Difference between revisions of "Team:UCLA/Notebook/Recombinant Expression/20 May 2015"

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** Incubated shaking at 25 degrees Celsius for 30 hours.
 
** Incubated shaking at 25 degrees Celsius for 30 hours.
 
*** Will check culture at 1500 tomorrow for an OD600 of at least 0.3. Target OD600 before glycerol stock preparation is 0.4 (optimal cell content/competency OD).
 
*** Will check culture at 1500 tomorrow for an OD600 of at least 0.3. Target OD600 before glycerol stock preparation is 0.4 (optimal cell content/competency OD).
 +
=Julian's Work=
 +
*I split the successful starter culture between 2 150 mL cultures in LB with Chlor diluted 1000X; so 5mL culture in each
 +
**grew one culture at 30C and one at 37C both overnight. I did not check OD
 +
**In todd's lab we never checked OD and got good results so I am seeing how this works here and will do a comparison test. If you dont care about OD this reduces timing issues with culturing
 +
*Both cultures worked. I then spun down the cells at 5000g for 5 min. This formed a good pellet
 +
**I placed the pellet in the fridge at -80C

Latest revision as of 23:12, 22 May 2015

  • Starter culture failure: Electric boogaloo
    • As per usual, the colonies attempted to pick for stater culture creation yesterday were unsuccessful.
      • Most likely due to the plates being very old; having to withstand steep changes in temperature and environmental conditions.
    • Decided to simply re-plate the colonies on an LB agar plate supplemented with 50 ug/uL Ampicillin (created on 4/12). Plate was briefly warned to room temp, and 4 separate glycerol stocks were picked using glycerol stocks and streaked out on 4 separate plates.
    • Plates were incubated at 37 degrees Celsius overnight. Will check tomorrow.
  • Preparation of Zymo Mix n' Go Chemically Competent E. coli BL21(DE3) expression strains.
    • Picked colony from BL21(DE3) plates given to us by Dr. Mark Arbing of the UCLA Department of Chemistry & Biochemistry DOE Protein Expression Core Facility in Boyer Hall.
    • Inoculated colony in 10 mL LB broth, shaken overnight at 37 degrees Celsius for 24 hours.
    • Inoculated 500 uL of starter culture in 50mL of 4 degree Zymo broth.
    • Incubated shaking at 25 degrees Celsius for 30 hours.
      • Will check culture at 1500 tomorrow for an OD600 of at least 0.3. Target OD600 before glycerol stock preparation is 0.4 (optimal cell content/competency OD).

Julian's Work

  • I split the successful starter culture between 2 150 mL cultures in LB with Chlor diluted 1000X; so 5mL culture in each
    • grew one culture at 30C and one at 37C both overnight. I did not check OD
    • In todd's lab we never checked OD and got good results so I am seeing how this works here and will do a comparison test. If you dont care about OD this reduces timing issues with culturing
  • Both cultures worked. I then spun down the cells at 5000g for 5 min. This formed a good pellet
    • I placed the pellet in the fridge at -80C