Difference between revisions of "Team:Valencia UPV/Notebook"

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<h2>Elements</h2>
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<p>Just an assorted selection of elements.</p>
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<h2>Notebook
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<br />
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<div class="row">
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<div class="12u">
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<h3 style="color:green">5 June 2015</h3>
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<p>We had 2 cultures from the last day, corresponding to other 2 colonies of ligation. </p>
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<h3>Text</h3>
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<p>This is <b>bold</b> and this is <strong>strong</strong>. This is <i>italic</i> and this is <em>emphasized</em>.
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This is <sup>superscript</sup> text and this is <sub>subscript</sub> text.
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This is <u>underlined</u> and this is code: <code>for (;;) { ... }</code>. Finally, <a href="#">this is a link</a>.</p>
  
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<p><i>Agrobacterium</i> culture of promoter less: Luciferase + Renilla </p>
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<h3>Heading with a Subtitle</h3>
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<p>Lorem ipsum dolor sit amet nullam id egestas urna aliquam</p>
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<p>Nunc lacinia ante nunc ac lobortis. Interdum adipiscing gravida odio porttitor sem non mi integer non faucibus ornare mi ut ante amet placerat aliquet. Volutpat eu sed ante lacinia sapien lorem accumsan varius montes viverra nibh in adipiscing blandit tempus accumsan.</p>
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<h4>Heading with a Subtitle</h4>
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<p>Nunc lacinia ante nunc ac lobortis. Interdum adipiscing gravida odio porttitor sem non mi integer non faucibus ornare mi ut ante amet placerat aliquet. Volutpat eu sed ante lacinia sapien lorem accumsan varius montes viverra nibh in adipiscing blandit tempus accumsan.</p>
  
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<p>Minipreps</p>
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<h2>Heading Level 2</h2>
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<h3>Heading Level 3</h3>
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<h4>Heading Level 4</h4>
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<h5>Heading Level 5</h5>
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<h6>Heading Level 6</h6>
  
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<p>Digestion with BamHI and EcoRV</p>
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<hr />
  
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<p>Agarose gel 1%</p>
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</section>
  
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<p>FOTO</p>
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</div>
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</div>
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<p>How to ask and make primers?</p>
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<ul><li>Select the sequence to amplify and save in FASTA format.</li>
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<li>gbCloning, go to Tools-Domesticator-1º Category</li>
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<li>Add FASTA and select parts.</li>
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<li>On the protocol we have the primers </li>
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<li>The oligos they give us:</li>
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<ul class="ul_2"><li>4 first nucleotides: so the enzyme can recognize without problems</li>
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<li>6 following bingind sites.</li>
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<li>1 extra nucleotide.</li>
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<li>4 overhangs. </li>
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</ul></ul>
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<p>Meeting with Daniel Ramón (Biopolis). </p>
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<p>Ligation with part 2 and 24 of task sheet.</p>
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<div class="table-wrapper"><table class="alt">
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<tr><td>PIF6 + PhyB; &Omega;1</td><td>Etr8 CMV+Bxb1_T35S; &alpha;1</td></tr>
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<tr><td>1µL (GB892) PIF; &alpha;1</td><td>1µL 1097 (Etr8 CMV) pUPD2</td></tr>
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<tr><td>1µL (GB88E) PhyB; &alpha;2</td><td>1µL Bxb1; pUPD2</td></tr>
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<tr><td>1µL &Omega;1 </td><td>1µL Tnos PuPD</td></tr>
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<tr><td>1.2µL Buffer ligase</td><td>1µL &alpha;1</td></tr>
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<tr><td>1µL Bsmb1</td><td>5.8µL H<sub>2</sub>O</td></tr>
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<tr><td>6.8µL H<sub>2</sub>O</td><td></td></tr>
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</div></table>
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<p>Digestions:</p>
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<div class="table-wrapper"><table class="alt">
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<tr><td>(GB160) 35S:Renilla:tNOS-35S:P19:tNOS</td><td>EcoRV</td><td>2475, 381, 4601</td></tr>
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<tr><td>(GB896) Luc:PIF6:PhyB</td><td>EcoRV</td><td>11608, 3942</td></tr>
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</div></table>
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</br><h3 style="color:green">6 June 2015</h3>
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<p>Transform to <i>E. coli</i> from PIF+Phy and BxbI and make petri dish cultures.</p>
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<p>Digest of 160, 289 and the two ligations, PIF+phy and Etr8+BxbI. </p>
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<p>Agarose gel. </p>
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<div class="table-wrapper"><table class="alt">
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<tr><td>GB160</td><td>289</td><td>PIF+PhyB</td><td>BxbI </td></tr>
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<tr><td>ok</td><td>no</td><td>?</td><td>?</td></tr>
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</div></table>
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<p>FOTO</p>
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</br><h3 style="color:green">7 June 2015</h3>
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<p>We?ve got white colonies from PIF+Phy and Bxb1!</p>
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<p>Pick two colonies from each construction.</p>
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</br><h3 style="color:green">8 June 2015</h3>
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<p>Minipreps of the 4 liquid cultures and digestion to see the band patterns.</p>
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<p>Digestion:</p>
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<div class="table-wrapper"><table class="alt">
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<tr><td>Etr8(CMV):Bxb1:Tnos; &alpha;1</td><td>EcoRI</td><td>6345, 238</td></tr>
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<tr><td>EPIF6 + PhyB-PV16; &Omega;1</td><td>BamHI</td><td>6686, 1439, 2685, 2237</td></tr>
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</div></table>
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<p>Agarose gel was made:</p>
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<div class="table-wrapper"><table class="alt">
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<tr><td>Bxb1 (C1)</td><td>Bxb1 (C2)</td><td>EPIF6 + PhyB-PV16 (C1)</td><td>EPIF6 + PhyB-PV16 (C2)</td><td></td></tr>
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<tr><td>ok</td><td>ok</td><td></td><td></td><td></td></tr>
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</div></table>
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<p>FOTO</p>
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<p>Repeat digestion because we are not sure of the last digestions.</p>
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<p>We don?t have sure the toggle, so we decide to repeat the digestion with other enzyme tomorrow, noticing that the
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colony 2 has better bands pattern.</p>
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<p>Optimized ligation:</p>
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<div class="table-wrapper"><table class="alt">
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<tr><td>PIF-Phy-Luc-Renilla-P19</td></tr>
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<tr><td>1 µL vector</td></tr>
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<tr><td>0.8 µL dilution ½ GB160</td></tr>
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<tr><td>1.7 µL PIF:PhyB</td></tr>
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<tr><td>4.15 µL H<sub>2</sub>O</td></tr>
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<tr><td>Ratio 1:2 vector insert</td></tr>
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</div></table>
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<p>As BxbI was good at the digestion we put 1 µL of LB and 1 µL of Kanamicyne on the tube where it had grown and store at
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37ºC to glycerinate later.</p>
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<p>We design primers to binding domain (BD) and PIF.</p>
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<ul><li>Problem: domesticator is introduced in an old pUPD2. The new one has different bases. </li>
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<li>Change manually the pUPD2 bases in the program (Benchling).</li>
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</ul></ul>
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</br><h3 style="color:green">9 June 2015</h3>
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<p>Digestion of the ligation of yesterday containing: EPIF6-PhyB-VP16 (C1 y C2)</p>
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<div class="table-wrapper"><table class="alt">
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<tr><td>EPIF6-PhyB-VP16</td><td>PvuII (green buffer)</td><td>3663, 9472pb</td></tr>
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</div></table>
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<p>Agarose gel 1%:</p>
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<div class="table-wrapper"><table class="alt">
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<tr><td>EPIF6-PhyB-VP16 (C1)</td><td>EPIF6-PhyB-VP16 (C2)</td></tr>
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<tr><td>no</td><td>no</td></tr>
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</div></table>
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<p>FOTO</p>
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<p>We see three bands: 7000, 4000, 1900pb</p>
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<p>Transform optimized ligation PIF-Phy-Luc-Renilla-P19 and make petri dish cultures.</p>
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Revision as of 13:01, 10 September 2015

Valencia UPV iGEM 2015

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