Biomod/2013/OSU/protocol/gel prep

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<img src="http://openwetware.org/images/1/15/Ohiomod2013.jpg" height="60%" width="60%" style="margin-left:-40px;"> <div id="navbar" style="margin-left:auto; margin-right:auto;text-align:center;width:650px;height:auto;" > <ul id="homebutton" class="navigation-bar"> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU">Home</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/about">About</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/abstract">Abstract</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/goals">Goals</a></li>

<li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/about">Description</a> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/experiments">Project</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/experiments">Life of Project</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/protocol">Protocols</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/labnotes">Lab Notes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/meetingnotes">Meeting Notes</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/resources">Resources</a></li> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/results">Results</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/results">Experimental Results</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/data">Data</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/futurework">Future Work</a></li> </li> </ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/media">Media</a> <ul> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/projectvideo">Project Video</a><li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/animations">Animations</a><li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/videoblogs">Video Blog</a></li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/photos">Photos</a></li> </ul> </li> <li><a href="http://openwetware.org/wiki/Biomod/2013/OSU/team">Team</a></li> </ul>

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<div id="protocol-content" style="width:65%;margin-left:auto;margin-right:auto;"> <h1>Gel Preparation</h1> <h2>Prepare a 2% agarose gel (125 ml slab)</h2> <ul> <li>Weight 2.5 agarose into beaker</li> <li>Fill up to 125 g with 0.5x TBE-Puffer</li> <li>Boil in microwave until agarose is dissolved (2 min)</li> <li>Some water may evaporate through heat. Add ddH2O until back up to 125 grams (apply water to sides of hot beaker)</li> <li>Cool till hand warm</li> <li>Add 1 ml of 1.375 MgCl2 solution</li> <li>Add 7 μL ethidium bromid from 10 mg/ml stock solution. (use gloves for this protocol. minimize exposure)</li> <li>Fill gel tray and install desired comb.</li> <li>Once solid, fill gel box with TBE/11 mM MgCL2 buffer.</li> <li>Remove comb.</li> </ul>

<h2>Gel Loading</h2> <ul> <li>Sample: mix 12 μL of each DNA sample with 3 μL 6x loading dye. Load into lanes</li> <li>Unfolded Plasmid: mix 1.2 μL of phage DNA (100 nM) with 10.8 μL ddH2O and 3 μL 6x-loading dye. </li> <li>Load into lanes</li> <li>DNA ladder: add 6 μL of 1kb DNA ladder (New England Biolabs)</li> </ul>

<h2>Run Gel</h2> <ul> <li>Put gel box into ice water bath</li> <li>Apply constant 70 V across the gel.</li> <li>Run for 3-4 hours.</li> </ul>

<h2>Imaging</h2> <ul> <li>Can image using UV illuminator in dark room. Get picture>exposure>take picture>save under C: share files.</li> </ul>

<h2>Crunch-n-Squeeze Purification</h2> <ul> <li>Use UV transilluminator for band visulaization, and cut out the desired band with razor bands.</li> <li>Put slice of gel into tube.</li> <li>Spin down the debris.</li> <li>Cut off debris containing itp of the tub.</li> <li>Invert tip into a freeze'n'squeeze spin column (Biorad).</li> <li>Spin 12 min at 12,000G in tabletop centrifuge.</li> <li>Throw away the top portion of the squeeze spin column.</li> <li>Label and store purified DNA at 4°C</li> </ul>

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<td id="email">Contact us at <a href="mailto:theohiomod@gmail.com"><font color="white">theohiomod@gmail.com</a></td>

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Biomod/2013/OSU/protocol/gel prep

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