Team:UC Davis/notebook/assembly.html
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<li><a href="https://2010.igem.org/Team:UC_Davis/notebook/luxpathway.html" class="help">Lux Activated "Off" Pathway</a></li> | <li><a href="https://2010.igem.org/Team:UC_Davis/notebook/luxpathway.html" class="help">Lux Activated "Off" Pathway</a></li> | ||
</ul> | </ul> | ||
+ | |||
+ | <p class="header"><b>Sample Assembly Workplan</b></a><p> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2010/2/29/Initiatorconstruct.jpg"><p> | ||
+ | |||
+ | Because the initiator is one of the smaller constructs, we have decided to use this as an example to illustrate the utmost basic workplan in greater detail. <p> | ||
+ | |||
+ | <a name="startup"><h1>Starting Up</h1></a> | ||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/hydration.html" class="help">Hydrated</a> parts: BBa_B0034, BBa_C0051, BBa_B0015, BBa_R0082.</li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">Transformed</a> these parts into DH5α competent cells.</li> | ||
+ | </ul><p><br /> | ||
+ | |||
+ | <a name="rbsc0051"><h1>RBS to BBa_C0051</h1></a> | ||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/culture.html" class="help">Cultured</a> cells that have parts: BBa_B0034 and BBa_C0051</li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/miniprep.html" class="help">Miniprepped</a> BBa_B0034 and BBa_C0051</li> | ||
+ | </ul> | ||
+ | |||
+ | <table border="0" margin="1" width="650px" padding="5px"> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/f/f6/RBSplasmidclosedxx.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with SpeI & Pst</li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/2/29/RBSplasmidx.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help">Run on gel</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/2/29/RBSplasmidx.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/d/d3/C0051plasmidclosed.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with Xba & Pst</li></ul> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/3/3d/C0051plasmidcut.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li>Separate fragments through <a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help"> gel electrophoresis</a></li><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/c/c5/C0051insert1.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table><p><br /> | ||
+ | |||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/ligation.html" class="help">Ligate</a> the RBS and BBa_C0051 fragments together and <a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">transform</a> on carb plates.</li> | ||
+ | <li>Depending on the outcome of visible colonies on the experimental and control plates, a <a href="https://2010.igem.org/Team:UC_Davis/protocols/pcrscreen.html" class="help">PCR screening</a> may be needed. | ||
+ | </ul><br /> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2010/b/bb/RBSc0051.jpg"><br /> | ||
+ | |||
+ | Assembly notes | ||
+ | <ul> | ||
+ | <li>It is most practical to cut RBS as a vector because RBS is less than 100 bp long as an insert, making it hard to see and extract on the gel.</li> | ||
+ | <li>The sticky ends from the XbaI and SpeI, when ligated together, will form an 8 bp scar. Thus, XbaI and SpeI will no longer be able to cut here.</li> | ||
+ | </ul><br /> | ||
+ | |||
+ | <a name="stop"><h1>'RBS & BBa_C0051' to Terminator</h1></a> | ||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/culture.html" class="help">Cultured</a> cells that have parts: 'RBS & BBa_C0051', BBa_B0015</li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/miniprep.html" class="help">Miniprepped</a> 'RBS & BBa_C0051' and BBa_B0015</li> | ||
+ | </ul> | ||
+ | |||
+ | <table border="0" margin="1" width="650px" padding="5px"> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/9/9a/Stopplasmid.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with EcoRI & XbaI</li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/7/70/Xstopopenvector.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help">Run on gel</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/7/70/Xstopopenvector.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/b/bb/RBSc0051.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with EcoRI & SpeI</li></ul> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/9/94/RBSc0051cut.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li>Separate fragments through <a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help"> gel electrophoresis</a></li><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/d/de/RBSc0051insertx.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table><p><br /> | ||
+ | |||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/ligation.html" class="help">Ligate</a> the fragments together and <a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">transform</a> on kan plates.</li> | ||
+ | <li>Depending on the outcome of visible colonies on the experimental and control plates, a <a href="https://2010.igem.org/Team:UC_Davis/protocols/pcrscreen.html" class="help">PCR screening</a> may be needed. | ||
+ | </ul><br /> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2010/e/eb/RBSc0051stop.jpg"><br /><p> | ||
+ | |||
+ | Assembly notes <br /> | ||
+ | <ul> | ||
+ | <li>It is most practical to cut BBa_B0015 as the vector because it is only 130 bp long. It would be hard to see on a gel. </li> | ||
+ | </ul><br /> | ||
+ | |||
+ | <a name="r0082"><h1>'RBS & BBa_C0051 & stop' to BBa_R0082</h1></a> | ||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/culture.html" class="help">Cultured</a> cells that have parts: 'RBS & BBa_C0051 & stop', BBa_R0082</li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/miniprep.html" class="help">Miniprepped</a> 'RBS & BBa_C0051& stop' and BBa_R0082</li> | ||
+ | </ul> | ||
+ | |||
+ | <table border="0" margin="1" width="650px" padding="5px"> | ||
+ | <tr> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/c/cc/Promoterclosed.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with SpeI & PstI</li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/2/21/Promoterplasmidopenx.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help">Run on gel</a></li> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/2/21/Promoterplasmidopenx.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | <td> | ||
+ | <table border="0" margin="1" width="320px" padding="5px"> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/e/eb/RBSc0051stop.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/digestion.html" class="help">Digest</a> with XbaI & PstI</li></ul> </td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/4/46/RBSc0051stopcut.jpg"></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><ul><li>Separate fragments through <a href="https://2010.igem.org/Team:UC_Davis/protocols/gelelectrophoresis.html" class="help"> gel electrophoresis</a></li><li><a href="https://2010.igem.org/Team:UC_Davis/protocols/gelextraction.html" class="help">Gel extract</a></li></ul></td> | ||
+ | </tr> | ||
+ | <tr> | ||
+ | <td><img src="https://static.igem.org/mediawiki/2010/6/61/RBSc0051stopinsertx.jpg"></td> | ||
+ | </tr> | ||
+ | </table> | ||
+ | </td> | ||
+ | </tr> | ||
+ | </table><p><br /> | ||
+ | |||
+ | <ul> | ||
+ | <li><a href="https://2010.igem.org/Team:UC_Davis/protocols/ligation.html" class="help">Ligate</a> the fragments together and <a href="https://2010.igem.org/Team:UC_Davis/protocols/transformation.html" class="help">transform</a> on carb plates.</li> | ||
+ | <li>Depending on the outcome of visible colonies on the experimental and control plates, a <a href="https://2010.igem.org/Team:UC_Davis/protocols/pcrscreen.html" class="help">PCR screening</a> may be needed. | ||
+ | </ul><br /> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2010/3/34/Initiatorplasmid.jpg"><br /> | ||
+ | |||
+ | Assembly notes | ||
+ | <ul> | ||
+ | <li>The promoter had to be cut as a vector because it is only 108 bp by itself.</li> | ||
+ | </ul><br /> | ||
<p> | <p> |
Revision as of 04:37, 27 October 2010
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