E coli and GFP

Hello All,

A big thank you to everyone for their suggestions on how to analyse GFP
in E coli. We've looked at the cultures with confocal and flow, fixed
and unfixed and found that by changing the fixation (student now
understands the importance of making fresh PFA not diluting from a 40%
stock!) we are now able to see great fluorescence in one mutant and v
low level fluorescence in the other.

Thanks again
Kathy

> -----Original Message-----
> From: Kathy Heel [mailto:kheel@meddent.uwa.edu.au]
> Sent: 22. august 2006 09:42
> To: Cytometry Mailing List
> Subject: E coli and GFP
> 
> Hello All,
> 
> We have a student trying to look at E coli transformed with a GFP
vector
> on a FACSCalibur. After inserting the vector, the E coli are grown for

> 1-7 days then fixed in 2% formaldehyde/2% glucose in PBS for 30
minutes
> and run on the Calibur. Problem is...I only see minimal (if any) GFP 
> fluorescence. Can anyone give us any tips on how to improve our 
> detection?
> 
> Thanks in advance
> Kathy
> 
> Dr Kathryn Heel-Miller
> Associate Lecturer
> Biomedical Imaging & Analysis Facility (M510)
> The University of Western Australia
> 35 Stirling Highway
> Crawley, Western Australia 6009
> Ph +61 8 9346 4525
> Fax +61 8 9346 3469
> Mob +61 416 085 960
> www.biaf.uwa.edu.au/biaf/
>