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Team:Yale/Our Project/Protocols/disappearance curve
From 2010.igem.org
our project
protocols
Copper-Bathocuproinedisulfonic acid standard absorbance curve measurement
1. Autoclave 3 X 50 mL LB in 125 mL flask. Allow to cool and add Amp (50 uL of 1000X stock).2. Night before, inoculate 5 mL LB+Amp with pBS-140 from a colony on a freshly streaked plate.
3. Measure OD600 of overnight cultures.
4. Dilute overnight culture to OD600 = 0.0125 into each of the 50 ml LBs with 2mM concentration of copper.
• OD=0.0125 corresponds to 4 generations before stationary phase.
5. Establish the following culture variables in the three flasks:
A. Bacteria grow with 0 IPTG added (control)
B. Allow bacteria to grow for 3hrs without addition of 2mM IPTG
C. Bacteria grow with 2mM IPTG from t = 0
6. At t=0, take 1 mL aliquot of all cultures and measure OD of bacteria at 600nm. Record.
•Use either LB media as blank.
7. Transfer media from cuvette to 1mL epindorff tubes and centrifuge at 13,200 rpms for 2 minutes. Collect supernatant and store as A0, B0, C0 respectively. Store at -20 C.
• Supernatant should have copper and no bacteria.
8. Collect time points every 30 minutes repeating steps 5 & 6.
Measuring Copper Concentration
9. Add x amount of Bathocuproinedisulfonic to each supernatant and mix.
10. Using LB as a blank, measure absorbance of supernatant at 470nm. Record data.
