IGEM:British Columbia/2009/Notebook/Biosensor Sensitivity/2009/05/27: Difference between revisions

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* Will try using DH5alpha cells instead of DB3.1 cells to make sure it isn't the cells being resistant to current stock of chloramphenicol.
* Will try using DH5alpha cells instead of DB3.1 cells to make sure it isn't the cells being resistant to current stock of chloramphenicol.


==Testing of Chloramphenicol (yet again)==
* Goal: The DB3.1 cells may be chloramphenicol resistant (not likely, but still it's a possibility).
* Logic here is that if DH5 alpha cells grow in the presence of chloramphenicol, then we know that the DB3.1 cells weren't the problem and that the chloramphenicol is the problem.
* So, test with DH5alpha cells to see if it's the antibiotic that doesn't work.
* Steps are as follows:
# Aliquoted remaining LB broth into 50 mL Falcon tubes.
# 10 mL LB Broth was aliquoted each into two test tubes.
# 10 µL of 25 mg/mL stock Chloramphenicol was added to one of the test tubes, labeled as "LB + Chlor + DH5alpha". Other tube labeled as "LB Only + DH5alpha"
# Innoculated DH5alpha cells from glycerol freezer stocks into both test tubes.
# Incubated in Lagally Lab shaker at 37ºC overnight at 6:03PM.


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Revision as of 18:15, 27 May 2009

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Results of Transformation of Kan and Tet Construction Plasmids

  • Kan and Tet construction plasmid transformation did not succeed.
  • No colony growth was observed on the Kan and Tet plates.
  • Possible Reasons:
    • May have been due to fewer colonies than usual being plated (300 µL, rather than 500 µL).
    • May also have to do with the amount of DNA that we used - only 1 µL rather than 2 µL.
  • Paul will bring over test plasmids that contain Kan and Tet resistance markers that we can use as a transformation control.

Results of Chloramphenicol Testing

  • Growth observed in both LB Broth and LB Broth + Chloramphenicol tubes.
    • This time round, chloramphenicol was either spread plated after cooling LB Agar, or added to LB broth at 1X concentration.
  • Measured ODs by adding 2mL of bacterial culture into plastic cuvettes
  • OD > 2 for both
Media OD660
LB Broth + Chloramphenicol 2.485
LB Broth Only 2.112
  • Cuvettes were rinsed with tap water and returned to the cuvette storage styrofoam box.
  • OD readings are bad as it means that either the cells are chloramphenicol resistant or that the method of preparation of chloramphenicol media isn't correct.
  • Possible Causes:
    • Concentration of chloramphenicol not high enough?
    • Possibly need warmer temperatures for dissolving antibiotic into the media - but that may not be relevant as the dissolved antibiotic would just ppt out if it needed high temperatures for dissolution
  • Will try using DH5alpha cells instead of DB3.1 cells to make sure it isn't the cells being resistant to current stock of chloramphenicol.

Testing of Chloramphenicol (yet again)

  • Goal: The DB3.1 cells may be chloramphenicol resistant (not likely, but still it's a possibility).
  • Logic here is that if DH5 alpha cells grow in the presence of chloramphenicol, then we know that the DB3.1 cells weren't the problem and that the chloramphenicol is the problem.
  • So, test with DH5alpha cells to see if it's the antibiotic that doesn't work.
  • Steps are as follows:
  1. Aliquoted remaining LB broth into 50 mL Falcon tubes.
  2. 10 mL LB Broth was aliquoted each into two test tubes.
  3. 10 µL of 25 mg/mL stock Chloramphenicol was added to one of the test tubes, labeled as "LB + Chlor + DH5alpha". Other tube labeled as "LB Only + DH5alpha"
  4. Innoculated DH5alpha cells from glycerol freezer stocks into both test tubes.
  5. Incubated in Lagally Lab shaker at 37ºC overnight at 6:03PM.


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