Measurement/InterLab/Plate Reader
Plate Reader and CFU Protocol
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Please note: The data submission deadline was July 27. Data is now under review by the Measurement Committee.
Method Overview
Measurements must be made in a plate reader, as the plate reader with a 96 well format is ideal as it provides a convenient format for multiple measurements. The methods are written from the perspective of using a 96 well format.
The first objective is to obtain your standard curves for fluorescence based on the sodium fluorescein reference material provided. Because the sodium fluorescein should fluoresce in the same way across different labs, measuring a standard curve of fluorescein values will allow us to calibrate the values obtained from your plate reader so that it can be compared with data from other labs. The standard curve must be obtained under EXACTLY the same instrument conditions that you will use when you do your cell based expression assays. This includes all settings that affect the amplitude of the signal collected: filters or monochromator settings; slit widths; gain settings; plates or cuvette type used; measurement from top or bottom (in plates); number of reads (integration time); orbital averaging (available in some plate readers). You may not know right now what settings will be suitable when you do your cell based assays. The objective is to fix the obvious settings now, such as monochromator/ filter, top or bottom reads. The key settings that affect sensitivity are slit width and/or gain. You MUST therefore collect now several standard curves under different sensitivity settings.
When doing cell based assays you should as far as possible settle on a single setting, but this may not be possible as it may not have sufficient dynamic range. By having a series of standard curves collected with different sensitivity you can use one (of a limited number) of the settings to obtain appropriate data (i.e. within range on the instrument). Because you will have a standard curve to match this setting, you can still transform into absolute units.
Recommended Filters: Excitation of 485nm, Emission 530/30 (or as close as possible to that range).
Plate Reader and Colony Forming Unit (CFU) Protocol Details
We have provided detailed instructions, included background material, in the PDF below. We recommend that each team downloads this protocol when carrying out the experiments and copy any notes on those pages and in your lab notebook.
Required Data Forms
Each team is required to fill out the following data forms (one Excel sheet, four online Forms).
Form I asks questions about who conducted the InterLab on your team, biosafety, and general overview questions. Forms II and III are for recording information about the calibration and cell measurements from the plate reader protocol. Form IV is for recording your CFU results (the experiment from pages 13-14 in the protocol PDF).
Excel File
Click this button to download the Excel form you will use to collect your plate reader data.
Once you have entered the data into your Excel file, please rename it by replaced the text "YourTeamName" with your team's name. For example: InterLab_2018_iGEMHQ.xlsx
Click on the button below to upload your completed Excel form to Dropbox. If you cannot access Dropbox, please email your completed Excel file to measurement [AT] igem [DOT] org.
Online Forms I, II, III, and IV
The online forms are now closed. Thank you for participating in the InterLab study!
iGEM Foundation
The International Genetically Engineered Machine (iGEM) Foundation is an independent, non-profit organization dedicated to the advancement of synthetic biology, education and competition, and the development of an open community and collaboration. Visit us at igem.org
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