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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#Install plate reader into 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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#Username = fluorescence ; Password = password
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#Choose the Cary Eclipse folder
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##For GFP, select 485 (excitation) and 515 (emission) for optimal reading (with 100 gain)
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#Choose the Advanced Reads application for the fluorimeter
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#Select wells filled
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#Set Up
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#Place the well inside the machine, and Click OK when ready.
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#For GFP, set Excitation wavelength = 395 nm, Emmission = 509 nm
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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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#For RFP, set Excitation wavelength = 584 nm, Emmission = 607 nm
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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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#Exitation slit = 5 (may try 20)
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##Under data drop down menue, look at ratio or number of bacteria also.
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#Emmission slit = 5 (may try 20)
 
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#Average time = 0.1000
 
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#Options
 
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#Excitation filter = Auto
 
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#Emmission filter = open
 
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#PMT voltage = Medium (may try High)
 
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#Optional: Replicates = 3, change under Samples
 
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#Accessories
 
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#Well Plate
 
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#Click Well Plate Reader
 
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#Auto Zero
 
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#Select wells
 
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#Click OK
 
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#Insert plate, Notch matches up to corner
 
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#Click Start
 
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#Ignore Data Save Warning
 
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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
 

Revision as of 14:21, 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)
    1. For GFP, select 485 (excitation) and 515 (emission) for optimal reading (with 100 gain)
  2. Select wells filled
  3. Place the well inside the machine, and Click OK when ready.
  4. To change color scale, click edit matrix, blue scale, custom, scale drop down menue, custom scale, select colors, and click OK.
  5. 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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