Team:Toronto/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:
  • lambert-law
  • 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.
Table1.

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.

LUDOX_CL_X 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.
graph graph1

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).

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