Team:Yale/Our Project/Protocols/disappearance curve

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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 (corresponding to 4 generations before stationary phase) into each of the 50 ml LBs with 2mM concentration of copper.
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, using either LB media as a blank. Record.
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.
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.