Preparing an Overnight Culture of E. coli
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Preparing an Overnight Culture of E. coli 2014
description
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. - PowerPoint PPT Presentation
Transcript of 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.
• 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
