Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/27 July 2015"

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(→‎Starting Honeybee Protein Expression)
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*#Continue to incubate at 37 C and shake overnight.
 
*#Continue to incubate at 37 C and shake overnight.
 
*# In the morning, spin down the cells, decant the supernatant, and freeze the pellet at -80 for storage.
 
*# In the morning, spin down the cells, decant the supernatant, and freeze the pellet at -80 for storage.
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*THE LB WE USED TO GROW OUR CELLS WAS CONTAMINATED! :( WILL REPEAT TOMORROW

Revision as of 23:35, 27 July 2015

Starting Honeybee Protein Expression

  • Following a similar protocol to the one performed on 5/18.
  • Growing up cells in 100ml of LB.
    • We are doing a smaller volume because we are running low on bugbuster lysis reagents, and we want to be able to do at least one more reaction after this one.
  • Instead of making a starter culture, we are simply inoculating a colony into a 2L beveled culture flask.
    • I would have used a 1L one, but there were none available.
  • I started incubating the culture at 12:35 pm 7/27, but I forgot to add the Kanamycin antibiotic until 1 pm. (100 ul of 1000x).
  • Here are the steps for today's growth protocol.
    1. Inoculate one colony in 100 ml of LB in an autoclaved beveled flask.
    2. Add appropriate antibiotics.
    3. Grow and shake at 37 until OD reaches between 0.4 and 0.6.
    4. At that point, add IPTG to final concentration of 0.5 mM
    5. Continue to incubate at 37 C and shake overnight.
    6. In the morning, spin down the cells, decant the supernatant, and freeze the pellet at -80 for storage.
  • THE LB WE USED TO GROW OUR CELLS WAS CONTAMINATED! :( WILL REPEAT TOMORROW