Difference between revisions of "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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<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;">
<h1>Preparing Custom Grids for TEM</h1>
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<h1>Operating TEM</h1>
  
<h2>Coat grids with a thin collodion plastic film</h2>
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<h2>Set-Up</h2>
<h3>Materials</h3>
 
 
<ul>
 
<ul>
<li>Collodion</li>
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<li>Make sure nitrogen tank is at least 2/3 full</li>
<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>
<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>
<li>tweezers, forceps</li>
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<li>load grid sample side up, can tap to get in place</li>
<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>
<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>
 +
<li>Mouse ball to move stage </li>
 +
<li>Perform z axis wobbler to get image to stay in place  </li>
 +
<li> Set locations on stage  </li>
 +
<li> Set integrate time to about >4-5 seconds so you don't burn out the camera </li>
 +
<li> flip camera switch and cover viewing area. MAKE SURE THE CAMERA IS OUT AT ALL OTHER TIMES!  </li>
 +
<li> Click for live image. use focus knob. set intensity to cover enter histogram to middle. FINAL IMAGE BABY </li>
 +
<li> Save it. </li>
 
</ul>
 
</ul>
  
<h3>Procedure</h3>
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<ul>
 
<li>Completely fill basin with ddH2O right to the rim.</li>
 
<li>Use razor blade to shorten a 200er pipet tip to create a 3mm diameter aperture.</li>
 
<li>Pipet out 50 μL collodion solution.</li>
 
<li>Eject the collodion from approx 10 cm onto the calm water surface. CAUTION: collodion is toxic! </li>
 
<li>Ensure proper ventilation and hold breath while handling the collodion!</li>
 
<li>A collodion film will spread across the water surface and harden. Wait for 5 min.</li>
 
<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>
 
<li>Position vellum paper on top of girds. Let soak with water.</li>
 
<li>Put collodion film around the circumference of the vellum paper by using the razor blade.</li>
 
<li>Carefully pick up the vellum paper at one corner/edge and pull out of the water.</li>
 
<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>
 
<li>Ready!</li>
 
</ul>
 
  
 
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<img src="http://openwetware.org/images/9/9e/Protocol_01.gif" width="129" height="125" alt=""></a></td> <td rowspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/gel_prep"> <img src="http://openwetware.org/images/4/48/Protocol_02.gif" width="99" height="123" border="0" alt=""></a></td> <td align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/negative_staining"> <img src="http://openwetware.org/images/e/ed/Protocol_03.gif" width="116" height="122" border="0" alt=""></a></td> <td rowspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/custom_tem_grids"> <img src="http://openwetware.org/images/a/ae/Protocol_04.gif" width="93" height="123" border="0" alt=""></a></td> <td colspan="2" rowspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/drugloading"> <img src="http://openwetware.org/images/e/e9/Protocol_05.gif" width="132" height="123" border="0" alt=""></a></td> <td colspan="2" rowspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/TIRF"> <img src="http://openwetware.org/images/f/fd/Protocol_06.gif" width="137" height="123" border="0" alt=""></a></td> <td rowspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/MTT"> <img src="http://openwetware.org/images/2/20/Protocol_07.gif" width="94" height="123" border="0" alt=""></a></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="1" height="122" alt=""></td> </tr> <tr> <td rowspan="3"> <img src="http://openwetware.org/images/0/0b/Protocol_08.gif" width="116" height="3" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="1" height="1" alt=""></td> </tr> <tr> <td rowspan="2"> <img src="http://openwetware.org/images/5/59/Protocol_09.gif" width="99" height="2" alt=""></td> <td colspan="2" rowspan="2"> <img src="http://openwetware.org/images/e/e4/Protocol_10.gif" width="211" height="2" alt=""></td> <td colspan="2" align="left" valign="top"> <a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol/cellanalysis"> <img src="http://openwetware.org/images/3/3c/Protocol_11.gif" width="108" height="1" border="0" alt=""></a></td> <td colspan="2" rowspan="2"> <img src="http://openwetware.org/images/8/83/Protocol_12.gif" width="137" height="2" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="1" height="1" alt=""></td> </tr> <tr> <td colspan="2"> <img src="http://openwetware.org/images/7/77/Protocol_13.gif" width="108" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="1" height="1" alt=""></td> </tr> <tr> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="129" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="99" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="116" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="93" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="118" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="14" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="94" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="43" height="1" alt=""></td> <td> <img src="http://openwetware.org/images/5/52/Spacer.gif" width="94" height="1" alt=""></td> <td></td> </tr> </table> </center>

<div id="protocol-content" style="width:65%;margin-left:auto;margin-right:auto;"> <h1>Operating TEM</h1>

<h2>Set-Up</h2> <ul> <li>Make sure nitrogen tank is at least 2/3 full</li> <li>Make sure TEM is on - REd light and 2 white lights on</li> <li>Set high filament to 80 kV</li> <li>Make sure column valve is closed whenever putting sample in/out0</li> <li>vellum paper (Pergament-Papier)</li> <li>load grid sample side up, can tap to get in place</li> <li>then load with registry pin down. Turn about 5 degrees counter clock-wise. Vacuum should be automatic 50 seconds</li> <li>After around 50 seconds, red light goes off, turn the pin the rest of the 180 degrees and slowly guide into column</li> <li>To remove sample, pull out straight and then turn clockwise 180 degrees </li> <li>Once sample is in, open column valves and turn on filament </li> <li>Use intensity knob to spread beam </li> <li>Mouse ball to move stage </li> <li>Perform z axis wobbler to get image to stay in place </li> <li> Set locations on stage </li> <li> Set integrate time to about >4-5 seconds so you don't burn out the camera </li> <li> flip camera switch and cover viewing area. MAKE SURE THE CAMERA IS OUT AT ALL OTHER TIMES! </li> <li> Click for live image. use focus knob. set intensity to cover enter histogram to middle. FINAL IMAGE BABY </li> <li> Save it. </li> </ul>


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