Endy:Measuring Screening Plasmid on MOFLO: Difference between revisions

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#*This is the highest settings that eliminate [[Endy:MOFLO/Dark noise|"dark noise"]].  Additionally, these setting are acceptable for the full range of arabinose induction for the current Screening Plasmid series. (I13534,I13537,I13538).
#*This is the highest settings that eliminate [[Endy:MOFLO/Dark noise|"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?) can be turned off to reduce noise in the readings.
#If you are not sorting the droplet maker (technical name?) can be turned off to reduce noise in the readings.
==Controls==
==Controls==
#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 [[Endy:FACS calibration with beads|bead calibration page]].  Be sure to save the bead calibration data. [[Image:MOFLObeads.PNG|thumb|none|Beads should look approximately like this with good alignment.]]
[[Image:MOFLObeads.PNG|thumb|right|Beads should look approximately like this with good alignment.]]
 
#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 [[Endy:FACS calibration with beads|bead calibration page]].  Be sure to save the bead calibration data.  
==Experimental samples==
#Run negative control
#Run blank media
#Experiment specific controls (often inlcudes the empty screening plasmid or construct expressiong GFP only, see specific protocol for details.)
<br style="clear:both" />
==Aquiring samples==
#Back flush the collection tube to clear out the previous sample
#*Open and close the valve a few times in order to clear it out better.
#Vortex sample
#Load sample and collect cells (number depends on protocol)
#
===Notes===
*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).


#Run the empty plasmid again, followed by the negative control.
==Sorting samples==
#*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).
#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 experimental samples. (have been collecting ~30K cells / sample)
#Run the sample in "Aquire mode" first in order to save a file of the cell population (the files are not saved while sorting).

Revision as of 17:55, 13 March 2006

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).
  2. If you are not sorting the droplet maker (technical name?) can be turned off to reduce noise in the readings.

Controls

Beads should look approximately like this with good alignment.
  1. 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.
  2. Run negative control
  3. Run blank media
  4. Experiment specific controls (often inlcudes the empty screening plasmid or construct expressiong GFP only, see specific protocol for details.)


Aquiring samples

  1. Back flush the collection tube to clear out the previous sample
    • Open and close the valve a few times in order to clear it out better.
  2. Vortex sample
  3. Load sample and collect cells (number depends on protocol)

Notes

  • 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).

Sorting samples

  1. Load cells as decribed above, you may want to run bleach followed by water (?) in between samples if you are concerned about contamination.
  2. Run the sample in "Aquire mode" first in order to save a file of the cell population (the files are not saved while sorting).
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