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Microplate Reader Quick Start |
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- It's always best to start from single colony picks that had been grown overnight in liquid culture to saturation.
- A typical start culture dilution is 2:200, 1:200 or 1:250. The most diluted one (1:250) would provide the best result for measuring lag phase times.
- Plan out which wells will contain what sample. Randomizing a bit can help average out temperature differences throughout the plate; you can use a simple "JumpTwoRowsDown-Jump#ofReplicatesToRight" algorithm for positioning each replicate in the 96-well plate. Printable 96 well plate templates are available online. Generally, each condition should be done in quadruplicate (or triplicate depending on space concerns). DON'T FORGET TO INCLUDE MEDIA BLANKS (minimum of one!)
- Sign up and make reservation for TECAN microplate reader here TECAN microplate
- Select the appropriate type of plate for your application: black walled, clear bottom for fluorescence (white walled, clear bottom is generally for luminescence and completely clear is generally for other applications, like measuring OD600).
- Ensure that you have adequate controls for your experiment. At the very least, have your negative control in the same media grown under the same conditions.
- Turn on the plate reader - there is a power button in the back next to the power cord - and then turn on the Magellan software on the computer next to it. Not doing it in this order may cause the software to fail to recognize the machine.
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> | Best Practices
- Follow typical best practice for experimental design. Utilize technical replicates from the same biological sample. Have biological replicates spread across different days, appropriate controls, etc.
- It's always best to start from single colony picks that had been grown overnight in liquid culture to saturation.
- A typical start culture dilution is 2:200, 1:200 or 1:250 from an overnight culture. The most diluted one (1:250) would provide the best result for measuring lag phase times.
- It is advisable to spread out samples across the plate, rather than clustering samples together or along the same column/row. This reduces the impact of biases due to evaporation, temperature, or other affects across the plate. Randomization is close to (but not always entirely) ideal. See this paper – the concepts broadly apply.
- Remember to record the positions of each sample for later analysis and to include at least one blank.
- Select the appropriate type of plate for your application: black walled, clear bottom for fluorescence; white walled, clear bottom for luminescence; and completely clear for other applications, like measuring OD600;
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- Select the option that says "raw data" and eventually you will find yourself at the configuration screen.
- Once the protocol is started an Excel worksheet will open up automatically to record the data.
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< | For OD600 measurements and growth rates go to Determining Growth Rates |
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> | Plate reader operation |
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- If your plate reader is controlled by a Windows computer (like our’s), turn off WiFi and ensure that the computer is up to date prior to starting your experiment. Windows Update will reboot the computer mid-experiment and kill your run.
- Ensure the computer is on and the plate reader is plugged in prior to powering on the plate reader.
- Depending on the software you use (ex. iControl), using the scroll wheel may change a numerical setting. Be cognisant of this and take care not to inadvertently adjust settings
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< | For fluorescence measurements go to Fluorescence Measurements |
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> | Determining Gain |
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< | --Main.GabrielSuarez - 14 Dec 2017 |
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> | ‘Gain’ is the sensitivity of a plate reader to the emission of whatever you are measuring. It is generally important to set this yourself – and especially so for time series – as automatically adjusting gain will render each set of measurements in a time course (or individual run) as incomparable to each other. This value will differ from experiment to experiment depending on the plate reader, the plate used, the measurement in question, media used, and others. A higher gain will improve the dynamic range when measuring dim samples; however, too high of a gain will result in saturation of the detector. The best gain setting one which is a bit below the saturation mark for the brightest anticipated measurement. In certain rare situations where no single gain setting per fluorophore is appropriate, it may be necessary to use two gain settings. The same wells measured at two different gains should linearly relate to each other. |
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Standard wavelengths
Different fluorescent markers have different absorption and emission spectra. While there are resources that detail the spectra, such as FPbase, the most appropriate wavelengths to use for measurements may not be immediately apparent. Provided here is a list of commonly used fluorphores and appropriate wavelengths to use.
| Target |
Absorption |
Emission |
Citation |
Notes |
| Cell Density |
600 |
600 |
Myers 2013 |
Use 660 with mRFP |
| Cell Density w/ mRFP |
660 |
660 |
Pedelacq 2006 |
Use 660 with mRFP |
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| mBFP |
| mCFP |
| mYFP |
| mRFP |
| mCherry |
| E2Chrimson |
| mScarlet |
Our Plate Reader Protocols
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