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Revision as of 17:00, 31 October 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
1. OD600 Reference point
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.
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
- Day 3 Overnight culture
Fehlenden Text aus der Drive einfügen!!!!!!