Synergy Machine Protocol

Synergy Machine Protocol

1. All Wells should contain 200 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 OR move to step 4.
4. Click on Create New Protocol. This option is available under "File", New Task.
5. Select Standard Protocol type
6. Click on Procedure
7. Under Shake, select Orbital Shake for 3 seconds
8. Under Read, select Fluorescence from drop down menu
   1. Enter Emission and Excitation wavelengths
      1. If you are reading more than one type of fluorescence, click the appropriate number of types above the wavelength values.
   2. For RFP, select 585 nm (excitation) and 615 nm (emission) for optimal reading (with 100 gain)
   3. For GFP, select 485 (excitation) and 515 (emission) for optimal reading (with 100 gain)
   4. If GFP and RFP are combined in the same well, use a different method.
      1. Use the wavelengths of 460nm (excitation) and 490 nm(emission) for GFP, and 585 nm(excitation) and 615 nm (emission) for RFP, to minimize noise/interference. However, some of the GFP reading will be from bleeding over of RFP.
9. Under Read, select Absorbance from the drop down menu and enter 590 nm
10. Once those values have been submitted, drag tasks into proper order. First Shake, then read Fluorescence, then read Absorbance.
    1. If you are measuring GFP and RFP, measure GFP before RFP.
11. Click OK, and from the banner menu, select File, New Task, then Read Now (selecting the procedure you created)
12. Place the well inside the machine, and Click OK when ready.
13. 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.
14. Note- if only a few wells give a value of OVRFLW, try lowering the gain from 100 to 95 or 90 (or lower if necessary). Enter the value on the page with the wavelengths for fluorescence. If all wells give a value of OVRFLW, this means the wavelengths are too close together. Try increasing the difference between the excitation and emission wavelengths.
15. 
    1. 485 nm/515 nm (100 gain) are the optimal exitation/emission wavelengths for use with GFP.
16. 
    1. 585 nm/615 nm (100 gain) are the optimal excitation/emission wavelengths for use with RFP, as they resulted in the highest fluorescence readings.
17. 
    1. 460 nm (excitation) and 490 nm (emission) are the optimal wavelengths to use with GFP and RFP in the same well, in order to minimize noise/interference from RFP at GFP wavelengths.