Difference between revisions of "Team:DTU-Denmark/InterLab"
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<h1>InterLab</h1> | <h1>InterLab</h1> | ||
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<h3>Introduktion</h3> | <h3>Introduktion</h3> | ||
<hr> | <hr> | ||
| − | <h3> | + | To quote the official Interlab Measurement Study site, “reliable and repeatable measurement is a key component to all engineering disciplines” [indsæt kilde?]. |
| + | |||
| + | 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 | ||
| + | |||
| + | <h3>0. Transformation of competent cells</h3> | ||
<hr> | <hr> | ||
<h3>Results and discussion</h3> | <h3>Results and discussion</h3> | ||
Revision as of 21:57, 23 August 2018
InterLab
Introduktion
To quote the official Interlab Measurement Study site, “reliable and repeatable measurement is a key component to all engineering disciplines” [indsæt kilde?]. 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
Results and discussion
Thursday 21/6/2018
Competent cells were prepared for the interlab studies using the given the iGem 2018 interlab platereader protocol. We did not need to create our own competent cells as we already had high-efficiency dh5α cells ready.
Friday 22/6/2018
Today we transformed our prepared competent cells by using the iGem 2018 interlab study transformation.
Tuesday 26/6/2018
Luquid LB media was prepared using the "Luria-Bertani (LB) Medium Preparation" protocol for the interlab studies in the morning, as we did not have enough for the next step. Interlab is put on hold until we have a platereader.
Monday 2/7/2018
We picked 2 colonies from each of our transformation plates and inoculated them in the apropriate LB and Chloramphenicol medium. The cells were left to grow overnight at 37 degrees Celsius.
Tuesday 3/7/2018
Today we finally had the platereader available and continued on the protocol from the 21st of June. We encountered som problems with our fluorescein samples and decided to redo them. We also decided to redo the overnight cultures as we would not have the time to use them today
Wednesday 4/7/2018
As we finally had all our controls finished, we cpntinued with the "Cell measurement" protocol. Our initial absorbance for our samples were way too high. We then finalized this part of the study and continued with the "Colony Forming Units" protocol.
Thursday5/7/2018
Data was extracted from our platereader and this, together with the CFU data, was sent off to finalize the interlab study. Another thing on our long list that can be ticked off.