Difference between revisions of "Team:DTU-Denmark/InterLab"
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| − | + | After performing the calibrations with a pleasing result, we measured OD600 and fluorescence of the transformed cells at 0 and 6 hours. Measurements of given times were created and calculations with the values we obtained from the standard curves and reference points gave us the following data: | |
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Revision as of 22:43, 26 August 2018
InterLab
To quote the official Interlab Measurement Study site, “reliable and repeatable measurement is a key component to all engineering disciplines”.
As a first step in our wetlab efforts we decided to embark on the fifth Interlab Measurement Study. In previous studies, it was shown that the variability between different lab measurements could be reduced by GFP fluorescence expression against a known concentration of a fluorescent molecule [minder det for meget om det der står på siden?]. However, the number of cells in the sample also holds a large impact on the variability.
Therefore, this year’s interlab study is about reducing the variability in fluorescence measurements between labs by normalizing to CFU or absolute cell count. To this, we firstly needed a plate reader that is capable of measuring both absorbance and fluorescence. We used the Biotek Synergy MX plate reader. Specifications of this model important to the protocol in use was:
- It could measure both absorbance and fluorescence
- It had pathlength correction
- Temperature settings went from 4 to 65°C with a precision of ± 0.5°C at 37°C.
- Bandpass width was 530/20
- Excitation was 485 nm
- It used top optics
0. Transformation of competent cells
Luckily, we did not need to create our own competent cells as we already had high-efficiency dh5α cells at hand. As we had a sufficient amount of colonies on our plates, we picked two colonies from each plate and inoculated them in LB medium + chloramphenicol O/N at 37°C and 220 rpm.
1. Calibration 1: OD600 Reference point - LUDOX Protocol
Afterwards, we measured the LUDOX CL-X (45% colloidal silica suspension) as a single point reference to gain a conversion factor to transform the absorbance to a OD600 measurement. Plate readers, in general, are volume dependent and this calibration was therefore necessary. We obtained the following data:
| LUDOX CL-X | H2O | |
|---|---|---|
| Replicate 1 |
0.051 | 0.036 |
| Replicate 2 | 0.052 | 0.035 |
| Replicate 3 | 0.054 | 0.037 |
| Replicate 4 | 0.052 | 0.037 |
| Arith. Mean | 0.052 | 0.036 |
| Corrected Abs600 | 0.016 | |
| Reference OD600 | 0.063 | |
| OD600/Abs600 | 3.938 |
2. Calibration 2: Particle Standard Curve - Microsphere Protocol
Next we prepared a dilution series of monodisperse silica microspheres to measure the Abs600 in our plate reader. Since there is a known amount of particles per volume and their size and optical characteristics are similar to those of cells, we could use the measurement to construct a standard curve. Our results were as following:
| Number of Particles | 2,35E+08 | 1,18E+08 | 5,88E+07 | 2,94E+07 | 1,47E+07 | 7,35E+06 | 3,68E+06 | 1,84E+06 | 9,19E+05 | 4,60E+05 | 2,30E+05 | 0 |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Replicate 1 | 0,994 | 0,575 | 0,311 | 0,163 | 0,101 | 0,091 | 0,042 | 0,04 | 0,043 | 0,038 | 0,036 | 0,036 |
| Replicate 2 | 0,97 | 0,567 | 0,271 | 0,162 | 0,106 | 0,064 | 0,051 | 0,042 | 0,039 | 0,038 | 0,037 | 0,042 |
| Replicate 3 | 1,092 | 0,615 | 0,28 | 0,244 | 0,104 | 0,082 | 0,053 | 0,041 | 0,039 | 0,035 | 0,037 | 0,038 |
| Replicate 4 | 1,199 | 0,454 | 0,283 | 0,148 | 0,096 | 0,069 | 0,048 | 0,041 | 0,039 | 0,037 | 0,036 | 0,036 |
| Arith. Mean | 1,064 | 0,553 | 0,286 | 0,179 | 0,102 | 0,077 | 0,049 | 0,041 | 0,04 | 0,037 | 0,037 | 0,038 |
| Arith. Std.Dev. | 0,104 | 0,069 | 0,017 | 0,044 | 0,004 | 0,012 | 0,005 | 0,001 | 0,002 | 0,001 | 0,001 | 0,003 |
| Arith. Net Mean | 1,026 | 0,515 | 0,248 | 0,141 | 0,064 | 0,039 | 0,011 | 0,003 | 0,002 | -0,001 | -0,002 |
Particles / OD
4. Cell measurement of transformed cells: OD600 and fluorescence
After performing the calibrations with a pleasing result, we measured OD600 and fluorescence of the transformed cells at 0 and 6 hours. Measurements of given times were created and calculations with the values we obtained from the standard curves and reference points gave us the following data: