IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/06/01

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Results of Chloramphenicol Testing

  • Chloramphenicol plates did not work.
  • Growth was observed on both the LB-only and LB+Chloramphenicol plates.
  • Both DB3.1 and DH5alpha cells grew on both sets of plates.
  • Could possibly be an issue with the concentration. See here.
    • This page states that chloramphenicol plates should be made at 100 µg/mL concentration rather than 25 µg/mL.
    • To do this, we would need to plate 80 µL on each LB plate rather than 20 µL.
  • New plates will be made in the afternoon.

Testing of Chloramphenicol at 100 µg/mL Concentration

  • Used two LB-agar plates
    • One for LB-only control
    • One for LB + Chlor-100 test
  • Plated 80 µL of 25 mg/mL chloramphenicol onto 20 mL LB agar plate for a final concentration of 100 µg/mL
  • Let dry for 1 hour.
  • Plates were divided in two, and both DH5alpha and DB3.1 were streaked, each on one half of the plate.
  • The plates were then incubated in the Lagally Lab 37ºC incubator at 1:32PM ON.
  • Will check them tomorrow to see if the higher concentration worked.

Results of Test Plasmid pNHG1 with DH5α and DB3.1

  • DB3.1 and DH5α both grew on Tet and Kan plates
  • Both cells were able to pick up a resistant plasmid and express the resistance gene
  • Indicates that the failure of the Tet Construction plasmid and Kan Construction plasmid may be caused by the loss of function in the plasmid

Results of 09/05/29 DH5α and BW27783 Competency Test

  • Neither DH5α or BW27783 showed any growth when plated onto an Amp plate
  • Indicates that the cells did not become competent
  • Will transform both strains again with pUC19 (Amp resistance) to make sure of the result
  • 5mL of DH5 and 5mL of BW27783 was innoculated and grown overnight in a 30°C incubator to make new competent cells tomorrow if a test cells do not grow

Transformation of DH5α and BW27783

  • Thaw 100uL of DH5α and BW27783 glycerol stocks on ice
  • Add 2uL of pUC19 plasmid to each of the stocks
  • Keep on ice for 30min
  • Heat shock at 42°C for 60 seconds
  • Keep on ice for 3 minutes
  • Add 400uL of LB, the LB added to the BW27783 cells was contaminated
  • Recover at 37°C for 2 hours
  • Spread plate cells on Amp plates