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Measuring whole GFP bacteria


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#1 k_josefin

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Posted 23 February 2009 - 08:15 AM

Hi there!

Been getting some GFP E. coli. Can see them under microscope fairly well, but when I try to measure with a fluorometer I get nothing.

I've tried to scan for the best wavelength, but it finds neither an excitation peak nor emission peak.

I've tried to wash them too, they're now suspended in Ringer's solution. No effect.


What can I do?!?

#2 perneseblue

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Posted 23 February 2009 - 08:18 AM

what do you know about this strain? Perhaps the GFP gene is under an inducible promoter?
May your PCR products be long, your protocols short and your boss on holiday

#3 k_josefin

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Posted 24 February 2009 - 12:08 AM

I know so much that you need IPTG to induce the GFP production, but that was added (1mM). And as I mentioned, I see the bacteria using fluorescence microscopy. I did the microscopy just prior to measuring in fluorometer, so it all seems very strange to me :)

#4 molgen

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Posted 25 February 2009 - 08:11 AM

What exactly do you want to measure? The efficiency of the transformation/expresion under different conditions……

What kind of fluorometer are you using? Spectrophotometer/flowcitometer…….

The more details you give the better we can try to help

#5 k_josefin

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Posted 02 March 2009 - 01:55 AM

What exactly do you want to measure? The efficiency of the transformation/expresion under different conditions……

What kind of fluorometer are you using? Spectrophotometer/flowcitometer…….

The more details you give the better we can try to help



Hi!

Sorry for being short on the details =)

I want to measure bacterial growth, but try to avoid the staining procedure since I want to measure the same sample over longer a longer period. I thought that GFP producing bacteria would be good for this. I also thought that using flourometric methods instead of turbidity I would increase the sensitivity of the assay.

The flourometer I'm using is a spectrophotometer type from Varian (model name Eclipse). It can scan for both excitation and emission peaks. I can also read multiwell plates with it, which was my plan when I strated this.




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