Team:Yale/Protocols/cuabsorbance

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Revision as of 03:31, 28 September 2010

Copper Absorbance Protocols

Copper-Bathocuproinedisulfonic acid Standard Absorbance Curve

1. Make stock solutions of x concentration of Copper (mg/ml or M) in both LB and minimal media.
   • Make solutions by making one concentrated solution precisely and doing dilution series (10-1 or 10-2)
2. Dissolve Bathocuproinedisulfonic in x amount of water for a final concentration of x (mg/ml or M)
3. Take absorbance (470nm) of blank in order to calibrate spectrophotometer.
   • Blank should be LB or minimal media without copper.
4. Mix x amount of reagent to copper solution and shake gently to ensure complete mixing. Add 1mL to cuvette
   • Wipe sides of cuvette with kim wipe to ensure better absorbance data
5. Measure absorbance of each copper solution at (470nm)
6. Record results.

Copper Disappearance as function of Bacterial Growth Curve

1. Start growing bacteria at OD600 = 0.0125 in x ml of LB or minimal medium with x concentration of copper.
   • OD=0.0125 corresponds to 4 generations before stationary phase.
2. At t=0, take x aliquot of liquid culture and split into two. For one aliquot, measure OD of bacteria at 600nm. Record.
   • Use either LB or minimal media as blank.
3. For 2nd aliquot, centrifuge at x rpm for 5 minutes and collect supernatant.
   • Supernatant should have copper and no bacteria.
4. Add x amount of Bathocuproinedisulfonic to the supernatant and mix.
5. Measure absorbance of supernatant at 470nm. Record data.
   • Use LB or minimal media as a blank.
6. Take OD and Abs measurements every 30min or 1 hr for 6 hours and record data.

Hypothetical Data

Hypothetical Cu absorbance curve

Hypothetical Cu toxicity curve