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. 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 Protocol Details
We have provided detailed instructions, included background material, in the PDF below (InterLab_2017_Plate_Reader_Protocol.pdf). 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. Once you have completed your protocol, you can then fill out the Google Forms by clicking on the buttons at the bottom of this page (Day 1, Day 2, and Day 3). These Plate Reader Forms is a form version of the protocol and asks for specific details from your experiments. If you cannot access the Google Forms, please email us at measurement AT igem DOT org and we will send you an alternative form to fill out.
All teams must use a plate reader to measure GFP and finish the following items to fulfill the InterLab:
- Download the Excel File below (TeamName_InterLab_2017_Measurements.xls) and fill it out with your data. Rename the file [TeamName] to reflect your team's name.
- Email your completed Excel files to measurement AT igem DOT org (Due by September 29)
- Fill in the three Plate Reader Forms (Day 1, Day 2, and Day 3) provided at the bottom of this page (Due by September 29)
- Edit the InterLab study page on your team wiki (edit this page in your wikis: http://2017.igem.org/Team:[TeamName]/InterLab) (Due by November 1, Wiki Freeze day)
Plate Reader Forms
Download the Protocol PDF file here: InterLab_2017_Plate_Reader_Protocol.pdf
Download the Excel file here: TeamName_InterLab_2017_Measurements.xls
Download the Dilution Calculation Sheet here: 2017 Interlab Dilution Calculation Sheet.xlsx