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Team:Tokyo Tech/Projects/Urea-cooler/method
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| - | <li>A single colony of cells transformed with mock, | + | <li>A single colony of cells transformed with engineered plasmids (mock,<i>rocF</i> or <i>rocF</i>+Arg box) was inoculated into 3 mL of LB with appropriate antibiotics and grown to saturation at 37℃.</li> |
<li>The saturated culture was diluted 50-fold, grown till the log phase (OD<sub>600</sub> = 0.5).</li> | <li>The saturated culture was diluted 50-fold, grown till the log phase (OD<sub>600</sub> = 0.5).</li> | ||
<li>The culture was induced with 1 mM IPTG at 37℃ for 1 hour. </li> | <li>The culture was induced with 1 mM IPTG at 37℃ for 1 hour. </li> | ||
| - | <li> | + | <li>1.5 mL of culture was centrifuged at 9,000 rpm for 1 minute and the supernatant fluid was used as a sample for urea concentration assay. </li> |
</ol> | </ol> | ||
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<h2 id="assay">2. Urea concentration assay</h2> | <h2 id="assay">2. Urea concentration assay</h2> | ||
<p>Urea concentrations of the samples were determined colorimetrically with <a href="http://www.clonagen.com/clonagen/ab52e63f-4e38-4465-b325-5fd126415f1a/quantichrom_urea_assay_kit_product.aspx">DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems</a>.<br /> | <p>Urea concentrations of the samples were determined colorimetrically with <a href="http://www.clonagen.com/clonagen/ab52e63f-4e38-4465-b325-5fd126415f1a/quantichrom_urea_assay_kit_product.aspx">DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems</a>.<br /> | ||
| - | + | Detailed methods are as follows. | |
<ol> | <ol> | ||
<li>10 µL of the supernatant fluid from each sample, 10 µL blank(LB),and 10 µL standard (10 mg/dL urea LB) were transferred to wells of clear bottom 96-well plates. </li> | <li>10 µL of the supernatant fluid from each sample, 10 µL blank(LB),and 10 µL standard (10 mg/dL urea LB) were transferred to wells of clear bottom 96-well plates. </li> | ||
<li>200 µL working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.</li> | <li>200 µL working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.</li> | ||
| + | <li>The mixture was incubated for 20 munites at room temperature.</li> | ||
<li>Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as<br /> | <li>Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as<br /> | ||
<img src="https://static.igem.org/mediawiki/2011/5/55/Kit_equation.png" alt="equation" />.<br /> | <img src="https://static.igem.org/mediawiki/2011/5/55/Kit_equation.png" alt="equation" />.<br /> | ||
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| - | < | + | <h4 id="Overall1">Standard curve for coloring reaction in urea assay</h4> |
<p>To generate standard curve, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate in the same way as the samples. Standard curve is shown in Fig.1.<br /> | <p>To generate standard curve, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate in the same way as the samples. Standard curve is shown in Fig.1.<br /> | ||
| - | <img src="https://static.igem.org/mediawiki/2011/9/9c/Standard_curve.png" alt="Standard curve for coloring reaction in urea assay | + | <img src="https://static.igem.org/mediawiki/2011/9/9c/Standard_curve.png" alt="Standard curve for coloring reaction in urea assay" width="400px" /> <br /> |
Fig.1 Standard curve for coloring reaction in urea assay | Fig.1 Standard curve for coloring reaction in urea assay | ||
Latest revision as of 03:17, 27 October 2011
Urea assay method
1. Preparation of samples for urea concentration assay
- A single colony of cells transformed with engineered plasmids (mock,rocF or rocF+Arg box) was inoculated into 3 mL of LB with appropriate antibiotics and grown to saturation at 37℃.
- The saturated culture was diluted 50-fold, grown till the log phase (OD600 = 0.5).
- The culture was induced with 1 mM IPTG at 37℃ for 1 hour.
- 1.5 mL of culture was centrifuged at 9,000 rpm for 1 minute and the supernatant fluid was used as a sample for urea concentration assay.
2. Urea concentration assay
Urea concentrations of the samples were determined colorimetrically with DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems.
Detailed methods are as follows.
- 10 µL of the supernatant fluid from each sample, 10 µL blank(LB),and 10 µL standard (10 mg/dL urea LB) were transferred to wells of clear bottom 96-well plates.
- 200 µL working reagent for coloring reaction from DIUR-500 -QuantiChrom™ Urea Assay Kit was added and the wells were taped lightly to mix.
- The mixture was incubated for 20 munites at room temperature.
- Optical density at 450 nm was read and urea concentration (mg/dL) of the sample was calculated as
.
ODSAMPLE, ODBLANK and ODSTANDARD are OD450 values of sample, standard and blank, respectively.
Standard curve for coloring reaction in urea assay
To generate standard curve, 0, 0.5, 1.0, 2.5, 7.5, 10 mg/dL urea LB were assayed in triplicate in the same way as the samples. Standard curve is shown in Fig.1.
Fig.1 Standard curve for coloring reaction in urea assay
