Difference between revisions of "Team:Valencia UPV/Notebook"
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<div class="row"> | <div class="row"> | ||
<div class="12u"> | <div class="12u"> | ||
| + | <section class="box"> | ||
| − | + | <h3 style="color:green">5 June 2015</h3> | |
| − | + | <p>We had 2 cultures from the last day, corresponding to other 2 colonies of ligation. </p> | |
| − | + | <p><i>Agrobacterium</i> culture of promoter less: Luciferase + Renilla </p> | |
| − | + | <p>Minipreps</p> | |
| − | + | <p>Digestion with BamHI and EcoRV</p> | |
| − | + | <p>Agarose gel 1%</p> | |
| − | + | <p>FOTO</p> | |
| − | + | <p>How to ask and make primers?</p> | |
| − | + | <ul><li>Select the sequence to amplify and save in FASTA format.</li> | |
| − | + | <li>gbCloning, go to Tools-Domesticator-1º Category</li> | |
| − | + | <li>Add FASTA and select parts.</li> | |
| − | + | <li>On the protocol we have the primers </li> | |
| − | + | <li>The oligos they give us:</li> | |
| − | + | <ul class="ul_2"><li>4 first nucleotides: so the enzyme can recognize without problems</li> | |
| − | + | <li>6 following bingind sites.</li> | |
| − | + | <li>1 extra nucleotide.</li> | |
| − | + | <li>4 overhangs. </li> | |
| − | + | </ul></ul> | |
| − | + | <p>Meeting with Daniel Ramón (Biopolis). </p> | |
| − | + | <p>Ligation with part 2 and 24 of task sheet.</p> | |
| − | + | <div class="table-wrapper"><table class="alt"> | |
| − | + | <tr><td>PIF6 + PhyB; Ω1</td><td>Etr8 CMV+Bxb1_T35S; α1</td></tr> | |
| − | + | <tr><td>1µL (GB892) PIF; α1</td><td>1µL 1097 (Etr8 CMV) pUPD2</td></tr> | |
| − | + | <tr><td>1µL (GB88E) PhyB; α2</td><td>1µL Bxb1; pUPD2</td></tr> | |
| − | + | <tr><td>1µL Ω1 </td><td>1µL Tnos PuPD</td></tr> | |
| − | + | <tr><td>1.2µL Buffer ligase</td><td>1µL α1</td></tr> | |
| − | + | <tr><td>1µL Bsmb1</td><td>5.8µL H<sub>2</sub>O</td></tr> | |
| − | + | <tr><td>6.8µL H<sub>2</sub>O</td><td></td></tr> | |
| − | + | </div></table> | |
| − | + | <p>Digestions:</p> | |
| − | + | <div class="table-wrapper"><table class="alt"> | |
| − | + | <tr><td>(GB160) 35S:Renilla:tNOS-35S:P19:tNOS</td><td>EcoRV</td><td>2475, 381, 4601</td></tr> | |
| − | + | <tr><td>(GB896) Luc:PIF6:PhyB</td><td>EcoRV</td><td>11608, 3942</td></tr> | |
| − | + | </div></table> | |
| − | + | </br><h3 style="color:green">6 June 2015</h3> | |
| − | + | <p>Transform to <i>E. coli</i> from PIF+Phy and BxbI and make petri dish cultures.</p> | |
| − | + | <p>Digest of 160, 289 and the two ligations, PIF+phy and Etr8+BxbI. </p> | |
| − | + | <p>Agarose gel. </p> | |
| − | + | <div class="table-wrapper"><table class="alt"> | |
| − | + | <tr><td>GB160</td><td>289</td><td>PIF+PhyB</td><td>BxbI </td></tr> | |
| − | + | <tr><td>ok</td><td>no</td><td>?</td><td>?</td></tr> | |
| − | + | </div></table> | |
| − | + | <p>FOTO</p> | |
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| − | + | </br><h3 style="color:green">7 June 2015</h3> | |
| − | + | <p>We?ve got white colonies from PIF+Phy and Bxb1!</p> | |
| − | + | <p>Pick two colonies from each construction.</p> | |
| Line 146: | Line 147: | ||
| − | + | </br><h3 style="color:green">8 June 2015</h3> | |
| − | + | <p>Minipreps of the 4 liquid cultures and digestion to see the band patterns.</p> | |
| − | + | <p>Digestion:</p> | |
| − | + | <div class="table-wrapper"><table class="alt"> | |
| − | + | <tr><td>Etr8(CMV):Bxb1:Tnos; α1</td><td>EcoRI</td><td>6345, 238</td></tr> | |
| − | + | <tr><td>EPIF6 + PhyB-PV16; Ω1</td><td>BamHI</td><td>6686, 1439, 2685, 2237</td></tr> | |
| − | + | </div></table> | |
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| − | + | <p>Agarose gel was made:</p> | |
| − | + | <div class="table-wrapper"><table class="alt"> | |
| − | + | <tr><td>Bxb1 (C1)</td><td>Bxb1 (C2)</td><td>EPIF6 + PhyB-PV16 (C1)</td><td>EPIF6 + PhyB-PV16 (C2)</td><td></td></tr> | |
| − | + | <tr><td>ok</td><td>ok</td><td></td><td></td><td></td></tr> | |
| − | + | </div></table> | |
| − | + | <p>FOTO</p> | |
| − | + | <p>Repeat digestion because we are not sure of the last digestions.</p> | |
| − | + | <p>We don?t have sure the toggle, so we decide to repeat the digestion with other enzyme tomorrow, noticing that the | |
| − | + | colony 2 has better bands pattern.</p> | |
| − | + | <p>Optimized ligation:</p> | |
| − | + | <div class="table-wrapper"><table class="alt"> | |
| − | + | <tr><td>PIF-Phy-Luc-Renilla-P19</td></tr> | |
| − | + | <tr><td>1 µL vector</td></tr> | |
| − | + | <tr><td>0.8 µL dilution ½ GB160</td></tr> | |
| − | + | <tr><td>1.7 µL PIF:PhyB</td></tr> | |
| − | + | <tr><td>4.15 µL H<sub>2</sub>O</td></tr> | |
| − | + | <tr><td>Ratio 1:2 vector insert</td></tr> | |
| − | + | </div></table> | |
| − | + | <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 | |
| − | + | 37ºC to glycerinate later.</p> | |
| − | + | <p>We design primers to binding domain (BD) and PIF.</p> | |
| − | + | <ul><li>Problem: domesticator is introduced in an old pUPD2. The new one has different bases. </li> | |
| − | + | <li>Change manually the pUPD2 bases in the program (Benchling).</li> | |
| − | + | </ul></ul> | |
| − | + | </br><h3 style="color:green">9 June 2015</h3> | |
| − | + | <p>Digestion of the ligation of yesterday containing: EPIF6-PhyB-VP16 (C1 y C2)</p> | |
| − | + | <div class="table-wrapper"><table class="alt"> | |
| − | + | <tr><td>EPIF6-PhyB-VP16</td><td>PvuII (green buffer)</td><td>3663, 9472pb</td></tr> | |
| − | + | </div></table> | |
| Line 246: | Line 247: | ||
| − | + | <p>Agarose gel 1%:</p> | |
| − | + | <div class="table-wrapper"><table class="alt"> | |
| − | + | <tr><td>EPIF6-PhyB-VP16 (C1)</td><td>EPIF6-PhyB-VP16 (C2)</td></tr> | |
| − | + | <tr><td>no</td><td>no</td></tr> | |
| − | + | </div></table> | |
| − | + | <p>FOTO</p> | |
| − | + | <p>We see three bands: 7000, 4000, 1900pb</p> | |
| − | + | <p>Transform optimized ligation PIF-Phy-Luc-Renilla-P19 and make petri dish cultures.</p> | |
| − | + | </section> | |
</div> | </div> | ||
</div> | </div> | ||
Revision as of 12:58, 10 September 2015
We had 2 cultures from the last day, corresponding to other 2 colonies of ligation. Agrobacterium culture of promoter less: Luciferase + Renilla Minipreps Digestion with BamHI and EcoRV Agarose gel 1% FOTO How to ask and make primers? Meeting with Daniel Ramón (Biopolis). Ligation with part 2 and 24 of task sheet. Digestions: Transform to E. coli from PIF+Phy and BxbI and make petri dish cultures. Digest of 160, 289 and the two ligations, PIF+phy and Etr8+BxbI. Agarose gel. FOTO We?ve got white colonies from PIF+Phy and Bxb1! Pick two colonies from each construction. Minipreps of the 4 liquid cultures and digestion to see the band patterns. Digestion: Agarose gel was made: FOTO Repeat digestion because we are not sure of the last digestions. We don?t have sure the toggle, so we decide to repeat the digestion with other enzyme tomorrow, noticing that the
colony 2 has better bands pattern. Optimized ligation: 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
37ºC to glycerinate later. We design primers to binding domain (BD) and PIF. Digestion of the ligation of yesterday containing: EPIF6-PhyB-VP16 (C1 y C2) Agarose gel 1%: FOTO We see three bands: 7000, 4000, 1900pb Transform optimized ligation PIF-Phy-Luc-Renilla-P19 and make petri dish cultures.Notebook
5 June 2015
PIF6 + PhyB; Ω1 Etr8 CMV+Bxb1_T35S; α1 1µL (GB892) PIF; α1 1µL 1097 (Etr8 CMV) pUPD2 1µL (GB88E) PhyB; α2 1µL Bxb1; pUPD2 1µL Ω1 1µL Tnos PuPD 1.2µL Buffer ligase 1µL α1 1µL Bsmb1 5.8µL H2O 6.8µL H2O
(GB160) 35S:Renilla:tNOS-35S:P19:tNOS EcoRV 2475, 381, 4601 (GB896) Luc:PIF6:PhyB EcoRV 11608, 3942 6 June 2015
GB160 289 PIF+PhyB BxbI ok no ? ? 7 June 2015
8 June 2015
Etr8(CMV):Bxb1:Tnos; α1 EcoRI 6345, 238 EPIF6 + PhyB-PV16; Ω1 BamHI 6686, 1439, 2685, 2237
Bxb1 (C1) Bxb1 (C2) EPIF6 + PhyB-PV16 (C1) EPIF6 + PhyB-PV16 (C2) ok ok
PIF-Phy-Luc-Renilla-P19 1 µL vector 0.8 µL dilution ½ GB160 1.7 µL PIF:PhyB 4.15 µL H2O Ratio 1:2 vector insert 9 June 2015
EPIF6-PhyB-VP16 PvuII (green buffer) 3663, 9472pb
EPIF6-PhyB-VP16 (C1) EPIF6-PhyB-VP16 (C2) no no