Biomod/2013/OSU/protocol

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(New page: {{OhioMOD2013}} <html> <div id="protocol" style="width:65%;margin-left:auto;margin-right:auto;"> <h2>Protocol</h2> <ul> <li>E. Coli bacteria is given “scaffold” virus which allows the...)
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<h2>Protocol</h2>
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<li>E. Coli bacteria is given “scaffold” virus which allows the bacteria to multiply scaffold virus while growing itself</li>
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<li><a href="#">Folding Reaction</a></li>
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<li>CADnano is used to design shape and used to determine what staples must be ordered to obtain desired shape</li>
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<li><a href="#">Gel Preparation</a></li>
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<li>The staples are organized and collected into “pre-stocks”</li>
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<li><a href="#">Negative Staining for TEM</a></li>
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<li>“Pre-stocks” are combined into working stocks</li>
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<li><a href="#">Preparing Custom Grids for TEM</a></li>
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<li>The staples are mixed with the scaffolds in high ion solution and placed in thermal ramp</li>
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<li><a href="#">PEG Purification</a></li>
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<li>After thermal ramp, the now folded structures are ran through an agarose gel with EtBr for purification</li>
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<li><a href="#">Atomic Force Microscopy</a></li>
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<li>The purified structures are cut from the gel</li>
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<li>The structures are then mixed with 2 mM daunorubicin and incubated for 40 hours</li>
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<li>The solution is then centrifuged and the precipitate is re-suspended in a PBS solution with 10 mM MgCl¬2</li>
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<li>The absorbance of the supernatant is then measured on a plate reader to determine the concentration of daunorubicin.</li>
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<li>The suspension with the structures is then diluted with cell medium to have various concentrations</li>
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<li>This is then added to the resistant cells</li>
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<li>After 12 hours, the structures are then washed with PBS to remove Daunorubicin and the structures</li>
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<li>Next the cells are incubated for 48 hours in after which they are added to an imaging plate and observed using the TIRF microscope</li>
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<li>Brightfield microscope images are taken in addition to 640 nm wavelength images to visualize dead cells (7-AAD dye)</li>
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<li>The number of live/dead cells can be determined from these images</li>
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<li>Additionally cell viability is investigated by placing structures with cells in a 96 well plate</li>
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<li>The same procedure is followed, except after the 48 hour incubation, a MTT assay is run</li>
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<li>This involves adding MTT solution (5 mg/mL) and then reading absorbance on a plate reader to quantify cell viability</li>
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Revision as of 22:04, 6 October 2013


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