Synergy Machine Protocol

Synergy Machine Protocol

1. Open Gen 5 software
2. Click on Existing protocol OR move to step 3
3. Click on Create New Protocol
4. Select Standard Protocol type
5. Click on Procedure
6. Under Shake, select Orbital Shake for 3 seconds
7. 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)
8. Under Read, select Absorbance from the drop down menu and enter 590 nm
9. 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.
10. Click OK, and from the banner menu, select File, New Task, then Read Now (selecting the procedure you created)
11. Place the well inside the machine, and Click OK when ready.
12. 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.
13. 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.
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    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.