Problem: My WT has the highest fluorescence data than strains with empty vector and GFP-containing.
Protocol:
1. I inoculated all my GFP - M.organophilum strains into a Choi medium - a medium predominantly containing phosphate, methanol and other trace elements.
2. I grew them into an O.D approximately at 2.0.
3. I centrifuged each of the culture and discarded all the supernatant and then resuspended it into fresh media and centrifuged again, for three times. I did this to minimize or eliminate possible sources like byproducts during culture and also to eliminate kanamycin in the media.
4. After resuspension, I pipetted 200uL of each of the culture into both black and transparent 96 well plates.
5. I did the reading using different combination of excitation/emission wavelength along with its respective bandwidth. The ff. are:
Excitation(bandwidth)/Emission(bandwidth)
495(10)/510(25)
485(20)/510(25)
465(35)/510(25)
495(10)/530(25)
485(20)/530(25)
465(35)/530(25)
495(10)/535(10)
485(20)/535(10)
465(35)/535(10)
Hello, Rey.
For GFP measurement, it says excitation peak at the range of 395 nm wave length. Your excitation range of 495-485 is far beyond the GFP peak range. The range you are set is for yellow or orange fluorescence.
Set the excitation (395) and emission ranges (509) again for WT and GFP cells. Prepare each cell stock, disperse, dilute cells for optimum readouts. Make a mixed cells of WT and GFP cells together in a tube. by keeping the cell concentration right. See whether you get expected values from three tubes freshly prepared.
Good luck.
Hi Prof. Lee, our fluorometer is filter based so I can only select which wavelength I can use based on our capability. I did another experiment using 365nm but the fluorescent readings fell way below than my fluorescent readings at 488nm. According to the equipment setting, the wavelength max for EGFP was at 480-490nm for excitation and 505-510 for emission, so I set it accordingly. However I will try to do the mixing and do SDS-PAGE if the GFP are really expressed.
Hello Christian,
I used a flat black plate so I guess it shouldnt be fluorescent.
Hi, Rey.
i) Can you just check bacteria pellet under a glass slide with a coverslip with a drop of PBS buffer without capturing air bubbles. See under a fluorescent microscope 40~63X objectives. You should see GFP under an appropriate filter block. Once you see the GFP light, then next step.
ii) Wash your cells in PBS instead culture medium. Bac culture medium contains lots of things undefined. Take measurement of GFP along with your previous way of washing cells in culture medium. Compare them. Use only black-walled plate. I guess you are using a spectrofluorimeter. It need a pre-warming to get a stable reading.
Regards.
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