Endy:Basic data analysis on a plate reader
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When analyzing time series data from the plate reader there are some basic things you probably want to do to analyze your data. These suggestions assume you are interested in growth data and fluorescence levels.
- Use an excel macro to build tables of the time series of absorbance and fluorescence labels for each well. An example is here.
- Subtract the media background from the absorbance and fluorescence labels. To be able to do this, you need to run a media blank on your plate. Typically, the absorbance of a media well will be 0.036+/-0.01 and the fluorescence of media (e.g M9 or EZ) using the GFP separated label will be ~1800+/-100.
- Convert absorbance data into OD600 values. Using the calibration data found here, we know that the formula for conversion is estimated by OD600 = 3.113(Absorbance) − 0.0158.
- The data plots that are likely to be important are -
- OD600 vs. time - growth curve
- Fluorescence vs. time - fluorescence time course
- Fluorescence/OD600 vs. time - should be proportional to reporter protein concentration per cell.
- Fluorescence vs. OD600 - if different samples grow at very different rates, it can be more informative to show the way the fluorescence varies with OD rather than with time.
- It is not very easy to write a macro to accomplish this and allow for varying numbers of wells different blanks etc. If anyone has any partial macros please post them here.
- When exporting data from Microsoft Excel, note that Excel will only export values with the level of precision seen on the spreadsheet rather than the true precision with which the value is known. For example, if you measure a well to have absorbance 0.0345900990345890453 and your spreadsheet shows 0.0346 then 0.0346 will show up in the .csv file (not 0.0345900990345890453). This can be an issue especially for absorbance measurements.