Endy:Measuring Screening Plasmid on MOFLO

Setup the machine

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).
If you are not sorting the droplet maker (technical name?), the stream can be turned off to reduce noise in the readings.
Threshold for SSC should be set to as small a distance as possible above zero to avoid cutting out cells
- You should only see a single population on the FSC/SSC plot.
FSC can't be adjusted when set on log scale
SSC 418
Pressure differential should be set so counts are less than 30,000
Make sure the parameter scale for FL1/7 is set to log

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 bead calibration page. Be sure to save the bead calibration data.
Run negative control
Run blank media
Empsty screening plasmid in high induction (to get a "high" GFP/RFP control)
Experiment specific controls (often inlcudes the empty screening plasmid or construct expressiong GFP only, see specific protocol for details.)

Include links to control runs with M9, SuppM9, Negative control, etc. Include the eps (events per second) results as well.

Back flush the collection tube to clear out the previous sample
- Open and close the yellow valve a few times in order to clear it out better.
Vortex sample
Load sample and collect cells (number depends on protocol)
Close the red valve and yellow valve and wait for 5 seconfs before removing the tube, this lets the pressure go down and prevent the fluid from splashing in your face. (most of the time)
Begin back flush immediately after removing sample (the longer it runs the better).

If the cells begin arriving very slowely check that the laser power is still high. If it has declined karate chop the laser and it will fix it. (i'm serious).

Load cells as decribed above, you may want to run bleach followed by water (?) in between samples if you are concerned about contamination.
Run the sample in "Aquire mode" first in order to save a file of the cell population (the files are not saved while sorting).
When finished aquiring stop the cell from flowing by closing the yellow valve, then switch to sorting mode and define a sort region.
Print the page with the sort region since as far as I know right now that's the only way to document it (there has to be a way, ask about it).
Reset the sort count on the machine behind you
Insert the collection tube with fresh media, be careful not to touch the plates! (technical name)
Open the yellow valve
Collect X cells (defined by specific protocol)
Begin back flush immediately