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. Some transformed cells stop expressing rpf fairly early.

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)

Procedure 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 (35 o 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 Morning, Day before Lab 6

Time vs Growth