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

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<h2 style="font-weight:bold">Valencia UPV Team</h2>
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<h2><b>Notebook</b></h2>
 
<p>Be patient, we are under construction</p>
 
<p>Be patient, we are under construction</p>
 
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<li><a href="#main" id="button01" class="button">Abstract</a></li>
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<li><a href="#scroll1" class="button">1</a></li>
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<li><a href="#scroll2" class="button">2</a></li>
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<li><a href="#scroll3" class="button">3</a></li>
 
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<h3 style="color:green">5 June 2015</h3>
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<header class="major">
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<h2>Section 1<br />
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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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<p><i>Agrobacterium</i> culture of promoter less: Luciferase + Renilla </p>
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<p>Hello world</p>
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<p>Minipreps</p>
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<p>Digestion with BamHI and EcoRV</p>
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<p>Agarose gel 1%</p>
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<p>FOTO</p>
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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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<header id="scrollsect1" class="major">
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<h3 style="text-align:left">Sub section 1<br />
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</h3>
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</header>
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<p>Hello world</p>
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</section>
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<h2>Section 2<br />
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</h2><hr>
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<p>Hello world</p>
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Revision as of 14:09, 13 September 2015

Valencia UPV iGEM 2015

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Section 2

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