Hi experts,
I have cloned the DsRED gene into a plant vector under 35S promoter and used it for direct seed transformation of Chenopodium quinoa. When I checked the potential transformed seedlings (3 days old) for fluorescence under the red filter, I observed fluorescence in both control and potential transformants alike. Also, I can see mild fluorescence under the GFP filter too, even though there is no GFP in my plasmid. Could this be due to some auto-fluorescence? If so, how can I differentiate it from DsRED fluorescence? I could see no fluorescence in the newly formed roots which are white. Thoughts?
thanks in advance
From Article Imaging of the Blue, Green, and Red Fluorescence Emission of...
"Blue-green fluorescence derives from ferulic acids covalently bound to cell walls, and the red and far-red fluorescence comes from chlorophyll (Chl) a in the chloroplasts of green mesophyll cells."also: https://en.wikipedia.org/wiki/Chlorophyll_fluorescence
Actually, some secondary metabolites have also their fluorescence
Suja George In fluorescence microscopy, the lamp intensity and exposure time are the most critical parameters one should look at. If you use either higher lamp intensity (50% or more) or longer exposure time (> 1 sec), then probably you will see fluorescence everywhere. This is due to presence of flavonoids and other reductive compounds present in the cell that shows autofluorescence.
You should define the parameter in your case, try lamp intensity of 12-25% and exposure time 200-500 ms, under these conditions you would see the fluorescence only in transformed variety and not in the control lines.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence