http://2010.igem.org/wiki/index.php?title=Team:MIT_gateway&feed=atom&action=historyTeam:MIT gateway - Revision history2024-03-29T05:49:57ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:MIT_gateway&diff=188466&oldid=prevSupacalafrglstic at 16:54, 27 October 20102010-10-27T16:54:42Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:MIT_mge">Genetic Engineering</a></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:MIT_mge">Genetic Engineering</a></li></div></td></tr>
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</table>Supacalafrglstichttp://2010.igem.org/wiki/index.php?title=Team:MIT_gateway&diff=138980&oldid=prevSupacalafrglstic at 23:46, 24 October 20102010-10-24T23:46:01Z<p></p>
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</table>Supacalafrglstichttp://2010.igem.org/wiki/index.php?title=Team:MIT_gateway&diff=134345&oldid=prevSupacalafrglstic at 16:41, 24 October 20102010-10-24T16:41:55Z<p></p>
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</table>Supacalafrglstichttp://2010.igem.org/wiki/index.php?title=Team:MIT_gateway&diff=134325&oldid=prevSupacalafrglstic at 16:40, 24 October 20102010-10-24T16:40:49Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><tr><td><div class="bodybaby"><del class="diffchange diffchange-inline">bacterial biobrick construction</del></div></td></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><tr><td><div class="bodybaby"><ins class="diffchange diffchange-inline">Gateway Cloning</ins></div></td></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><tr><td><br>The Mammalian team used Gateway cloning to assemble its composite parts.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><tr><td><br>The Mammalian team used Gateway cloning to assemble its composite parts.<br><br></div></td></tr>
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</table>Supacalafrglstichttp://2010.igem.org/wiki/index.php?title=Team:MIT_gateway&diff=134317&oldid=prevSupacalafrglstic at 16:40, 24 October 20102010-10-24T16:40:25Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><div style="width:<del class="diffchange diffchange-inline">28%; height: 100%</del>; margin: 10px; position: relative; <del class="diffchange diffchange-inline">right</del>: <del class="diffchange diffchange-inline">10px</del>; display:block; float:right; background-color: <del class="diffchange diffchange-inline">#cee8f0</del>;"<del class="diffchange diffchange-inline">><br</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><<del class="diffchange diffchange-inline">ul </del>id="<del class="diffchange diffchange-inline">sidenav</del>"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">li</del>><<del class="diffchange diffchange-inline">a href="https://2010.igem.org/Team:MIT_bact"</del>>Bacterial Protocol</<del class="diffchange diffchange-inline">a</del>><<del class="diffchange diffchange-inline">br</del>><<del class="diffchange diffchange-inline">/li</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><div style="width:<ins class="diffchange diffchange-inline">250px</ins>; margin: 10px; position: relative; <ins class="diffchange diffchange-inline">top</ins>: <ins class="diffchange diffchange-inline">-4px; left:-11px</ins>; display: block; float:right<ins class="diffchange diffchange-inline">; padding: 7px</ins>; background-color: <ins class="diffchange diffchange-inline">white</ins>;"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><ul><li><a href="https://2010.igem.org/Team:MIT_bconst">Biobrick Construction</a<del class="diffchange diffchange-inline">><br</del>></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">dl </ins>id="<ins class="diffchange diffchange-inline">nav</ins>"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><li><a href="https://2010.igem.org/Team:MIT_bexp">Bacterial Experiments</a></li></ul></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><<ins class="diffchange diffchange-inline">dt</ins>><<ins class="diffchange diffchange-inline">b</ins>>Bacterial Protocol</<ins class="diffchange diffchange-inline">b</ins>><<ins class="diffchange diffchange-inline">/dt</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><<del class="diffchange diffchange-inline">li</del>><<del class="diffchange diffchange-inline">a href="https://2010.igem.org/Team:MIT_mmethods"</del>>Mammalian Protocol</<del class="diffchange diffchange-inline">a</del>></<del class="diffchange diffchange-inline">li</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><<ins class="diffchange diffchange-inline">dd</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><ul><li><a href="https://2010.igem.org/Team:MIT_mmethods">Microfluidics</a></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><ul></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><li><a href="https://2010.igem.org/Team:MIT_mge">Genetic Engineering</a></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><li><a href="https://2010.igem.org/Team:MIT_bconst">Biobrick Construction</a></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del><li <del class="diffchange diffchange-inline">class="sel"</del>><a href="https://2010.igem.org/Team:MIT_gateway">Gateway Cloning</a></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><li><a href="https://2010.igem.org/Team:MIT_bexp">Bacterial Experiments</a></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> </del></ul></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins></ul></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div></<del class="diffchange diffchange-inline">ul</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><<ins class="diffchange diffchange-inline">/dd</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><<del class="diffchange diffchange-inline">br</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><<ins class="diffchange diffchange-inline">dt><b</ins>>Mammalian Protocol</<ins class="diffchange diffchange-inline">b</ins>></<ins class="diffchange diffchange-inline">dt</ins>></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> <dd></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><ul></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><li><a href="https://2010.igem.org/Team:MIT_mmethods">Microfluidics</a></li></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><li><a href="https://2010.igem.org/Team:MIT_mge">Genetic Engineering</a></li></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins><li><a href="https://2010.igem.org/Team:MIT_gateway">Gateway Cloning</a></li></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins></ul></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins></<ins class="diffchange diffchange-inline">dd</ins>></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> </ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><<ins class="diffchange diffchange-inline">/dl</ins>></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><div id="unique" style="padding:<del class="diffchange diffchange-inline">5px</del>; font-size: 14px; border: 1px solid black; margin:<del class="diffchange diffchange-inline">5px</del>;"></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><table width=<del class="diffchange diffchange-inline">70%</del>><tr><td><div class="bodybaby">bacterial biobrick construction</div></td></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><div id="unique" style="padding:<ins class="diffchange diffchange-inline">0px</ins>; font-size: 14px; border: 1px solid black; margin:<ins class="diffchange diffchange-inline">0px; background-color:transparent</ins>;"></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><table width=<ins class="diffchange diffchange-inline">650px style="background-color: white; margin-top:5px; padding: 10px;"</ins>></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><tr><td><div class="bodybaby">bacterial biobrick construction</div></td></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><tr><td><br>The Mammalian team used Gateway cloning to assemble its composite parts.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><tr><td><br>The Mammalian team used Gateway cloning to assemble its composite parts.<br><br></div></td></tr>
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</table>Supacalafrglstichttp://2010.igem.org/wiki/index.php?title=Team:MIT_gateway&diff=109692&oldid=prevSupacalafrglstic at 21:57, 18 October 20102010-10-18T21:57:49Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><div style="width:<del class="diffchange diffchange-inline">24</del>%; <del class="diffchange diffchange-inline">position</del>: <del class="diffchange diffchange-inline">relative</del>; <del class="diffchange diffchange-inline">top</del>: <del class="diffchange diffchange-inline">45px</del>; right: <del class="diffchange diffchange-inline">45px</del>; display:block; float:right; background-color: #cee8f0;"><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><div style="width:<ins class="diffchange diffchange-inline">28</ins>%; <ins class="diffchange diffchange-inline">height</ins>: <ins class="diffchange diffchange-inline">100%</ins>; <ins class="diffchange diffchange-inline">margin</ins>: <ins class="diffchange diffchange-inline">10px; position: relative</ins>; right: <ins class="diffchange diffchange-inline">10px</ins>; display:block; float:right; background-color: #cee8f0;"><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><ul id="sidenav"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><ul id="sidenav"></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:MIT_bact">Bacterial Protocol</a><br></li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li><a href="https://2010.igem.org/Team:MIT_bact">Bacterial Protocol</a><br></li></div></td></tr>
</table>Supacalafrglstichttp://2010.igem.org/wiki/index.php?title=Team:MIT_gateway&diff=109674&oldid=prevSupacalafrglstic at 21:52, 18 October 20102010-10-18T21:52:27Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The resulting ENTRTM-vectors are kanamycin resistant. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The resulting ENTRTM-vectors are kanamycin resistant. <br></div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br><br><div class="<del class="diffchange diffchange-inline">known</del>" id="<del class="diffchange diffchange-inline">general</del>">Known Issues</div><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br><br><div class="<ins class="diffchange diffchange-inline">bodybaby</ins>" id="<ins class="diffchange diffchange-inline">known</ins>">Known Issues</div><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><ul><li> The reaction is very efficient. You can obtain about 200 colonies of which about 95 % are correct.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><ul><li> The reaction is very efficient. You can obtain about 200 colonies of which about 95 % are correct.</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li> BP reactions work better with linear templates like PCR-products. If you want to use plasmids, linearize them first with a suitable restriction enzyme.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li> BP reactions work better with linear templates like PCR-products. If you want to use plasmids, linearize them first with a suitable restriction enzyme.</li></div></td></tr>
</table>Supacalafrglstichttp://2010.igem.org/wiki/index.php?title=Team:MIT_gateway&diff=109672&oldid=prevSupacalafrglstic at 21:51, 18 October 20102010-10-18T21:51:59Z<p></p>
<table style="background-color: white; color:black;">
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="outline"></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="outline"></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <a href="#<del class="diffchange diffchange-inline">general</del>">1 Gateway Cloning</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <a href="#<ins class="diffchange diffchange-inline">gateway</ins>">1 Gateway Cloning</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <a href="#<del class="diffchange diffchange-inline">miniprep</del>">2 Commented Protocol</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <a href="#<ins class="diffchange diffchange-inline">commented</ins>">2 Commented Protocol</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">virtual</del>">2.1 Design PCR-Primers</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">design</ins>">2.1 Design PCR-Primers</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">digest</del>">2.2 Gel Purify PCR Product</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">gel</ins>">2.2 Gel Purify PCR Product</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">antarctic</del>">2.3 Confirm PCR Product</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">confirm</ins>">2.3 Confirm PCR Product</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">gel</del>">2.4 Measure DNA</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">measure</ins>">2.4 Measure DNA</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">extract</del>">2.5 Calculate PCR Product Needed</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">calculate</ins>">2.5 Calculate PCR Product Needed</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">transform</del>">2.6 Calculate volume of DONRTM needed</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">volume</ins>">2.6 Calculate volume of DONRTM needed</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">gel</del>">2.7 Prepare Gateway Reaction</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">prepare</ins>">2.7 Prepare Gateway Reaction</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">gel</del>">2.8 Retrieve 5x BP Clonase II</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">retrieve</ins>">2.8 Retrieve 5x BP Clonase II</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">gel</del>">2.9 Add BP-Clonase to Gateway Reaction</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">add</ins>">2.9 Add BP-Clonase to Gateway Reaction</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">gel</del>">2.10 Incubate</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">incubate</ins>">2.10 Incubate</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">gel</del>">2.11 Add ProteinaseK</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">proteinasek</ins>">2.11 Add ProteinaseK</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">gel</del>">2.12 Transform Bacteria</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">transform</ins>">2.12 Transform Bacteria</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<del class="diffchange diffchange-inline">gel</del>">2.13 Plate Bacteria</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> &nbsp;&nbsp;&nbsp;&nbsp;<a href="#<ins class="diffchange diffchange-inline">plate</ins>">2.13 Plate Bacteria</a><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <a href="#<del class="diffchange diffchange-inline">miniprep</del>">3 Known Issues</a><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <a href="#<ins class="diffchange diffchange-inline">known</ins>">3 Known Issues</a><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <a href="#cite">4 Citation</a><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <a href="#cite">4 Citation</a><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></td><tr><td><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></div></td><tr><td><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><div class="bodybaby" id="<del class="diffchange diffchange-inline">general</del>">Gateway Cloning</div><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><div class="bodybaby" id="<ins class="diffchange diffchange-inline">gateway</ins>">Gateway Cloning</div><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><div class="bodybaby" id="<del class="diffchange diffchange-inline">general</del>">Commented Protocol</div><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><div class="bodybaby" id="<ins class="diffchange diffchange-inline">commented</ins>">Commented Protocol</div><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b class="bolded" id="miniprep">Design PCR-Primers</b><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b class="bolded" id="miniprep">Design PCR-Primers</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The Gateway® clones have a reading frame which should be kept. Design primers that the PCR product starts with a ATG and ends with a STOP-codon or the last aminoacid (if you want to make a fusion protein). Primer3plus is a powerful tool helping you to pick primers with the right annealing temperature which should be 60°C. Try to avoid self similarity and other things as usual, but because you are very limited in the position of the primers (its start and stop), I only care about annealing temperature and give it a try. Then just add to the primer which binds the start codon the attB1.1-sequence at his 5' End . To the primer which binds the stop codon or the last aminoacid add the attB2.1-sequence at his 5' End . The open reading frame is indicated and you should change the last two NN to code for an aminoacid of your choice. Good luck for the PCR! Because of the long 5' overhang and the restrictions on picking the primers, getting the PCR to work can be tricky.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The Gateway® clones have a reading frame which should be kept. Design primers that the PCR product starts with a ATG and ends with a STOP-codon or the last aminoacid (if you want to make a fusion protein). Primer3plus is a powerful tool helping you to pick primers with the right annealing temperature which should be 60°C. Try to avoid self similarity and other things as usual, but because you are very limited in the position of the primers (its start and stop), I only care about annealing temperature and give it a try. Then just add to the primer which binds the start codon the attB1.1-sequence at his 5' End . To the primer which binds the stop codon or the last aminoacid add the attB2.1-sequence at his 5' End . The open reading frame is indicated and you should change the last two NN to code for an aminoacid of your choice. Good luck for the PCR! Because of the long 5' overhang and the restrictions on picking the primers, getting the PCR to work can be tricky.<br><br></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>attB3 GGG-GAC-AAC-TTT-GTA-TAA-Taa-agt-tgN <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>attB3 GGG-GAC-AAC-TTT-GTA-TAA-Taa-agt-tgN <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Gel Purify PCR Product</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">gel</ins>">Gel Purify PCR Product</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Purification of the PCR-product is needed to get rid of smaller side-products, which remove primer-dimers which can result in false positive colonies. Remember that you want to clone DNA, so the cutting should be made on the weakest UV-light available and as fast as possible. And of course you NEVER make a picture of the gel before. Use the kid for gel-purification available in your lab. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Purification of the PCR-product is needed to get rid of smaller side-products, which remove primer-dimers which can result in false positive colonies. Remember that you want to clone DNA, so the cutting should be made on the weakest UV-light available and as fast as possible. And of course you NEVER make a picture of the gel before. Use the kid for gel-purification available in your lab. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Confirm PCR Product</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">confirm</ins>">Confirm PCR Product</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>You need a PCR product with the attB1.1 and attB2.2 and the DONRTM vector MUST have attP1 and attP2 sites, or it will not work.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>You need a PCR product with the attB1.1 and attB2.2 and the DONRTM vector MUST have attP1 and attP2 sites, or it will not work.<br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The amount of plasmids is not soo important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the How to measure DNA. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTRTM clones. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The amount of plasmids is not soo important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the How to measure DNA. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTRTM clones. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Measure DNA</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">measure</ins>">Measure DNA</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The amount of ENTRTM is not so important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the multiple Gateway® protocol. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTRTM clones. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The amount of ENTRTM is not so important as in a multiple Gateway® reaction, because it is more efficient. If you want to optimize you can calculate equimolar amounts of both plasmids as described in the multiple Gateway® protocol. Here we use double the amount of DEST-vector, because most of the ones we use are round and about double the size of the ENTRTM clones. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Calculate PCR Product Needed</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">calculate</ins>">Calculate PCR Product Needed</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>ng needed = (length of the PCR product in bp) x 0.0165 <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>ng needed = (length of the PCR product in bp) x 0.0165 <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Calculate volume of DONTR(TM) needed</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">volume</ins>">Calculate volume of DONTR(TM) needed</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>µl needed = 75 ng needed / (concentration in ng/µl)<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>µl needed = 75 ng needed / (concentration in ng/µl)<br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The DONR-vector should be tested for low background colonies (due to a mutated ccdB-gene) when transferred in DH5alpha-bacteria. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The DONR-vector should be tested for low background colonies (due to a mutated ccdB-gene) when transferred in DH5alpha-bacteria. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Prepare Gateway Reaction</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">prepare</ins>">Prepare Gateway Reaction</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>PCR-product&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;( ? ng)<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>PCR-product&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;( ? ng)<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>pDONRTM-vector&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(75 ng)<br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>pDONRTM-vector&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;(75 ng)<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>add water to a total volume of<b> 4µl </b><br><br> </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>add water to a total volume of<b> 4µl </b><br><br> </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Retrieve 5x BP-Clonase II</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">retrieve</ins>">Retrieve 5x BP-Clonase II</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>...From -20&deg;C. It is most efficiently mixed by pipetting up and down, do not vortex. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>...From -20&deg;C. It is most efficiently mixed by pipetting up and down, do not vortex. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Add BP-Clonase to Gateway Reaction</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">add</ins>">Add BP-Clonase to Gateway Reaction</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The enzymes looses 50% activity after 15 freeze-thaw cycles. The advantage of BP-ClonaseTMII is that it can be stored at -20 °C because it contains already the buffer. DO NOT LEAVE OUT.<br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The enzymes looses 50% activity after 15 freeze-thaw cycles. The advantage of BP-ClonaseTMII is that it can be stored at -20 °C because it contains already the buffer. DO NOT LEAVE OUT.<br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Incubate</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">incubate</ins>">Incubate</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Incubation over-night will enhance the reaction ca. 5-10 fold. This is especially important for PCR products over 5.000 bp. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Incubation over-night will enhance the reaction ca. 5-10 fold. This is especially important for PCR products over 5.000 bp. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Add ProteinaseK</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">proteinasek</ins>">Add ProteinaseK</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This step will enhance the reaction ca. 100 fold!!!!. This is different to the LR-reactions which are only enhanced 2 fold by adding the proteinase K!!!! </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This step will enhance the reaction ca. 100 fold!!!!. This is different to the LR-reactions which are only enhanced 2 fold by adding the proteinase K!!!! </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Transform Bacteria</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">transform</ins>">Transform Bacteria</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For electro competent cells use 1-2 µl, for chemical competent all. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>For electro competent cells use 1-2 µl, for chemical competent all. <br><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><b class="bolded" id="<del class="diffchange diffchange-inline">miniprep</del>">Plate Bacteria</b><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><b class="bolded" id="<ins class="diffchange diffchange-inline">plate</ins>">Plate Bacteria</b><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The resulting ENTRTM-vectors are kanamycin resistant. <br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The resulting ENTRTM-vectors are kanamycin resistant. <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br><br><div class="<del class="diffchange diffchange-inline">bodybaby</del>" id="general">Known Issues</div><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br><br><div class="<ins class="diffchange diffchange-inline">known</ins>" id="general">Known Issues</div><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><ul><li> The reaction is very efficient. You can obtain about 200 colonies of which about 95 % are correct.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><ul><li> The reaction is very efficient. You can obtain about 200 colonies of which about 95 % are correct.</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li> BP reactions work better with linear templates like PCR-products. If you want to use plasmids, linearize them first with a suitable restriction enzyme.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li> BP reactions work better with linear templates like PCR-products. If you want to use plasmids, linearize them first with a suitable restriction enzyme.</li></div></td></tr>
</table>Supacalafrglstichttp://2010.igem.org/wiki/index.php?title=Team:MIT_gateway&diff=109670&oldid=prevSupacalafrglstic at 21:50, 18 October 20102010-10-18T21:50:10Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 21:50, 18 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 120:</td>
<td colspan="2" class="diff-lineno">Line 120:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><div class="bodybaby" id="general">Known Issues</div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><div class="bodybaby" id="general">Known Issues</div><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> * </del> The reaction is very efficient. You can obtain about 200 colonies of which about 95 % are correct.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><ul><li> </ins> The reaction is very efficient. You can obtain about 200 colonies of which about 95 % are correct.<ins class="diffchange diffchange-inline"></li></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> * </del>BP reactions work better with linear templates like PCR-products. If you want to use plasmids, linearize them first with a suitable restriction enzyme.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><li> </ins>BP reactions work better with linear templates like PCR-products. If you want to use plasmids, linearize them first with a suitable restriction enzyme.<ins class="diffchange diffchange-inline"></li></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"> * </del>The obtained plasmids are big. To check for correct clones digest with Sty I and in parallel with Eco RI and Hind III. Compare the pattern of bands with the predicted band size to find the correct clones.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><li> </ins>The obtained plasmids are big. To check for correct clones digest with Sty I and in parallel with Eco RI and Hind III. Compare the pattern of bands with the predicted band size to find the correct clones.<ins class="diffchange diffchange-inline"></li></ul><br></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="bodybaby" id="cite">Citation</div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="bodybaby" id="cite">Citation</div><br></div></td></tr>
</table>Supacalafrglstichttp://2010.igem.org/wiki/index.php?title=Team:MIT_gateway&diff=109667&oldid=prevSupacalafrglstic at 21:49, 18 October 20102010-10-18T21:49:26Z<p></p>
<table style="background-color: white; color:black;">
<col class='diff-marker' />
<col class='diff-content' />
<col class='diff-marker' />
<col class='diff-content' />
<tr valign='top'>
<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 21:49, 18 October 2010</td>
</tr><tr><td colspan="2" class="diff-lineno">Line 117:</td>
<td colspan="2" class="diff-lineno">Line 117:</td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b class="bolded" id="miniprep">Plate Bacteria</b><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><b class="bolded" id="miniprep">Plate Bacteria</b><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The resulting ENTRTM-vectors are kanamycin resistant. <del class="diffchange diffchange-inline"><br></del><br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The resulting ENTRTM-vectors are kanamycin resistant. <br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><div class="bodybaby" id="general">Known Issues</div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br><br><div class="bodybaby" id="general">Known Issues</div><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> * The reaction is very efficient. You can obtain about 200 colonies of which about 95 % are correct.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> * BP reactions work better with linear templates like PCR-products. If you want to use plasmids, linearize them first with a suitable restriction enzyme.</ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"> * The obtained plasmids are big. To check for correct clones digest with Sty I and in parallel with Eco RI and Hind III. Compare the pattern of bands with the predicted band size to find the correct clones.</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="bodybaby" id="cite">Citation</div><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><div class="bodybaby" id="cite">Citation</div><br></div></td></tr>
</table>Supacalafrglstic