1 / 5

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.

Download Presentation

Preparing an Overnight Culture of E. coli

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related