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Team:Yale/LabNotebook/Cu
From 2010.igem.org
General description of goal of this part of the project.
Contents |
Oct 06, 2010; Making stock solutions
Generated the stock solutions for the Followed the standard curve protocol.
- Made a 100 mM stock solution of BCS.
- Made two CuSO4 dilution series, in LB and MM, from 10 mM Cu to 10 pM.
Oct 07, 2010; Bacterial growth assays, Cu ppt
| # | Sample ID | Date and Time | 1 (nm) | 1 (Abs) |
| 1 | o/n culture | 10/7/2010 11:16:10 AM | 600 | 0.854 |
| 2 | MM t0 | 10/7/2010 11:44:45 AM | 600 | 0.002 |
| 3 | LB t0 | 10/7/2010 11:31:06 AM | 600 | 0.089 |
| 4 | LB t1 | 10/7/2010 12:47:55 PM | 600 | 0.189 |
| 5 | MM t1 | 10/7/2010 12:45:13 PM | 600 | 0.002 |
| 6 | MM t2 | 10/7/2010 1:41:51 PM | 600 | 0.004 |
| 7 | LB t2 | 10/7/2010 1:43:38 PM | 600 | 0.450 |
| 8 | MM t3 | 10/7/2010 2:44:50 PM | 600 | 0.001 |
| 9 | LB t3 | 10/7/2010 2:46:09 PM | 600 | 0.761 |
| 10 | MM t4 | 10/7/2010 3:53:12 PM | 600 | 0.005 |
| 11 | LB t4 | 10/7/2010 3:54:36 PM | 600 | 1.064 |
Oct 12, 2010; Generating a standard curve, troubleshooting
Checked Abs of a dilution series of CuSO4, from 100 mM to 1 nM, in LB.
To a cuvette, added:
- 800 uL H2O
- 200 uL LB + Cu
- 2 uL 100 mM BCS
{insert data}
Saw no change between samples.
Revisited literature to see what we're missing.
[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T38-41J66VR-B&_user=483702&_coverDate=11/01/2000&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_rerunOrigin=scholar.google&_acct=C000022720&_version=1&_urlVersion=0&_userid=483702&md5=4dfd3ba70e1e7ab05c0fb6950583baed&searchtype=a#secx9 Lebeau et al, 2000] used this method:
- Measurement of Cu(II) reduction. Seven hundred seventy μl of BDA stock solution (5.7 mM) BDA in 5.2 M ammonium acetate/10% ethanol was mixed with 720 μL of CuSO4 solution (1.25 mM) in water and 10 μL of test compounds (15 mM) except quercetin (1.5 mM) dissolved in ethanol [37]. The complex formation (BDA/Cu(I)) was followed by optical density measurements made at 484 nm. The amount of reduced copper was determined from a calibration curve constructed using solutions of Cu(II) (0–300 μM) reduced by ascorbate (1 mM) in the presence of BDA (2.7 mM). The percent of ethanol in water for the dosage does not exceed 5.6%.
[http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4WBK7DP-4&_user=483702&_coverDate=09/01/2009&_rdoc=1&_fmt=high&_orig=search&_origin=search&_sort=d&_docanchor=&view=c&_acct=C000022720&_version=1&_urlVersion=0&_userid=483702&md5=98048357a77f175010bf42a5d109fdf4&searchtype=a#bbib25 Campos et al 2009] used this method:
- Different concentrations of copper sulfate (0.05–0.8 mM) and BCS (0.1–1.6 mM) were investigated by altering one variable at a time to optimize the assay. Final concentrations of 0.2 mM BCS and 0.1 mM CuSO4 gave enough absorption intensity at 490 nm with good linearity up to 2 mM trolox, as is shown below; therefore, they were established as optimal for the assay. EDTA (1.67 mM final concentration) is used as a stop reagent. When EDTA is added at this concentration before the sample, no color is developed [http://www.sciencedirect.com/science?_ob=MiamiCaptionURL&_method=retrieve&_udi=B6W9V-4WBK7DP-4&_image=fig1&_ba=1&_user=483702&_coverDate=09/01/2009&_rdoc=1&_fmt=full&_orig=search&_cdi=6692&_issn=00032697&_pii=S0003269709003418&view=c&_acct=C000022720&_version=1&_urlVersion=0&_userid=483702&md5=ea49e6dc6b4c77b6fb0ce00751e6aa39 (Fig. 1)]. On the other hand, we noticed that when a concentration of EDTA higher than 1.67 mM is used, a precipitate could appear in plasma samples.
Useful links
[http://meta.wikimedia.org/wiki/Help:Wikitext_examples Wiki formatting help]
[http://excel2wiki.net/ Excel table to Wiki format tool]
