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<div class="section-text container"> | <div class="section-text container"> | ||
1. OD | 1. OD | ||
− | <sub>600</sub> Reference point Results of OD | + | <sub>600</sub> Reference point <br> Results of OD |
<sub>600</sub> measurement of 1 ml LUDOX and 1 ml H | <sub>600</sub> measurement of 1 ml LUDOX and 1 ml H | ||
<sub>2</sub>O with dH | <sub>2</sub>O with dH | ||
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<img class="section-image" src="" alt="fluorescein Table"> | <img class="section-image" src="" alt="fluorescein Table"> | ||
<div class="section-sub-text"> | <div class="section-sub-text"> | ||
− | <b>Fig. 2:</b> Fluorescein fluorescence plate reader measurement with gain 48 & 70 in a 96 well plate. The concentration cuts in half from pure fluorescein in line 1 to | + | <b>Fig. 2:</b> Fluorescein fluorescence plate reader measurement with gain 48 & 70 in a 96 well plate. The concentration cuts in half from pure fluorescein in line 1 to 9.766*10<sup>-4</sup>> µl fluorescein diluted in PBS in line 11 and PBS only in line 12. All volumes are 100 µl. |
</div> | </div> | ||
</p> | </p> | ||
<div class="section-sub-text container"> | <div class="section-sub-text container"> | ||
<p>The task was to find the optimal plate reader settings for the cell measurement on Day 3 through a fluorescein | <p>The task was to find the optimal plate reader settings for the cell measurement on Day 3 through a fluorescein | ||
− | dilution | + | dilution in PBS and a 485 excitation & 531 emission fluorescence measurement. We came up with the problem, |
that the same gain settings could not be used for both, fluorescein and cell measurement as in each case | that the same gain settings could not be used for both, fluorescein and cell measurement as in each case | ||
one of the measurement only displayed 50% usable outcomes. Additionally, only a gain of 48 or less displayed | one of the measurement only displayed 50% usable outcomes. Additionally, only a gain of 48 or less displayed | ||
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<div class="section-sub-text"> | <div class="section-sub-text"> | ||
<b>Fig. 3 & 4:</b> This diagrams display the amount of fluorescence according to the fluorescein µM concentration | <b>Fig. 3 & 4:</b> This diagrams display the amount of fluorescence according to the fluorescein µM concentration | ||
− | in 100 µl | + | in 100 µl PBS per well. |
</div> | </div> | ||
</div> | </div> | ||
<div class="section-text container"> | <div class="section-text container"> | ||
− | <p>As values bigger than 100,000 are labeled as OVERFLOW and | + | <p>As values bigger than 100,000 are labeled as OVERFLOW and do not represent a usable number, the excel algorithm could not |
− | display them in | + | display them in usable curves. |
<p> | <p> | ||
</div> | </div> |
Revision as of 20:59, 1 November 2017
INTERLAB MEASUREMENT STUDY
Reliable and repeatable measurement is key to compare and analyse data from different labs all over the world. To support the effort to create detailed protocols to measure fluorescence and improve the possibility of comparing data, the iGEM Team NAWI_Graz 2017 decided to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology. The goal is to establish a GFP measurement protocol based on engineering principles.
Our tasks were to follow exactly the iGEM Plate Reader Protocol , fill in the prepared excel sheets and send the results to the iGEM headquarter.Results
Calibration Protocols
Results of OD 600 measurement of 1 ml LUDOX and 1 ml H 2O with dH 2O as blank.
2. Protocol fluorescein fluorescence standard curve:
The task was to find the optimal plate reader settings for the cell measurement on Day 3 through a fluorescein dilution in PBS and a 485 excitation & 531 emission fluorescence measurement. We came up with the problem, that the same gain settings could not be used for both, fluorescein and cell measurement as in each case one of the measurement only displayed 50% usable outcomes. Additionally, only a gain of 48 or less displayed a complete set of fluorescein values. But the producing company of our plate reader only recommends a gain range for 50-150. So not even gain 48 displays “usable" values. We found the possibility for wider detection ranges in the product help documents, but our plate reader model Synergy MX from BioTek did not offer this option. Problems with the dilution by pipetting could be excluded.
![[Fluorescein standard curve]](https://static.igem.org/mediawiki/2017/f/f4/Fl.png)
![[Standard curve (log)]](https://static.igem.org/mediawiki/2017/1/17/Fl_log.png)
As values bigger than 100,000 are labeled as OVERFLOW and do not represent a usable number, the excel algorithm could not display them in usable curves.
Cell Measurement Protocol
- Day 1 OD600 Measurement
- Day 2 Transformation
The standard dilution sheet only calculates volumes for a target of 10 ml. The official InterLab protocol wants a target volume of 12 ml but also the use of the dilution calculation sheet, so we made a decision and sticked clearly to the protocol and did not decide to change the sheet algorithm.
- Day 3 Cell growth, sampling and assay. Preparing and taking probes at T0h, T2h, T4h, T6h accordingly to the dilution sheet and plate reader protocol.
![Flourescence table](https://static.igem.org/mediawiki/2017/1/1f/Raw_fluorescence.png)
![Flourescence table](https://static.igem.org/mediawiki/2017/5/5f/Fltable.png)
Even though device 2 has the strongest fluorescence, device 4 grew better resulting in a higher OD 600.