---+ Microplate Reader Quick Start ---++ Best Practices 1 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. 2 It's always best to start from single colony picks that had been grown overnight in liquid culture to saturation. 3 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. 4 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. [[https://doi.org/10.2307/1942661][See this paper]] – the concepts broadly apply. 5 Remember to record the positions of each sample for later analysis and to include at least one blank. 6 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; ---++ Plate reader operation 1 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. 2 Ensure the computer is on and the plate reader is plugged in prior to powering on the plate reader. 3 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 ---++ Determining Gain ‘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. ---++ 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. One common error is to use excitation and emission wavelengths that are too close together for the filters on a machine. For example, do not use excitation 488 nm and emission 509 nm for GFP. Numbers in parentheses after a wavlength show the total size of the window of wavelengths transmitted by a filter (e.g., 450(20) nm means light gets through from 440 to 460 nm). | *Measurement* | *Absorption* | *Emission* | *Citation* | *Notes* | | Cell Density | 600 | 600 | [[https://doi.org/10.1186/2046-1682-6-4][Myers 2013]] | Use 660 with mRFP | | Cell Density w/ mRFP | 660 | 660 | [[https://doi.org/10.1038/nbt1172][Pedelacq 2006]] | Use 660 with mRFP | | *Fluorescent Protein* | *Excitation* | *Emission* | *Citation* | *Notes* | | BFP (mTagBFP) | 405 nm | 453 nm | [[https://doi.org/10.1101/2024.04.08.588465][Radde 2024]] | | | YFP | 436 nm | 480 nm | [[https://doi.org/10.1126/science.1122469][Hegreness 2006]] | | | GFP (sfGFP) | 485 nm | 528 nm | [[https://doi.org/10.1101/2024.04.08.588465][Radde 2024]] | | | CFP | 500 nm | 535 nm | [[https://doi.org/10.1126/science.1122469][Hegreness 2006]] | | | RFP (mRFP1) | 585 nm | 610 nm | [[https://doi.org/10.1101/2024.04.08.588465][Radde 2024]] | Be aware of absorption overlap with 600 nm for OD measurements | ---++ Our Plate Reader Protocols * [[https://barricklab.org/twiki/bin/view/Lab/ProtocolsGrowthRates][Microbial Growth Rates]] * [[https://barricklab.org/twiki/bin/view/Lab/ProtocolsIntrocellularROSDetection][Measuring Intracellular Reactive Oxygen Species]] * [[https://2019.igem.org/Team:Austin_UTexas/Experiments#Burden_Assay][Burden Assays]]
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Topic revision: r4 - 2024-04-17 - JeffreyBarrick