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| − | </style>
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| − |
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| − | <div align="center"><div id="cn-box" align="justify"><br/>
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| − |
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| − | <div align="center"><img class="img-title" alt="Notebook" src="https://static.igem.org/mediawiki/2014/2/2a/VUPVNotebook.png"></img></div><br/>
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| − |
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| − | <div class="box_above_notebook">
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| − |
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| − | Contents:
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| − | <ul style="margin-left: 1.5em;"> <li> <a href="#Constructions">Constructions</a></li></ul>
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| − | </div><a name="Constructions"></a></br></br><h3 class="section_notebook">Constructions</h3><p class="p_notebook">CONSTRUCTIONS</p>
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| − |
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| − | </br><h4 class="date_notebook">5 June 2015</h4>
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| − |
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| − |
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| − |
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| − | <p class="p_notebook">We had 2 cultures from the last day, corresponding to other 2 colonies of ligation. </p>
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| − |
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| − | <p class="p_notebook"><i>Agrobacterium</i> culture of promoter less: Luciferase + Renilla </p>
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| − |
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| − |
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| − |
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| − | <p class="p_notebook">Minipreps</p>
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| − |
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| − | <p class="p_notebook">Digestion with BamHI and EcoRV</p>
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| − |
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| − | <p class="p_notebook">Agarose gel 1%</p>
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| − |
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| − | <table class="table_notebook">
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| − |
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| − | <tr class="tr_notebook"><td class="td_notebook">Mw</td><td class="td_notebook">Ples</td><td class="td_notebook">4E</td><td class="td_notebook">4B</td><td class="td_notebook">3E</td><td class="td_notebook">3B 1kb</td></tr>
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| − |
| |
| − | </table>
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| − |
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| − |
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| − |
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| − | <p class="p_notebook">How to ask and make primers?</p>
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| − |
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| − | <ul class="ul_notebook"><li>Select the sequence to amplify and save in FASTA format.</li>
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| − |
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| − | <li>gbCloning, go to Tools-Domesticator-1º Category</li>
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| − |
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| − | <li>Add FASTA and select parts.</li>
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| − |
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| − | <li>On the protocol we have the primers </li>
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| − |
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| − | <li>The oligos they give us:</li>
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| − |
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| − | <ul class="ul_ul_notebook"><li>4 first nucleotides: so the enzyme can recognize without problems</li>
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| − |
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| − | <li>6 following bingind sites.</li>
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| − |
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| − | <li>1 extra nucleotide.</li>
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| − |
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| − | <li>4 overhangs. </li>
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| − |
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| − | </ul></ul>
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| − |
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| − | <p class="p_notebook">Meeting with Daniel Ramón (Biopolis). </p>
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| − |
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| − | <p class="p_notebook">Ligation with part 2 and 24 of task sheet.</p>
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| − |
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| − | <table class="table_notebook">
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| − |
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| − | <tr class="tr_notebook"><td class="td_notebook">Pif6 + PhyB; ?1</td><td class="td_notebook">Etr8 CMV_Bxb1_T35S</td></tr>
| |
| − |
| |
| − | <tr class="tr_notebook"><td class="td_notebook">1µL 892 (Pif α1)</td><td class="td_notebook">1µL 1097 (Etr8 CMV) Pupd2</td></tr>
| |
| − |
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| − | <tr class="tr_notebook"><td class="td_notebook">1µL 88E (Phy α2)</td><td class="td_notebook">1µL Bxb1 (PuPD)</td></tr>
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| − |
| |
| − | <tr class="tr_notebook"><td class="td_notebook">1µL ?1 </td><td class="td_notebook">1µL Tnos PuPD</td></tr>
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| − |
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| − | <tr class="tr_notebook"><td class="td_notebook">1.2µL Buffer ligase</td><td class="td_notebook">1µL α1</td></tr>
| |
| − |
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| − | <tr class="tr_notebook"><td class="td_notebook">1µL Bsmb1</td><td class="td_notebook">5.8µL H2O</td></tr>
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| − |
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| − | <tr class="tr_notebook"><td class="td_notebook">6.8µL H2O</td><td class="td_notebook"></td></tr>
| |
| − |
| |
| − | </table>
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| − |
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| − |
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| − |
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| − | <p class="p_notebook">If we make a digestion of 160 (35S:Renilla:tNOS-35S:P19:tNOS) with EcoRV, we obtain: 2475, 381, 4601 pb.</p>
| |
| − |
| |
| − | <p class="p_notebook">If we make a digestion of 896 (Luc:Pif6:PhyB)with EcoRV, we obtain: 11608, 3942 pb.</p>
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| − |
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| − |
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| − |
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| − | </br><h4 class="date_notebook">June 2015</h4>
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| − |
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| − |
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| − |
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| − | <p class="p_notebook">Transform to E.coli from Pif+Phy and Bxb1</p>
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| − |
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| − | <ul class="ul_notebook"><li>1.5µL of ligation</li>
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| − |
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| − | <ul class="ul_ul_notebook"><li>Cuvette on ice</li>
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| − |
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| − | <li>Competent cells + 1.5µL of ligation</li>
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| − |
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| − | <li>Pulse (electroporator) at 1500V</li>
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| − |
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| − | <li>Add 300µL shock medium and put Eppendorf 1h at 37ºC</li>
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| − |
| |
| − | </ul></ul>
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| − |
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| − | <p class="p_notebook">Culture on petri dishes the ligations.</p>
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| − |
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| − | <p class="p_notebook">Digest of 160, 289 and the two ligations, pif+phy and Etr8+BxbI. </p>
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| − |
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| − | <p class="p_notebook">Agarose gel.</p>
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| − |
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| − | <table class="table_notebook">
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| − |
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| − | <tr class="tr_notebook"><td class="td_notebook">6µL ladder</td><td class="td_notebook">160</td><td class="td_notebook">289</td><td class="td_notebook">ligation</td><td class="td_notebook">ligation</td><td class="td_notebook">Ladder</td></tr>
| |
| − |
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| − | <tr class="tr_notebook"><td class="td_notebook">1Kb</td></tr>
| |
| − |
| |
| − | </table>
| |
| − |
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| − | <p class="p_notebook">Storage of gel on: Basura en Arabidopsis – Igem – 2015 – 150606_Digestion_ToggleRojo</p>
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| − |
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| − |
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| − |
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| − | </br><h4 class="date_notebook">7 June 2015</h4>
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| − |
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| − |
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| − |
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| − | <p class="p_notebook">We’ve got white colonies! (from Pif+Phy and Bxb1)</p>
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| − |
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| − | <p class="p_notebook">Pick two colonies from each construction.</p>
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| − |
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| − | <p class="p_notebook">4 tubes</p>
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| − |
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| − | <ul class="ul_notebook"><li>3.5µL LB each tube</li>
| |
| − |
| |
| − | </ul>
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| − |
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| − | <p class="p_notebook">2) 2 tubes + 3.5µL Kanamycin (K)</p>
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| − |
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| − |
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| − |
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| − | </br><h4 class="date_notebook">8 June 2015</h4>
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| − |
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| − |
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| − |
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| − | <p class="p_notebook">Minipreps of the 4 liquid cultures and digestion to see the band patterns.</p>
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| − |
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| − | <p class="p_notebook">Digestion:</p>
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| − |
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| − | <table class="table_notebook">
| |
| − |
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| − | <tr class="tr_notebook"><td class="td_notebook">Etr8(CMV):Bxb1:Tnos; Ω1</td><td class="td_notebook">EcoRI</td><td class="td_notebook">6345, 238</td></tr>
| |
| − |
| |
| − | <tr class="tr_notebook"><td class="td_notebook">EPIF6 + PhyB-PV16; Ω1</td><td class="td_notebook">BamHI</td><td class="td_notebook">6686, 1439, 2685, 2237</td></tr>
| |
| − |
| |
| − | </table>
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| − |
| |
| − | </div>
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| − | <div id="space-margin"></div>
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| | </div> | | </div> |
