Team:Botchan Lab Tokyo/InterLab
Calibration!!!!!!!!
OD600 Reference point - LUDOX Protocol
We used LUDOX CL-X (45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform absorbance(Abs600) data from plate reader into a comparable OD600 measurement as would be obtained in a spectrophotometer. We showed the results in Table 1.
2 Particle Standard Curve - Microsphere Protocol
We used a dilution series of monodisperse silica microspheres and measured the Abs600 in plate reader. (We showed the results in Table 2.)
Over, we made a standard curve of particle concentration which can be used to convert Abs600 measurements to an estimated number of cells using this measurements. (We showed the results in Figure 1.)
3 Fluorescence standard curve - Fluorescein Protocol
In order to compare fluorescence output of test devices between teams, it is necessary for each team to create a standard fluorescence curve, using the small molecule fluorescein which has similar excitation and emission properties to GFP. (We showed the results in Table3 and Figure 2.)
Cell measurement !!!!!!
We measured Abs600and fluorescence of Escherichia coli DH5α transformants.
Colony Forming Units per 0.1 OD600 E.coli cultures
We did a dilution series to investigate the ration (culture: medium) which can transform the measured OD600 of our overnight cultures to 0.1 OD600.
We calculated “fixed 0.1OD600” (each score - blank).
From table8, we decided the best ratio:Sample1;positive1→10^5 and negative1→10^5:Sample2;positive2→10^5 and negative2→10^5. To check if this ratio is proper, we did a dilution at such ratio.
Next, we performed a dilution series with triplicates for each overnight culture and plated the 8 x 104, 8 x 105, and 8 x 106 dilutions. Assuming that one bacteria gives rise to one colony, we counted the number of colonies per plate and multiplied by the final dilution factor to obtain a CFU/mL value for a starting sample of OD600.
