Difference between revisions of "Team:TecMonterrey GDL/InterLab"
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Silica microspheres were provided by iGEM kits, these were meant to simulate cells in a dilution, and thus the protocol stated to make serial dilutions of the microsphere suspension in order to construct a standard curve for Abs600 which would allow conversion of Abs600 measurements into an estimated number of cells. Our curve results are shown in the following figure: | Silica microspheres were provided by iGEM kits, these were meant to simulate cells in a dilution, and thus the protocol stated to make serial dilutions of the microsphere suspension in order to construct a standard curve for Abs600 which would allow conversion of Abs600 measurements into an estimated number of cells. Our curve results are shown in the following figure: | ||
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Revision as of 02:39, 28 July 2018
InterLab
This year (2018) the iGEM Team TecMonterrey_GDL decided to contribute into the Interlab study measurements. The goal of the study is summarized in achieving a way to reduce lab-to-lab variability in fluorescence measurements via normalizing to absolute cell count or colony forming units. The approaches taken were indicated by the iGEM measurement team: 1. Converting between absorbance of cells to absorbance of a known concentration of microsphere silica beads 2. Counting colony-forming units (CFU) from the samples The team followed a series of protocols created to help standardize the measurement of GFP (green fluorescent protein), which is widely used as measurement marker. Specifications: The equipment we used was Cytation 3 by Biotek. It can measure both absorbance and fluorescence. And the pathlength correction was disabled. The temperature of the measurements was done at room temperature.
Calibration Protocol 1: OD600 Reference point - LUDOX Protocol
The first protocol’s goal was to obtain a conversion factor between absorbance measured and the OD600 measurement as it occurs in spectrophotometry measurements. This was made with the kit provided LUDOX CL-X (45% colloidal silica suspension) provided by iGEM and sterile mili Q water, reactants were added accordingly to the protocol and the obtained results are shown in the next table (Table 1):
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Table 1. Calibration Protocol 1: OD(600) Reference point - LUDOX Protocol
The corrected absorbance was was calculated by subtracting the mili Q water reading. The reference OD600 was provided to the team by iGEM. The correction factor was obtained by dividing the reference OD600 by corrected Abs600 obtained.
Calibration Protocol 2: Particle Standard Curve - Microsphere Protocol
Silica microspheres were provided by iGEM kits, these were meant to simulate cells in a dilution, and thus the protocol stated to make serial dilutions of the microsphere suspension in order to construct a standard curve for Abs600 which would allow conversion of Abs600 measurements into an estimated number of cells. Our curve results are shown in the following figure: