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Preparing an Overnight Culture of E. coli

Preparing an Overnight Culture of E. coli. 2014. Possible Problems. When there is a mixed culture of red and white (nonexpressing) cells, the white cells will grow faster than those that are using their resources producing red fluorescent protein (mFP) = less mFP produced for purification.

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Preparing an Overnight Culture of E. coli

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  1. Preparing an Overnight Culture of E. coli 2014

  2. Possible Problems • When there is a mixed culture of red and white (nonexpressing) cells, the white cells will grow faster than those that are using their resources producing red fluorescent protein (mFP) = less mFP produced for purification. • Some transformed cells stop expressing rpf fairly early.

  3. Materials • Sterile 125 mL flasks of LB/amp broth. • Vented cap to ensure adequate aeration during overnight shaking • Mini shaker table • Frozen cells transformed with pARA-R • Tube of sterile arabinose (500mg/mL)

  4. Procedure Morning, Day before Lab 6 • Aseptically, add 500 uL of transformed cells into each flask of LB/amp broth • Secure vented cap (do not wet the membrane) • Shake and incubate (35o C) for 2–3 hours • Following the 2–3 hours, add the appropriate volume of arabinose to each flask so the final concentration of arabinose is 5mg/100mL • Continue to shake overnight

  5. Time vs Growth

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