Team:UCLA/Notebook/Recombinant Expression/21 May 2015

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• Starter culture creation
  • Looked at plates streaked from [tubes Tokyo gBlock 3/4/5]
  • Each plate had a good amount of isolated colonies, well streaked.
  • Observation of the streaked colonies under gel box UV yielded several colonies with high fluorescence, enough to verify proper insert presence.
  • Colony morphology was also identical, which indicates lack of satellite colonies.
    • Picked an individual colony and in separate tubes prepared starter and inoculated 10mL LB supplemented with 1x Amp. Placed in 37 deg shaking at 1600. Will check cell growth at 0900 tomorrow]

• Zymo Mix N' Go Competency tested
  • Yesterday's 50mL culture of BL21(DE3) streaked from Mark Arbing and starter cultures made were grown to OD600 of 1.2 -- way above the 0.4-0.6 range recommend by Zymogen.
  • Competency was conducted regardless, resuspending cell pellets in competency buffer wafter wash buffering the cells from each culture.
  • David aliquotted the resulting DH5alpha and BL21(DE3) cells into 50uL samples, and froze in -80.

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Replating of a glycerol stock of a Tokyo G-Block aliquot #5. Cells were scraped from the stock and streaked onto an LB agar/Amp plate, and incubated overnight at 37 degrees. Single isolated colonies are visible after the 4th streak; cells fluoresce under UV box illumination

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Replating of a glycerol stock of a Tokyo G-Block aliquot #4. Cells were scraped from the stock and streaked onto an LB agar/Amp plate, and incubated overnight at 37 degrees. Single isolated colonies are visible after the 4th streak; cells fluoresce under UV Box illumination

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Replating of a glycerol stock of a Tokyo G-Block aliquot #5. Cells were scraped from the stock and streaked onto an LB agar/Amp plate, and incubated overnight at 37 degrees. Single isolated colonies are visible after the 4th streak; cells fluoresce under UV Box illumination

Julian's Work

• Today I did Cell Lysis
• I thawed the 30C pellets from the freezer. I followed the nature protocol for all cell lysis with some differences as noted below. <a href="http://www.nature.com/nprot/journal/v4/n3/abs/nprot.2008.250.html">Find protocol here</a>