Team:Yale/Protocols/cuabsorbance
From 2010.igem.org
These protocols are adapted from Blair and Diehl, 1961 and Zak, 1958.
Contents |
Copper-Bathocuproinedisulfonic acid Standard Absorbance Curve
- Make stock solutions of 10 mM to 10 pM [CuSO4] in both LB and minimal media.
- Make solutions by making one concentrated solution precisely and doing dilution series (10-1 or 10-2)
- Dissolve 0.0057 g Bathocuproinedisulfonic in 101 uL water for a final concentration of 10 mM
- Take absorbance (483nm) of blank to calibrate spectrophotometer.
- Blank should be LB or minimal media without copper.
- Mix 2 uL reagent to 198 uL copper solution and vortex. Add 200 uL to cuvette
- Wipe sides of cuvette with kim wipe to ensure better absorbance data
- Measure absorbance of each copper solution at (483nm)
- Record results.
2010 10 12 -- dissolved .0075 g Bathocuproinedisulfonic in 12 ul water for a final concentration of 100 mM of stock reagent.
Copper Disappearance as function of Bacterial Growth Curve
- Autoclave 50 mL LB and minimal medium in 125 mL flask. Allow to cool and add Amp (50 uL of 1000X stock).
- Night before, inoculate 5 mL LB+Amp and MM+Amp from a colony on a freshly streaked plate.
- Measure OD600 of overnight cultures.
- Dilute overnight culture to OD600 = 0.0125 in 50 ml of LB or minimal medium with x concentration of copper by diluting overnight culture.
- OD=0.0125 corresponds to 4 generations before stationary phase.
- At t=0, take 1 mL aliquot and measure OD of bacteria at 600nm. Record.
- Use either LB or minimal media as blank.
- For 2nd aliquot, centrifuge at max speed for 5 minutes and collect supernatant.
- Supernatant should have copper and no bacteria.
- Add x amount of Bathocuproinedisulfonic to the supernatant and mix.
- Measure absorbance of supernatant at 470nm. Record data.
- Use LB or minimal media as a blank.
- Take OD and Abs measurements every 30min or 1 hr for 6 hours and record data.