Difference between revisions of "Team:USMA-West Point/Experiments"

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<h1>Experiments</h1>
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<p>Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.</p>
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Experiments - iGem 01</title>
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Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.  
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<h5>Inspiration</h5>
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General Lab Etiquette
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Plating Cells
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<h3 id="element-f871fa233a4afac" class="preview-element preview-subtitle magic-circle-holder text-element quick-text-style-menu  allow-mobile-hide" data-menu-name="PREVIEW_SUBTITLE">Transformation of bacterial cells<br></h3>
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<ol><li class="font_7" style="text-align: justify;">OR vectors were purchased from OriGene and used to transform XL-10 Gold Ultracompetent cells by heat shocking at 42 °C<br></li><li class="font_7" style="text-align: justify;">Cells were cultured in LB broth to amplify vectors<br></li><li class="font_7" style="text-align: justify;">DNA gels were run with specific restrion enzymes to determine multiple cloaning sites<br></li><li class="font_7" style="text-align: justify;">Relevant documents<br><ol><li class="font_7" style="text-align: justify;"><a data-cke-saved-href="https://www.mcdb.ucla.edu/Research/Banerjee/protocols/maxiprep--Qiagen.pdf" href="https://www.mcdb.ucla.edu/Research/Banerjee/protocols/maxiprep--Qiagen.pdf">Qiagen MaxiPrep </a><br></li><li class="font_7" style="text-align: justify;"><a data-cke-saved-href="http://www.chem-agilent.com/pdf/strata/200314.pdf" href="http://www.chem-agilent.com/pdf/strata/200314.pdf">XL10-Gold Ultracompetent cells</a><br></li><li class="font_7" style="text-align: justify;"><a data-cke-saved-href="https://tools.thermofisher.com/content/sfs/manuals/Lipofectamine_2000_Reag_protocol.pdf" href="https://tools.thermofisher.com/content/sfs/manuals/Lipofectamine_2000_Reag_protocol.pdf">Lipofectamine 2000 Reagent</a><br></li><li class="font_7" style="text-align: justify;"><a data-cke-saved-href="https://www.neb.com/tools-and-resources/usage-guidelines/cloning-guide" href="https://www.neb.com/tools-and-resources/usage-guidelines/cloning-guide">DNA cloning</a><br></li><li class="font_7" style="text-align: justify;"><a data-cke-saved-href="https://www.neb.com/tools-and-resources/usage-guidelines/cloning-guide" href="https://www.neb.com/tools-and-resources/usage-guidelines/cloning-guide">PCR-based cloning</a><br></li><li class="font_7" style="text-align: justify;">GelPilot DNA Molecular Weight Markers<br></li><li class="font_7" style="text-align: justify;">QIAquick Gel Extraction Kit<br></li></ol></li></ol>
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<ol><li class="font_7" style="text-align: justify;">HT-22 cells were lysed in RIPA buffer with phosphate and protease inhibitors<br></li><li class="font_7" style="text-align: justify;">Protein concentration was measured for each OR<br></li><li class="font_7" style="text-align: justify;">Proteins were prepared with Laemmli sample buffer before loading into 4% Bis-Tris gels to measure protein purity and size<br></li><li class="font_7" style="text-align: justify;">Relevant documents<ol><li class="font_7" style="text-align: justify;"><a data-cke-saved-href="https://www.mcdb.ucla.edu/Research/Banerjee/protocols/maxiprep--Qiagen.pdf" href="https://www.mcdb.ucla.edu/Research/Banerjee/protocols/maxiprep--Qiagen.pdf"></a><a data-cke-saved-href="https://2017.igem.org/File:Western_Blotting_Protocol.docx" href="https://2017.igem.org/File:Western_Blotting_Protocol.docx">Western Blotting Protocol<a data-cke-saved-href="https://www.mcdb.ucla.edu/Research/Banerjee/protocols/maxiprep--Qiagen.pdf" href="https://www.mcdb.ucla.edu/Research/Banerjee/protocols/maxiprep--Qiagen.pdf"> </a><br></li></ol></li></ol>
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<ol><li class="font_7" style="text-align: justify;">HT-22 cells were fixed with 4% paraformaldehyde and immunohistochemistry with antibodies responsible for individual OR binding  followed by secondary antibodies for visualization<br></li></ol>
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<ol><li class="font_7" style="text-align: justify;">Nucleofected HT-22 cells were stimulated with individual odorants<br></li><li class="font_7" style="text-align: justify;">The neurons generated action potentials that were measured via a microelectrode array<br></li><li class="font_7" style="text-align: justify;">Relevant documents<ol><li class="font_7" style="text-align: justify;"><a data-cke-saved-href="https://2017.igem.org/File:Calcium_Dye_Fluorescence.docx" href="https://2017.igem.org/File:Calcium_Dye_Fluorescence.docx">Staining with Voltage Sensitive Dyes<a data-cke-saved-href="https://www.mcdb.ucla.edu/Research/Banerjee/protocols/maxiprep--Qiagen.pdf" href="https://www.mcdb.ucla.edu/Research/Banerjee/protocols/maxiprep--Qiagen.pdf"> </a><br></li></ol></li></ol>
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Latest revision as of 23:15, 1 November 2017

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USMA-West_Point


Experiments - iGem 01

usMA-WEST POINT


Experiments



PRotocols used for each step of the process



Methods



Transformation of bacterial cells



  1. OR vectors were purchased from OriGene and used to transform XL-10 Gold Ultracompetent cells by heat shocking at 42 °C
  2. Cells were cultured in LB broth to amplify vectors
  3. DNA gels were run with specific restrion enzymes to determine multiple cloaning sites
  4. Relevant documents
    1. Qiagen MaxiPrep
    2. XL10-Gold Ultracompetent cells
    3. Lipofectamine 2000 Reagent
    4. DNA cloning
    5. PCR-based cloning
    6. GelPilot DNA Molecular Weight Markers
    7. QIAquick Gel Extraction Kit


Western Blotting



  1. HT-22 cells were lysed in RIPA buffer with phosphate and protease inhibitors
  2. Protein concentration was measured for each OR
  3. Proteins were prepared with Laemmli sample buffer before loading into 4% Bis-Tris gels to measure protein purity and size
  4. Relevant documents
    1. Western Blotting Protocol


Confocal Microscopy



  1. HT-22 cells were fixed with 4% paraformaldehyde and immunohistochemistry with antibodies responsible for individual OR binding followed by secondary antibodies for visualization


Action Potential Measurements



  1. Nucleofected HT-22 cells were stimulated with individual odorants
  2. The neurons generated action potentials that were measured via a microelectrode array
  3. Relevant documents
    1. Staining with Voltage Sensitive Dyes


DEPARTMENT OF CHEMISTRY AND LIFE SCIENCE



BARTLETT HALL, ROOM 400, WEST POINT, NY 10996, USA


TEL: 845.938.3915