User:Justin M. Scott/Notebook/Col V Population Dynamics/Entry Base

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Solid Media Experiment

9/18/2012

  • Placed singles from an LB+CMP plate for BC208pACYC184 (Streaked 9/7/12) in 10mL of LB with 10µL of CMP (1000x), 10µL of AMP (1000x), and 10µL of STR (1000x)
  • Placed singles from an LB+NAL plate for Exp. Resist #4 (Streaked 9/7/12) in 10mL of LB with 10µL of NAL (1000x) and 10µL of AMP (1000x)
  • Placed singles from an LB+NAL+KN plate for BC212 (Streaked 9/7/12) in 10mL of LB with 10µL of NAL (1000x) and 20µL of KN (500x)
  • Incubated on roller drum at 37˚C and speed 5

9/19/2012

  • Spun 1mL of O.N. at 14000 rpm for 3 minutes.
  • Poured off supernatant
  • Resuspended pellet in 1mL of M63
  • Diluted resuspended pellet to 108
  • Pipetted 30µL drops of diluted BC208pACYC184 onto LB plates in an circular patter, 8 drops to a circle
  • Allowed droplet to soak into the media
  • Pipetted 30µL drops of diluted Exp. Resist #5 onto LB plates in an circular patter, 8 drops to a circle
  • Allowed droplet to soak into the media
  • Pipetted 30µL drops of diluted BC212 onto LB plates in an circular patter, 8 drops to a circle
  • Allowed droplet to soak into the media
  • Incubated at 37˚C
  • After 24 hours of incubation, replica plated on LB+STR+AMP+CMP, LB+NAL+AMP, LB+NAL+KN plates and a new LB plate
  • Incubated at 37˚C for 24 hours
  • Replica plated the LB plate onto LB+STR+AMP+CMP, LB+NAL+AMP, LB+NAL+KN plates and a new LB plate
  • Incubated at 37˚C for 24 hours
  • Added 300µL of liquid LB to the LB+STR+AMP+CMP, LB+NAL+AMP, LB+NAL+KN plates
  • Scraped plate with a glass spreader
  • Poured off liquid LB into a sterile beaker
  • Added 200µL of liquid LB to the LB+STR+AMP+CMP, LB+NAL+AMP, LB+NAL+KN plates
  • Scraped plate with a glass spreader
  • Poured off liquid LB into a sterile beaker
  • Added 100µL of liquid LB to the LB+STR+AMP+CMP, LB+NAL+AMP, LB+NAL+KN plates
  • Scraped plate with a glass spreader
  • Poured off liquid LB into a sterile beaker
  • Diluted the liquid in the sterile beaker to 107
  • Used droplet method for viable cell count onto new LB+STR+AMP+CMP, LB+NAL+AMP, LB+NAL+KN plates
  • Incubated at 37˚C overnight
  • Repeated for 72 hours




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