Difference between revisions of "Team:Heidelberg/InterLab"
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| − | With these replicates measured, we obtained a ratiometric conversion factor of 4.30 for our specific plate reader when measured 100 | + | With these replicates measured, we obtained a ratiometric conversion factor of 4.30 for our specific plate reader when measured 100 μL of the provided LUDOX-HS40 to water.</p> |
<h2>1.3 Graphing a fluorescein fluorescence standard curve</h2> | <h2>1.3 Graphing a fluorescein fluorescence standard curve</h2> | ||
Revision as of 10:16, 22 September 2017
Team:Heidelberg/internal tools
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From 2014.igem.org
1. Interlab Study
As a first step to contribute to this year's iGEM competition we decided to participate in iGEM's fourth International Interlaboratory Study along with many other teams from around the world. This study is organized by iGEM's measurement committee in an effort to establish standardized, reliable and repeatable measurement tools for the iGEM community and the synthetic biology community as a whole. This year's iGEM InterLab study is about establishing a standardized protocol for the measurement of GFP using a plate reader. To start things off we needed a plate reader that is qualified to measure GFP fluorescence. Namely, Tecan Infinite M200 Pro plate reader. Additionally, we needed competent E.coli DH5α. These were prepared from glycerol stocks. Together with the material iGEM had provided we were ready for work. Throughout our experiments, we tested 8 plasmids (2 controls and 6 test devices) by measuring the OD600 and the fluorescence of the cells carrying the constructs. The workflow can be separated into four segments. The first segment is the transformation of all plasmids into competent DH5α cells.
1.1 Transformation of 8 test devices into competent DH5α cells
The transformation was successful for all the plasmids and resulted in a sufficient amount of colonies on all plates. Therefore, two colonies from each plate were picked and inoculated in LB medium containing chloramphenicol over night for approximately 16-18 hours at 37°C and 220 rpm.
1.2 Measurement of LUDOX-HS40 OD600 Reference Point
The second segment is the measurement of the LUDOX-HS40 solution to obtain a ratiometric conversion factor which allows us to transform the absorbance data into a standard OD600 measurement. This is necessary because plate readers in general do not have a 1 cm path length which means that measurements are volume dependent. By doing so, we obtained following data.
Figure 1.1: Measurement of the LUDOX-HS40 and water in four replicates and calculation of the ratiometric conversion factor
With these replicates measured, we obtained a ratiometric conversion factor of 4.30 for our specific plate reader when measured 100 μL of the provided LUDOX-HS40 to water.
1.3 Graphing a fluorescein fluorescence standard curve
In the third segment we were instructed to measure a standard curve for fluorescence of fluorescein which can be used to correct our cell based readings to an equivalent fluorescein concentration. Following the instructions, we could obtain following results for the serial dilution of fluorescein in phosphate buffered saline.
Figure 1.2: Measurement of a standard curve with four replicates by serial dilution of fluorescein in phosphate buffered saline
Figure 1.3: Fluorescence is plotted against the fluorescein concentration
Figure 1.4: Fluorescence is plotted against the fluorescein concentration on a logarithmic scale
The data give us a more or less linear regression line when plotted in a logarithmic scale but behave more like a quadratic equation when plotted normally.
1.4 Measurement of OD600 and Fluorescence of Transformed Cells
In the last of the four segments the OD600 and the fluorescence of the transformed cells were measured according to the protocol after 0, 2, 4 and 6 hours. Measurements of all these time points gave us the following tables and calculations which were conducted with the values we obtained from the standard curves and the reference point.
Figure 1.5: Raw data
Figure 1.6: Raw data and measurements for the first two time points
Figure 1.7: Raw data and measurements for the last two time points
References
