bristol.ac.uk
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