Biomod/2013/OSU/protocol/custom tem grids

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(New page: {{OhioMOD2013}} <html> <div id="protocol-content" style="width:65%;margin-left:auto;margin-right:auto;"> <h1>Preparing Custom Grids for TEM</h1> <h2>Coat grids with a thin collodion plas...)
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/gel_prep">
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/negative_staining">
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/custom_tem_grids">
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<a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/drugloading">
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<div id="protocol-content" style="width:65%;margin-left:auto;margin-right:auto;">
<div id="protocol-content" style="width:65%;margin-left:auto;margin-right:auto;">
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<h1>Preparing Custom Grids for TEM</h1>
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<h1>Operating TEM</h1>
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<h2>Coat grids with a thin collodion plastic film</h2>
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<h2>Set-Up</h2>
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<h3>Materials</h3>
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<ul>
<ul>
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<li>Collodion</li>
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<li>Make sure nitrogen tank is at least 2/3 full</li>
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<li>minimum 20 cm diameter bowl/basin</li>
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<li>Make sure TEM is on - REd light and 2 white lights on</li>
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<li>TEM copper grids 200 or 400</li>
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<li>Set high filament to 80 kV</li>
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<li>Make sure column valve is closed whenever putting sample in/out0</li>
<li>vellum paper (Pergament-Papier)</li>
<li>vellum paper (Pergament-Papier)</li>
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<li>tweezers, forceps</li>
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<li>load grid sample side up, can tap to get in place</li>
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<li>razor blade</li>
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<li>then load with registry pin down. Turn about 5 degrees counter clock-wise. Vacuum should be automatic 50 seconds</li>
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<li>200 pipet</li>
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<li>After around 50 seconds, red light goes off, turn the pin the rest of the 180 degrees and slowly guide into column</li>
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<li>To remove sample, pull out straight and then turn clockwise 180 degrees </li>
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<li>Once sample is in, open column valves and turn on filament </li>
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<li>Use intensity knob to spread beam </li>
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<li>Mouse ball to move stage </li>
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<li>Perform z axis wobbler to get image to stay in place  </li>
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<li> Set locations on stage  </li>
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<li> Set integrate time to about >4-5 seconds so you don't burn out the camera </li>
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<li> flip camera switch and cover viewing area. MAKE SURE THE CAMERA IS OUT AT ALL OTHER TIMES!  </li>
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<li> Click for live image. use focus knob. set intensity to cover enter histogram to middle. FINAL IMAGE BABY </li>
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<li> Save it. </li>
</ul>
</ul>
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<h3>Procedure</h3>
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<ul>
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<li>Completely fill basin with ddH2O right to the rim.</li>
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<li>Use razor blade to shorten a 200er pipet tip to create a 3mm diameter aperture.</li>
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<li>Pipet out 50 μL collodion solution.</li>
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<li>Eject the collodion from approx 10 cm onto the calm water surface. CAUTION: collodion is toxic! </li>
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<li>Ensure proper ventilation and hold breath while handling the collodion!</li>
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<li>A collodion film will spread across the water surface and harden. Wait for 5 min.</li>
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<li>Use forcepts to position copper grids onto the collodion film. Put shiny (specular-reflecting) side of the grids facing the collodion. Arrange in a neat hexagonal or square pattern.</li>
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<li>Position vellum paper on top of girds. Let soak with water.</li>
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<li>Put collodion film around the circumference of the vellum paper by using the razor blade.</li>
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<li>Carefully pick up the vellum paper at one corner/edge and pull out of the water.</li>
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<li>Turn the vellum paper with the grids facing up, put into petri dish, partially cover and let dry overnight in a clean dust free area.</li>
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<li>Ready!</li>
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</ul>
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