Difference between revisions of "Measuring Fluorescence in Bacteria"

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#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.
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#All Wells should contain 250 microliters of cells in broth. Be sure to include wells containing broth with no cells- for negative control.
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#Centrifuge 5 min at 4000 RPM and resuspend bacterial pellet in 400 ul dH20
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#Open Gen 5 software by double clicking on the desktop shortcut.
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#Transfer 250 ul of resuspended bacteria to a well in a white microtiter plate
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#Click on Existing protocol
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#Transfer 250 ul of dH20 into one of the wells for use as a blank
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#Select Standard Protocol type
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#Choose the Advanced Reads application for the fluorimeter
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##For RFP, select 585 nm (excitation) and 615 nm (emission) for optimal reading (with 100 gain)
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#Ex. slit = Em. slit = 20, Ave. time = 0.1000, Excitation filter = Auto, Emmission filter = open, PMT voltage = High, Replicates = 3
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##For GFP, select 485 (excitation) and 515 (emission) for optimal reading (with 100 gain)
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#For GFP, set Excitation wavelength = 395 nm, Emmission = 509 nm
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#Select  wells filled
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#For RFP, set Excitation wavelength = 584 nm, Emmission = 607 nm
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#Place the well inside the machine, and Click OK when ready.
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#After measuring with the fluorimeter, transfer each of the samples to a clear microtiter plate and measure A600 on the microtiter plate reader
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#To change color scale, click edit matrix, blue scale, custom, scale drop down menue, custom scale, select colors, and click OK.
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#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.
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##Under data drop down menue, look at ratio or number of bacteria also.

Latest revision as of 14:22, 20 May 2015

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.
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