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Team:Yale/Protocols/cuabsorbance
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# Make stock solutions of 10 mM to 10 pM [CuSO4] in both LB and minimal media. | # 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) | #* Make solutions by making one concentrated solution precisely and doing dilution series (10-1 or 10-2) | ||
| - | # Dissolve Bathocuproinedisulfonic in | + | # Dissolve 0.0057 g Bathocuproinedisulfonic in 101 uL water for a final concentration of 10 mM |
| - | # Take absorbance ( | + | # Take absorbance (483nm) of blank to calibrate spectrophotometer. |
#* Blank should be LB or minimal media without copper. | #* Blank should be LB or minimal media without copper. | ||
| - | # Mix | + | # 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 | #* Wipe sides of cuvette with kim wipe to ensure better absorbance data | ||
| - | # Measure absorbance of each copper solution at ( | + | # Measure absorbance of each copper solution at (483nm) |
# Record results. | # Record results. | ||
Revision as of 14:17, 6 October 2010
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].
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.
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.
Hypothetical Data
