Endy:Measuring Screening Plasmid on MOFLO

Setup the machine

1. Set PMT voltages to FL1 = 525, FL7 = 650.
   • This is the highest settings that eliminate "dark noise". Additionally, these setting are acceptable for the full range of arabinose induction for the current Screening Plasmid series. (I13534,I13537,I13538).

Experimental samples

1. The droplet maker can be turned off to reduce noise in the signal if you are not sorting the cells.
2. Run beads (~6 drops in 500ml) - this is used to verify that the lasers are aligned as well as to serve as a calibration between runs, you can read more about this in the analysis techniques. Be sure to save the bead calibration data.

   

   Beads should look approximately like this with good alignment.

3. Run the empty plasmid again, followed by the negative control.
   • This allows you to determine the "noise region", since you shouldn't trust readings that are within the range of autoflourescence of your cells. Also, it makes sure that the method being used to clear the tubing between samples is working well. If you still see many positive cells in the negative control then you can try running bleach between samples. (This is maybe more important between different terminators then between different arabinose concentrations).
4. Run the experimental samples. (have been collecting ~30K cells / sample)