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Protocol: -Glycerol Stock:

  1. Add 1 culture from selection plate into 7.5 mL of LB Broth with 50 μg/mL of KAN
  2. Incubated culture overnight at 37°C with 225 rpm
    • Started at 5pm 22/04/2019.
    • Stopped at 8:30 am 23/04/2019
  3. Mix 0.5 mL of overnight culture with 0.5 mL of 50% glycerol in LB broth.
  4. Store glycerol stocks in -80°C freezer

-Expression culture:

  1. Use the remaining 5 mL of the overnight culture (mentioned in the glycerol stock protocol)
  2. Inoculate 200 mL of LB Broth with 50 μg/mL of KAN with the 5 mL overnight culture.
  3. Incubated culture for 2 hours at 37°C with 225 rpm.
  4. Add 205 μL of 1 M IPTG to the culture.
  5. Continue to incubate the culture at 37°C with 225 rpm for 4.5 hours.
  6. Split culture into four 50 mL falcon tubes and centrifuge at 3750 rpm for 30 mins.
  7. Remove supernatant and store pellet in -20°C freezer.

Results: The pellets after expression were all very pink. Protein production appears to be successful.