Team:Yale/Protocols/cuabsorbance

From 2010.igem.org

These protocols are adapted from [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6THP-44MRNW6-6&_user=10&_coverDate=02%2F28%2F1961&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_searchStrId=1480563724&_rerunOrigin=google&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=632629e9ae5f9ee1c85669826a545068&searchtype=a Blair and Diehl, 1961] and [http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T57-47RK1T9-35G&_user=10&_coverDate=07%2F31%2F1958&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000050221&_version=1&_urlVersion=0&_userid=10&md5=8943c9df67da1c9de1aa68b4aff690c8&searchtype=a Zak, 1958].

Copper-Bathocuproinedisulfonic acid Standard Absorbance Curve

1. 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)
2. Dissolve 0.0057 g Bathocuproinedisulfonic in 101 uL water for a final concentration of 10 mM
3. Take absorbance (483nm) of blank to calibrate spectrophotometer.
   • Blank should be LB or minimal media without copper.
4. 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
5. Measure absorbance of each copper solution at (483nm)
6. 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

1. Autoclave 50 mL LB and minimal medium in 125 mL flask. Allow to cool and add Amp (50 uL of 1000X stock).
2. Night before, inoculate 5 mL LB+Amp and MM+Amp from a colony on a freshly streaked plate.
3. Measure OD600 of overnight cultures.
4. 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.
5. At t=0, take 1 mL aliquot and measure OD of bacteria at 600nm. Record.
   • Use either LB or minimal media as blank.
6. For 2nd aliquot, centrifuge at max speed for 5 minutes and collect supernatant.
   • Supernatant should have copper and no bacteria.
7. Add x amount of Bathocuproinedisulfonic to the supernatant and mix.
8. Measure absorbance of supernatant at 470nm. Record data.
   • Use LB or minimal media as a blank.
9. 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