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Team:Tokyo Tech/Projects/Urea-cooler/method
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<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 /> | ||
Each sample was assayed in triplicate. | Each sample was assayed in triplicate. | ||
Revision as of 14:12, 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
.
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, 10mg/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
