Difference between revisions of "Team:Toronto/InterLab"
| Line 3: | Line 3: | ||
<body> | <body> | ||
| + | <h1>INTERLAB</h1> | ||
<u><b>Calibration 1: OD600 Reference point</b></u> | <u><b>Calibration 1: OD600 Reference point</b></u> | ||
<ul> | <ul> | ||
| Line 15: | Line 16: | ||
<p>Table shows absorbance measurements (at 600 nm) for LUDOX CL-X and dd-H2O using a plate reader. The corrected Abs<sub>600</sub> is the difference between the LUDOX CL-X reading and dd H2O reading. Reference OD<sub>600</sub> is a measurement by a spectrophotometer (provided on iGEM excel sheet). OD<sub>600</sub>/Abs<sub>600</sub> is the correction factor to convert Abs<sub>600</sub> to OD<sub>600</sub>, calculated by dividing Reference OD<sub>600</sub> by Abs<sub>600</sub>.</p> | <p>Table shows absorbance measurements (at 600 nm) for LUDOX CL-X and dd-H2O using a plate reader. The corrected Abs<sub>600</sub> is the difference between the LUDOX CL-X reading and dd H2O reading. Reference OD<sub>600</sub> is a measurement by a spectrophotometer (provided on iGEM excel sheet). OD<sub>600</sub>/Abs<sub>600</sub> is the correction factor to convert Abs<sub>600</sub> to OD<sub>600</sub>, calculated by dividing Reference OD<sub>600</sub> by Abs<sub>600</sub>.</p> | ||
<img src="https://static.igem.org/mediawiki/2018/d/d0/T--Toronto--7_25_2018_Interlab_LUDOX_CL_X.png" alt="LUDOX_CL_X"> | <img src="https://static.igem.org/mediawiki/2018/d/d0/T--Toronto--7_25_2018_Interlab_LUDOX_CL_X.png" alt="LUDOX_CL_X"> | ||
| + | <br /> | ||
| + | <br /> | ||
<u><b>Calibration 2: Particle Standard Curve</u></b> | <u><b>Calibration 2: Particle Standard Curve</u></b> | ||
Revision as of 01:12, 26 July 2018
INTERLAB
Calibration 1: OD600 Reference point- Purpose of this calibration: To transform absorbance data to a OD600 measurement, calculate a plate-reader specific (Tecan Infinite 200 Pro) conversion factor for OD600 from Abs600 calculated for Ludox CL-X on a mass spectrophotometer.
- Beer-Lambert’s law of absorbance dictates that optical path length plays a fundamental role in determining absorbance:
- This is necessary because cuvettes (used in photometer) have a fixed path optical path length when using light scattering to measure absorbance as opposed to the varying path lengths of wells in a 96-well plate which change as the volume of sample added in them changes.
- Results: Cell density readings can thus be converted to OD600 by multiplying correction factor value, 4.138.
Table shows absorbance measurements (at 600 nm) for LUDOX CL-X and dd-H2O using a plate reader. The corrected Abs600 is the difference between the LUDOX CL-X reading and dd H2O reading. Reference OD600 is a measurement by a spectrophotometer (provided on iGEM excel sheet). OD600/Abs600 is the correction factor to convert Abs600 to OD600, calculated by dividing Reference OD600 by Abs600.
Calibration 2: Particle Standard Curve
Construct a standard curve of Abs600 for microsphere particle concentration Purpose of calibration: iGEM distributed microspheres that mimic the size, shape and volume of cells which have a known amount of microspheres per volume. This calibration was required to generate a Particle Standard Curve which helped us determine the number of cells (as modelled by microspheres) in a sample.
- Use standard curve to convert Abs600 measurements to an estimate of number of cells.
- Monodisperse silica microspheres are used in the calibration because they have similar size and optical characteristics as cells.
Figure 1. Calibration of particle count to absorbance measured by plate-reader (a) A particle standard curve of Abs600 for known particle count/100ul measurements. (b) Graphical depiction of logarithmic scaling of particle standard curve in Figure 1.(a).
