IGEM:IMPERIAL/2007/Projects/Cell by date/Implementation/Calibrating Fluorescence: Difference between revisions
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===Summary=== | ===Summary=== | ||
Protein quantification is not new to measuring relative fluorescence in cells, but for our experiment it would be a bonus to be able to relate the relative measurements to that of the actual molecule numbers per cell. This would facilitate comparison of results from the plate reader with measurements that would be taken across the various parameters as described (eg. Temperature change, for example). This process of calibrating GFP counts using the GFP-separated labels on the fluorometer plate reader to GFP concentration is adapted from protocols described by [[Endy:Victor3_plate_reader/Calibrating_the_GFP-separated_label|Endy Lab]]. | Protein quantification is not new to measuring relative fluorescence in cells, but for our experiment it would be a bonus to be able to relate the relative measurements to that of the actual molecule numbers per cell. This would facilitate comparison of results from the plate reader with measurements that would be taken across the various parameters as described (eg. Temperature change, for example). This process of calibrating GFP counts using the GFP-separated labels on the fluorometer plate reader to GFP concentration is adapted from protocols described by [[Endy:Victor3_plate_reader/Calibrating_the_GFP-separated_label|Endy Lab]]. | ||
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===Materials Required=== | ===Materials Required=== | ||
====Equipment==== | ====Equipment==== | ||
1. | 1. | ||
2. | 2. | ||
3. | 3. | ||
====Reagents==== | ====Reagents==== | ||
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2. | 2. | ||
3. | 3. | ||
===Protocol=== | ===Protocol=== | ||
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2. | 2. | ||
3. | 3. | ||
===Calculations=== | ===Calculations=== | ||
More info to be added. | More info to be added. | ||
Revision as of 06:38, 1 August 2007
Calibrating Fluorescence
Summary
Protein quantification is not new to measuring relative fluorescence in cells, but for our experiment it would be a bonus to be able to relate the relative measurements to that of the actual molecule numbers per cell. This would facilitate comparison of results from the plate reader with measurements that would be taken across the various parameters as described (eg. Temperature change, for example). This process of calibrating GFP counts using the GFP-separated labels on the fluorometer plate reader to GFP concentration is adapted from protocols described by Endy Lab.
While calibration cannot be done directly to our experimental sample as the quantum efficiency of purified GFP in solution may be different from GFP in vivo, GFP from experimental samples to the GFP counts of lysed samples from the same original culture can be obtained by measuring GFP before and after cell lysis. Next, a standard curve that relates the concentration of purified GFP in non-fluorescent cell lysate to GFP counts can be measured. The combing of both sets of data would yield a clibration from GFP counts for experimental samples to GFP concentration.
[Insert image of calibration overview]
Materials Required
Equipment
1.
2.
3.
Reagents
1.
2.
3.
Protocol
1.
2.
3.
Calculations
More info to be added.
