Team:EPF-Lausanne/Tools/MITOMI

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(Running a MITOMI experiment)
 
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==Running a MITOMI experiment==
==Running a MITOMI experiment==
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<html>The protocol for the experiment can be found <a href="https://2011.igem.org/Team:EPF-Lausanne/Protocols/MITOMI_protein_DNA">  here. For more information on the chip fabrication please check our <a href="https://2011.igem.org/Team:EPF-Lausanne/Tools/Microfluidics/HowTo1"> Microfluidics page. </a></html>
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<html>The protocol for the experiment can be found <a href="https://2011.igem.org/Team:EPF-Lausanne/Protocols/MITOMI_protein_DNA">  here. </a></html> <html> For more information on the mold or chip fabrication please check our <a href="https://2011.igem.org/Team:EPF-Lausanne/Tools/Microfluidics/HowTo1"> Microfluidics </a> </html> pages.
[[File:EPFL2011_MITOMI_run_illustration.png|700px]]
[[File:EPFL2011_MITOMI_run_illustration.png|700px]]
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==MITOMI scans explanation==
==MITOMI scans explanation==
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Two scans of the chip are necessary for the analysis of the molecular interactions.  
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We've been using arrayWoRx scanner to acquire the images.
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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|300px| After incubation: CY5 fluorescence ]]
[[File:EPFL2011_MITOMIchip_beforewashDNA_illustration.png|300px| After incubation: CY5 fluorescence ]]
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Green fluorescence comes from the GFP-tag fused to the protein or from the incorporated Green Lysine.
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Red fluorescence is due to the CY5 labeling of the DNA.
    
    
* Second scan is taken after 10-15 minutes of  wash with PBS.
* 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_afterwashProt_illustration.png.png|300px| After PBS wash: GFP fluorescence ]]
[[File:EPFL2011_MITOMIchip_afterwashDNA_illustration.png|300px| After PBS wash: GFP fluorescence ]]
[[File:EPFL2011_MITOMIchip_afterwashDNA_illustration.png|300px| After PBS wash: GFP fluorescence ]]
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For data collection the fluorescence intensity measured under the button is normalised to the background and the DNA/protein ratio is calculated. This ratio is ploted against the intensity of the fluorescence of DNA in solution to make the saturation curves. And the Kd for each sequence is calculated from these curves.
 
[[File:EPFL2011_MITOMI_data_collection_points.JPG|400px]]
[[File:EPFL2011_MITOMI_data_collection_points.JPG|400px]]
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For data collection the fluorescence intensity measured under the button is normalised to the background and the DNA/protein ratio is calculated. This ratio is ploted against the intensity of the fluorescence of DNA in solution to make the saturation curves. And the Kd for each sequence is calculated from these curves by fitting the next equation:
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[[File:EPFL2011_MITOMI_Kd_fiting_formula.png|180px]]
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[[File:EPFL2011_MITOMI_Example_Saturation_curve_fited.png|400px]]
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{{:Team:EPF-Lausanne/Templates/Footer}}
{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 19:51, 28 October 2011