Difference between revisions of "Measuring Fluorescence in Bacteria"

Revision as of 16:56, 8 September 2010

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. Install plate reader into fluorimeter
6. Username = fluorescence ; Password = password
7. Choose the Cary Eclipse folder
8. Choose the Advanced Reads application for the fluorimeter
9. Set Up
10. For GFP, set Excitation wavelength = 395 nm, Emmission = 509 nm
11. For RFP, set Excitation wavelength = 584 nm, Emmission = 607 nm
12. Exitation slit = 20
13. Emmission slit = 20
14. Average time = 0.1000
15. Options
16. Excitation filter = Auto
17. Emmission filter = open
18. PMT voltage = High
19. Replicates = 3
20. Accessories
21. Well Plate
22. Click Well Plate Reader
23. Auto Zero
24. Select wells
25. Click OK
26. Insert plate, Notch matches up to corner
27. Click Start
28. Ignore Data Save Warning
29. After measuring with the fluorimeter, transfer each of the samples to a clear microtiter plate and measure A600 on the microtiter plate reader