Team:Washington/Protocols/osmY Assay


Revision as of 03:07, 1 October 2012 by Tjiang (Talk | contribs)

osmY Assay

  1. Make 500mL of M-9 media with 1x M9 salts, .1% glucose by mass, .5g casamino acids, and .001% biotin.
  2. Make 4 liquid culture tubes each of sfGFP-osmY (n-c), osmY-GFP (Bright) (c-n), iGEM2011-003 sfGFP-mamI 1A3, iGEM2011-009 plac Bright 1A3, and no cells. All of these cultures are in 2mLs of M-9 media. Leave these tubes overnight.
  3. The next day, take 700µL of media out of each culture tube.
    1. Pipet 200µL of that 700 into a well in a clear, flat-bottom 96 well plate.
    2. Pipet the other 500µL into a 1.5 mL eppendorf tube.
  4. Centrifuge the eppendorf tubes containing the media for 3 minutes at 3g.
  5. After spinning, remove the supernatent from each eppendorf tube and pipet it into a new eppendorf tube. Be sure not to disturb the cell pellets and keep the eppendorf tubes.
  6. Take the new eppendorf tubes with supernatent only and centrifuge them for 10 minutes at 17g.
  7. During this time, resuspend the cells in the old eppendorf tubes with 500µL of fresh M9-glucose media, the same media that the cells were grown in
  8. After resuspending, take 200µL of the supernatant containing the resuspended cells and pipet it into the same 96 well plate the original culture was pipetted into.
  9. Lastly, take the supernatent from the eppendorf tubes that have just been cenrifuged for 10 minutes and pipet 200µL from each tube into the 96 well plate. In total, 60 wells from the 96 well plate were used.