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Team:EPF-Lausanne/Tools/MITOMI
From 2011.igem.org
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Two scans of the chip are necessary for the analysis of the molecular interactions. | Two scans of the chip are necessary for the analysis of the molecular interactions. | ||
| - | * First scan is taken after 30-60 minutes of incubation, when the protein-DNA interactions reach thermodynamic stability. At this point the chamber containing DNA is open to allow diffusion and the valves separating each of the 756 units are closed, which prevents contamination. | + | * First scan is taken after 30-60 minutes of incubation, when the protein-DNA interactions reach thermodynamic stability. At this point the chamber containing DNA is open to allow diffusion and the valves separating each of the 756 units are closed, which prevents contamination. |
| - | + | ||
[[File:EPFL2011_MITOMIchip_beforewash_illustration.png|300px| After incubation: GFP fluorescence ]] | [[File:EPFL2011_MITOMIchip_beforewash_illustration.png|300px| After incubation: GFP fluorescence ]] | ||
| - | [[File:EPFL2011_MITOMIchip_beforewashDNA_illustration.png| | + | [[File:EPFL2011_MITOMIchip_beforewashDNA_illustration.png|300px| After incubation: CY5 fluorescence ]] |
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| + | * Second scan is taken after 10-15 minutes of wash with PBS. | ||
| + | [[File:EPFL2011_MITOMIchip_afterwashProt_illustration.png.png|300px| After PBS wash: GFP fluorescence ]] | ||
| + | [[File:EPFL2011_MITOMIchip_afterwashDNA_illustration.png|300px| After PBS wash: GFP fluorescence ]] | ||
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{{:Team:EPF-Lausanne/Templates/Footer}} | {{:Team:EPF-Lausanne/Templates/Footer}} | ||
Revision as of 07:57, 21 September 2011
MITOMI
Mitomi is a high troughput microfluidic system designed to mesure low affinity molecular interactions by mechanically trapping them.
Purpose: We applied this technology during our project for characterizing transcription factor binding energy landscapes in absolute terms by determining dissociation constants (Kd) for a comprehensive set of target DNA sequences.
Schematic of the Mitomi platform:
The control inputs control the valves that close the Detailed view of the chambers and of the movable membrane used for mechanical trapping, referred to as the 'Button' .
Advantages: The movable memrane shown in the scheme below enables measurements of transient interactions, exhibiting sub micromolar affinities which are generally problematic.
Running a MITOMI experiment
MITOMI scans explanation
Two scans of the chip are necessary for the analysis of the molecular interactions.
- First scan is taken after 30-60 minutes of incubation, when the protein-DNA interactions reach thermodynamic stability. At this point the chamber containing DNA is open to allow diffusion and the valves separating each of the 756 units are closed, which prevents contamination.
- Second scan is taken after 10-15 minutes of wash with PBS.
