Team:USMA-West Point/Experiments

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USMA-West_Point

<!DOCTYPE html> Experiments - iGem 01

usMA-WEST POINT


Experiments



PRotocols used for each step of the process



Methods



Nucleofection of bacterial cells



  1. OR vectors were purchased from OriGene and transfected into 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 Laemmle 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


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