IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/05/28

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Results of Tests on Chloramphenicol

  • Took tubes out of Lagally Lab 37ºC shaker at 11:00AM.
  • Growth/cloudiness observed in test tubes containing chloramphenicol and tubes containing NO chloramphenicol.
  • Briefly vortexed test tubes to homogenize them.
  • Pipetted 2mL of LB, LB+Chlor+DH5alpha and LB+DH5alpha into spectrophotometer cuvettes
  • Measured OD600 on eppendorf BioPhotometer in Lagally Lab
  • Results are as follows:
Sample OD600
LB onlyBlank
LB-Chloramphenicol + DH5a1.662
LB + DH5a1.694

Conclusions

  • This set of OD readings, combined with the set of DB3.1 cells' readings, suggest that the chloramphenicol isn't working.
  • Hence, today, will make new set of chloramphenicol, but make less in total to prevent wastage (in case this new batch also doesn't work).

Remade Chloramphenicol

  • Made 10mL of 25mg/mL Chloramphenicol
  • used 0.2521g Chloramphenicol powder
  • dissolved in 10mL of 100% Ethanol
  • splited solution into 10 aliquots of ~1mL each
  • stored in the -20°C freezer in the Lagally lab

Made more LB Plates

  • Made 2x 500mL of 37mg/mL LB agar
  • Brought liquid LB agar solution for autoclaving at 1400
  • Retreived liquid LB agar from the autoclave room at 1530
  • Pipetted 20mL of LB agar into each petri dish
  • Half of the LB plates made where bagged and stored in AMBL's 4ºC fridge.
  • Other half were sterile-parafilmed and put into the AMBL's 4ºC fridge.
  • Two chloramphenicol plates were also made by adding 40 µL of chloramphenicol into 40 mL of LB Agar, and then poured and stored in AMBL's 4ºC fridge. These will be used for testing chloramphenicol.
  • Unused petri dishes were sterile-parafilmed and brought back to the Lagally Lab.

Creating BW27783 and DH5α Competent Cells

  • Inoculate 5mL overnight culture and grow at 30°C.
  • Chilled the Centrifuge motor, CaCl2 and Centrifuge tubes
  • Dilute 0.5mL overnight culture into 50mL LB and incubate at 30°C for 3hours, shaking vigorously.
  • Harvested at BW27783 Abs600nm = 0.8 and DH5α Abs600 = 0.36.
  • Put the BW27783 in the 0.4°C fridge and incubated the DH5α for 1hour to raise the OD
  • Centrifuge at 1600g for 7 minutes (4°C).
  • Resuspend in 20mL cold filter sterilized 0.1M CaCl2.
  • Centrifuge at 1100g for 5 minutes, 4°C.
  • Resuspend in 12.5mL cold 0.1M CaCl2.
  • Keep on ice 40 minutes.
  • Centrifuge at 1100g for 5 minutes, 4°C.
  • Resuspend in 2-2.5mL cold 0.1M CaCl2.
  • Store over night at 4°
  • Will make glycerol stocks by adding 900uL of glycerol to 100uL of cells and store at -80°C.


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