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Revision as of 23:04, 1 October 2017
INTERLAB STUDY
Overview
Methods
Protocol
The iGEM Plate Reader Protocol was followed for both absorbance and fluorescence calibrations, and for cell measurements.Calibrations
Absorbance at 600nm was measured for LUDOX S40 and H2O, to calibrate the conversion factor optical density (OD600) and absorbance at 600nm (Abs600).
A fluorescein standard curve was constructed plotting fluorescence vs uM fluorescein of 2:1 serial dilutions of 50uM fluorescein.
Cell Measurements
Measurements were performed according to the iGEM Plate Reader Protocolprotocol.
E. coli DH5α Inoue competent cells were transformed according to the recommended iGEM Single Tube Transformation Protocol LINK. The serial dilutions of fluorescein were diluted according to the LINKiGEM Interlab Study Plate Reader Protocol.
Cells were grown overnight for 14 hours to a final optical density of ..... The cell cultures were then diluted according to the calculations in the dilution calculation sheet. Include sheet? . . .
At this point a measurement t=0 was taken, and the cell cultures were allowed to incubate for 6 hours, with 500mL aliquots taken every 2 hours for measurements. All aliquots were kept on ice, and transferred to the recommended format in the 96-well plates.
Flourescence Measurements:
| GFP IGEM | 4000065 |
|---|---|
| Top mirror | FITC |
| Bottom mirror | N/A |
| Exc. filter | FITC 485 |
| Using of excitation filter | Top |
| 2nd exc. filter | N/A |
| Using of 2nd excitation filter | Top |
| Ems. filter | FITC 535 |
| 2nd ems. filter | N/A |
| Measurement height | 6 mm |
| Number of flashes | 10 |
| Number of flashes integrated | 1 |
| PMT gain | 1 |
| Limits of excitation light | 1% |
| Range of excitation light | 100% |
| Reference AD gain | 2 |
| Reference signal | 388381 |
| Last edited | 9/21/17 11:34 |
| Last edited by | EnVision |
| Factory preset | No |
Absorbance 600 settings:
| Absorbance @ 600 | 2000005 |
|---|---|
| Exc. filter | Photometric 600 |
| Measurement height | 13 mm |
| Number of flashes | 10 |
| Number of flashes integrated | 1 |
| Limits of excitation light | 100% |
| Range of excitation light | 100% |
| Reference AD gain | 8 |
| Reference signal | 260565 |
| Continuous measurement | No |
| Last edited | 3/29/16 0:37 |
| Last edited by | EnVision |
| Factory preset | No |
Filters Used:
| FITC 485 | 102 |
|---|---|
| Filter type | Excitation |
| Description | X485 CWL=485nm BW=14nm Tmin=60% |
| Used with | Absorbance DELFIA - Time-resolved Fluorescence Fluorescence Intensity Fluorescence Polarization LANCE - Time-resolved Fluorescence |
| Last edited | 10/21/10 13:14 |
| Last edited by | EnVision |
| Factory preset | Yes |
| FITC 535 | 206 |
| Filter type | Emission |
| Description | M535 CWL=535nm BW=25nm Tmin=50% |
| Used with | Absorbance DELFIA - Time-resolved Fluorescence Fluorescence Intensity Fluorescence Polarization LANCE - Time-resolved FluorescenceLuminescence DELFIA - Time-resolved Fluorescence Fluorescence Intensity LANCE - Time-resolved Fluorescence |
| Last edited | 6/25/07 11:39 |
| Last edited by | Installation |
| Factory preset | Yes |
| Photometric 600 | 319 |
| Filter type | Excitation |
| Description | P600 CWL=600nm BW=8nm Tmin=40% |
| Used with | Absorbance DELFIA - Time-resolved Fluorescence Fluorescence Intensity Fluorescence Polarization LANCE - Time-resolved FluorescenceLuminescence DELFIA - Time-resolved Fluorescence Fluorescence Intensity LANCE - Time-resolved FluorescenceAbsorbance DELFIA - Time-resolved Fluorescence Fluorescence Intensity Fluorescence Polarization LANCE - Time-resolved Fluorescence |
| Last edited | 6/25/07 11:39 |
| Last edited by | Installation |
| Factory preset | Yes |
Results
Other plots including graphical interpretations of results
References:
