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Team:Macquarie Australia/Protocols/spectralanalysis
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<h3> Spectral Analysis </h3> | <h3> Spectral Analysis </h3> | ||
| - | Samples were irradiated (660 nm) for 15 minutes using an LED light under low ambient light conditions. Upon irradiation, single, 80 uL digests of each sample were transferred to a 384 well plate for analysis in a PHERAstar plate reader, over the range 350-1000 nm and at a resolution of 2 nm. The wells are shown below. We blanked the instrument using 80 uL of the 4KE3 sample. | + | <p>Samples were irradiated (660 nm) for 15 minutes using an LED light under low ambient light conditions. Upon irradiation, single, 80 uL digests of each sample were transferred to a 384 well plate for analysis in a PHERAstar plate reader, over the range 350-1000 nm and at a resolution of 2 nm. The wells are shown below. We blanked the instrument using 80 uL of the 4KE3 sample.<p> |
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<h2>Data Processing and Analysis</h2> | <h2>Data Processing and Analysis</h2> | ||
| - | Raw spectral data was exported directly from the PHERAstar Data Analysis Software to Excel. Absorption maxima were normalized against the absorption maximum and graphed, as seen in the results <a href="https://2012.igem.org/Team:Macquarie_Australia/Results#7">here</a> | + | <p>Raw spectral data was exported directly from the PHERAstar Data Analysis Software to Excel. Absorption maxima were normalized against the absorption maximum and graphed, as seen in the results <a href="https://2012.igem.org/Team:Macquarie_Australia/Results#7">here</a>.</p> |
Revision as of 03:52, 27 September 2012
Spectral Analysis of the BL21 Protein Samples
Incubation
Biliverdin (5 uL) was combined with 700 uL of sample in 5 2mL Eppendorf tubes. In addition, a negative control was prepared consisting of another 5 tubes of 700 uL of sample without BV. Each of these were wrapped together in foil and incubated overnight at 4 C. Overnight incubation was necessary here due to the slow reversion of the BP back to the red (660 nm) light absorbing conformation. Following irradiation at 660 nm we expect to see a change in absorbance at 720 nm due to a conformational change in the protein to the far-red light absorbing form.
Spectral Analysis
Samples were irradiated (660 nm) for 15 minutes using an LED light under low ambient light conditions. Upon irradiation, single, 80 uL digests of each sample were transferred to a 384 well plate for analysis in a PHERAstar plate reader, over the range 350-1000 nm and at a resolution of 2 nm. The wells are shown below. We blanked the instrument using 80 uL of the 4KE3 sample.
Wells in plate:
1: 1C3C #3 Irradiated
2: 1C3C #6 Irradiated
3: 1C3C #7 Irradiation
4: 1C3C #3 Control
5: 1C3C #6 Control
6: 1C3C #7 Control
7: Blank (4KE3)
Data Processing and Analysis
Raw spectral data was exported directly from the PHERAstar Data Analysis Software to Excel. Absorption maxima were normalized against the absorption maximum and graphed, as seen in the results here.
