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 Advanced Reads application for the fluorimeter
6. Ex. slit = Em. slit = 20, Ave. time = 0.1000, Excitation filter = Auto, Emmission filter = open, PMT voltage = High, Replicates = 3
7. For GFP, set Excitation wavelength = 395 nm, Emmission = 509 nm
8. For RFP, set Excitation wavelength = 584 nm, Emmission = 607 nm
9. After measuring with the fluorimeter, transfer each of the samples to a clear microtiter plate and measure A600 on the microtiter plate reader