Team:Yale/Protocols/cuabsorbance
From 2010.igem.org
(Difference between revisions)
Dspakowicz (Talk | contribs) (New page: Copper Absorbance Protocols __TOC__ ==Copper-Bathocuproinedisulfonic acid Standard Absorbance Curve== # Make stock solutions of x concentration of Copper (mg/ml or M) in both LB and mini...) |
Dspakowicz (Talk | contribs) (→Copper Disappearance as function of Bacterial Growth Curve) |
||
Line 26: | Line 26: | ||
#* Use LB or minimal media as a blank. | #* Use LB or minimal media as a blank. | ||
# Take OD and Abs measurements every 30min or 1 hr for 6 hours and record data. | # Take OD and Abs measurements every 30min or 1 hr for 6 hours and record data. | ||
+ | |||
+ | ===Hypothetical Data=== |
Revision as of 03:21, 28 September 2010
Copper Absorbance Protocols
Contents |
Copper-Bathocuproinedisulfonic acid Standard Absorbance Curve
- 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)
- Dissolve Bathocuproinedisulfonic in x amount of water for a final concentration of x (mg/ml or M)
- Take absorbance (470nm) of blank in order to calibrate spectrophotometer.
- Blank should be LB or minimal media without copper.
- 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
- Measure absorbance of each copper solution at (470nm)
- Record results.
Copper Disappearance as function of Bacterial Growth Curve
- 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.
- 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.
- For 2nd aliquot, centrifuge at x rpm 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.