Methods
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OR vectors were purchased from OriGene and transfected into XL-10 Gold Ultracompetent cells by heat shocking at 42 °C
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Cells were cultured in LB broth to amplify vectors
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DNA gels were run with specific restriction enzymes to determine multiple cloning sites
Nucleofection of bacteria cells
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Relevant Documents
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Qiagen Maxiprep Protocol
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XL10-Gold (R) Ultracompetent Cells
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Lipofectamine (R) 2000 Reagent
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DNA Cloning
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PCR-Based Cloning
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GelPilot (R) DNA Molecular Weight Markers
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Traditional Cloning Quick Guide, New England BioLabs (R)
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GelPilot (R) DNA Loading Dye, 5x
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QIAquick (R) Gel Extraction Kit
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Western Blotting
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HT-22 cells were lysed in RIPA buffer with phosphate and protease inhibitors
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Protein concentration was measured for each OR
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Proteins were prepared with Laemmle sample buffer before loading into 4% Bis-Tris gels to measure protein purity and size
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Relevant Documents
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Western Blotting Protocol
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Confocal Microscopy
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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
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Nucleofected HT-22 cells were stimulated with individual odorants
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The neurons generated action potentials that were measured via a microelectrode array
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Relevant Documents
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Staining with Voltage Sensitive Dyes
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