Measuring Fluorescence in Bacteria

Measuring Fluorescence in Bacteria

This is the procedure for measuring the function of RFP and GFP in bacterial cultures.

1. Inoculate 5 ml LB broth with the appropriate antibiotic in a 15 ml tube with the clone to be measured and grow overnight or up to 20 hours in shaking incubator. Be sure to also grow up cells that cannot fluoresce for use as a control.
2. Centrifuge 5 min at 4000 RPM and resuspend bacterial pellet in 400 ul dH20
3. Transfer 250 ul of resuspended bacteria to a well in a white microtiter plate
4. Transfer 250 ul of dH20 into one of the wells for use as a blank
5. Choose the Cary Eclipse folder
6. Choose the Advanced Reads application for the fluorimeter
7. Set Up
8. For GFP, set Excitation wavelength = 395 nm, Emmission = 509 nm
9. For RFP, set Excitation wavelength = 584 nm, Emmission = 607 nm
10. Exitation slit = 20
11. Emmission slit = 20
12. Average time = 0.1000
13. Options
14. Excitation filter = Auto
15. Emmission filter = open
16. PMT voltage = High
17. Replicates = 3
18. Accessories
19. Well Plate
20. Click Well Plate Reader
21. Auto Zero
22. Select wells
23. Click OK
24. Insert plate, Notch matches up to corner
25. Click Start
26. Ignore Data Save Warning
27. After measuring with the fluorimeter, transfer each of the samples to a clear microtiter plate and measure A600 on the microtiter plate reader