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 (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

Morning, Day before Lab 6

Time vs Growth