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