Team:EPF-Lausanne/Protocols

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== PDMS two layer device fabrication ==
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{{:Team:EPF-Lausanne/Templates/Header|title=Protocols}}
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made on 16.05.2011
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<br>
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'''''Materials:
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----
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<p>    </p>
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<p>• flow and control layer molds</p>
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<p>• Sylgard 184 silicone elastomer kit: Base part and a Curing agent</p>
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<p>• TMCS</p>
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<br>
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== Molecular Biology ==
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<p>'''''Method:</p>
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=== Products and stock preparation ===
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* [[Team:EPF-Lausanne/Protocols/Primers preparation|Primer preparation]]: initial dilution of ordered primers.
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* [[Team:EPF-Lausanne/Protocols/Competent cells|Competent Cells]]: prepare cells for plasmid uptake.
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* [[Team:EPF-Lausanne/Protocols/Competent cells. Protocol II|Competent Cells. Protocol II (Inoue method)]].
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* [[Team:EPF-Lausanne/Protocols/Glycerol stock|Glycerol stock]]: stock transformed cells at -80°C.
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* [[Team:EPF-Lausanne/Protocols/Agar Plates|Agar plate preparation]]: preparing agar plates for cell cultures.
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* [[Team:EPF-Lausanne/Protocols/Autoclave|Autoclave]]: to sterilize solutions and glassware.
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<p>  </p>
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=== Cloning, assembly, and mutations ===
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<p>• Place molds into a TMCS vapor chamber</p>
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* [[Team:EPF-Lausanne/Protocols/Transformation|Transformation]]: introduce foreign plasmid into competent cells.
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<p>• Control layer mixture: 20g Base + 4g Curing agent</p>
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* [[Team:EPF-Lausanne/Protocols/Plating|Plating]]: plate transformed cells
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<p>• Mix for 1 minute, degas for 2 minutes (standard protocol)</p>
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* [[Team:EPF-Lausanne/Protocols/Liquid cultures|Liquid cultures]]: when cells have grown on plates, put them in liquid cultures
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<p>• pour onto control layer mold and place mold in vacuum chamber</p>
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* [[Team:EPF-Lausanne/Protocols/Gibson assembly|Gibson assembly]].
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<p>• Flow layer mixture: 20g Base + 1g Curing agent</p>
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* [[Team:EPF-Lausanne/Protocols/TetR|Linear template- TetR]].
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<p>• Mix for 1 minute and degas for 2 minutes (standard protocol)</p>
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* [[Team:EPF-Lausanne/Protocols/TetR Extension PCR|tetR Overlap Extension PCR]]: induce specific mutations on tetR linear template.
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<p>• Spin coat onto flow layer at 2200-2400rpm for 35secs (so far you can use my protocol on a spin coater)</p>
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* [[Team:EPF-Lausanne/Protocols/Site-specific mutagenesis|tetR Site-specific mutagenesis]]: induce site-specific mutations on a plasmid containing tetR.
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<p>• Remove control layer mold from vacuum chamber, making sure no bubbles are left on the surface (remove with a toothpick if you see some )</p>
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* [[Team:EPF-Lausanne/Protocols/Gel_Electrophoresis|Agarose gel electrophoresis]]: DNA samples analysis (can be followed by band purification).
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<p>• Place the control and flow layer in a 80C convection oven and incubate for 30 minutes (timing is critical here!)</p>
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* [[Team:EPF-Lausanne/Protocols/Colony PCR|Colony PCR]]: Analyze the plated transformed cells, to see if Gibson assembly worked
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<p>• Remove casts from oven, cut out control layer, punch holes, and align to flow layer</p>
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* [[Team:EPF-Lausanne/Protocols/T7-ext|T7 extension PCR]]: Put T7 promoter upstream of a gene
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<p>• Put aligned device back into 80C oven and incubate for at least 90 minutes (and here you can increase the backing)</p>
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* [[Team:EPF-Lausanne/Protocols/bluntends|Blunt-End Cloning]]: Making Blunt Ends
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<p>• Remove devices from oven and punch flow layer holes</p>
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=== DNA recovery ===
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* [[Team:EPF-Lausanne/Protocols/Miniprep|Plasmid preparation - Miniprep]]: recover plasmids from a bacterial culture.
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* [[Team:EPF-Lausanne/Protocols/Gel purification|Gel purification]]: recover DNA separated by electrophoresis
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== Biochemistry ==
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* [[Team:EPF-Lausanne/Protocols/IPTG test|IPTG test]]: test expression of a gene downstream from Plac
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== Microfluidics ==
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* [[Team:EPF-Lausanne/Protocols/PDMS_two_layer_device_fabrication|PDMS two layer device fabrication]]
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* [[Team:EPF-Lausanne/Protocols/Master microfabrication for PDMS replica molding |Master microfabrication for PDMS replica molding]]
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* [[Team:EPF-Lausanne/Protocols/MITOMI_protein_DNA|MITOMI: Protein – DNA interactions]]
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* [[Team:EPF-Lausanne/Protocols/Klenow dsDNA synthesis|Klenow dsDNA synthesis]]
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== Worm culture ==
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* [[Team:EPF-Lausanne/Protocols/MG_Agar_Plates|Agar plate preparation]]: preparing MG Agar plates for worm cultures.
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{{:Team:EPF-Lausanne/Templates/Footer}}

Latest revision as of 15:35, 16 October 2011

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