Measuring Fluorescence in Bacteria

Measuring Fluorescence in Bacteria

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

1. All Wells should contain 250 microliters of cells in broth. Be sure to include wells containing broth with no cells- for negative control.
2. Open Gen 5 software by double clicking on the desktop shortcut.
3. Click on Existing protocol
4. Select Standard Protocol type
   1. For RFP, select 585 nm (excitation) and 615 nm (emission) for optimal reading (with 100 gain)
   2. For GFP, select 485 (excitation) and 515 (emission) for optimal reading (with 100 gain)
5. Select wells filled
6. Place the well inside the machine, and Click OK when ready.
7. To change color scale, click edit matrix, blue scale, custom, scale drop down menue, custom scale, select colors, and click OK.
8. After the machine has run, you can export the data to Excel using the Excel button on the banner of the window that pops up.
   1. Under data drop down menue, look at ratio or number of bacteria also.