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Team:Tokyo Tech/Projects/Urea-cooler/method
From 2011.igem.org
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<h1> Urea assay method </h1> | <h1> Urea assay method </h1> | ||
| - | <h2 id="Overall1">1. Preparation for urea concentration assay</h2> | + | <h2 id="Overall1">1. Preparation of samples for urea concentration assay</h2> |
<p> | <p> | ||
<ol> | <ol> | ||
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<li>The saturated culture was diluted 50-fold, grown till the log phase (OD600 = 0.5).</li> | <li>The saturated culture was diluted 50-fold, grown till the log phase (OD600 = 0.5).</li> | ||
<li>The culture was induced with 1mM IPTG at 37℃ for 1 hour. </li> | <li>The culture was induced with 1mM IPTG at 37℃ for 1 hour. </li> | ||
| - | <li> | + | <li>2mL of culture was centrifuged at 9,000 rmp for 1 minute and the supernatant fluid was used as a sample for urea concentration assay. </li> |
| + | </ol> | ||
| + | |||
| + | </p> | ||
| + | <h2 id="Overall1">2.Urea concentration assay</h2> | ||
| + | <p>Urea concentrations of the samples were determined colorimetrically with DIUR-500 -QuantiChrom™ Urea Assay Kit obtained from BioAssay Systems.<br /> | ||
| + | Each sample was assayed in triplicate. | ||
| + | <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>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>Optical density at 450nm was read and urea concentration (mg/dL) of the sample was calculated as </li> | ||
| + | |||
</ol> | </ol> | ||
Revision as of 11:04, 4 October 2011
Urea assay method
1. Preparation of samples for urea concentration assay
- A single colony of cells transformed with mock, pTrc-rocF or pTrc-rocF-Arg box was inoculated into 3mL of LB with kanamycin 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 1mM IPTG at 37℃ for 1 hour.
- 2mL of culture was centrifuged at 9,000 rmp 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.
Each sample was assayed in triplicate.
- 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.
- Optical density at 450nm was read and urea concentration (mg/dL) of the sample was calculated as
