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− | <b>Tab. 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 1:1024 in line 11 and | + | <b>Tab. 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 1:1024 fluorescein diluted with PBS in line 11 and PBS only in line 12. |
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Revision as of 20:40, 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
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 with 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 don’t represent a usable number, the excel algorithm can’t display them in a 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.