Difference between revisions of "Team:Valencia UPV/Notebook"
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Notebook</h2> | Notebook</h2> | ||
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+ | <div align="center"><div id="cn-box" align="justify"><br/> | ||
+ | |||
+ | <div align="center"><img class="img-title" alt="Notebook" src="https://static.igem.org/mediawiki/2014/2/2a/VUPVNotebook.png"></img></div><br/> | ||
+ | |||
+ | <div class="box_above_notebook"> | ||
+ | |||
+ | Contents: | ||
+ | <ul style="margin-left: 1.5em;"> <li> <a href="#Constructions">Constructions</a></li></ul> | ||
+ | </div><a name="Constructions"></a></br></br><h3 class="section_notebook">Constructions</h3><p class="p_notebook">CONSTRUCTIONS</p> | ||
+ | |||
+ | </br><h4 class="date_notebook">5 June 2015</h4> | ||
+ | |||
+ | |||
+ | |||
+ | <p class="p_notebook">We had 2 cultures from the last day, corresponding to other 2 colonies of ligation. </p> | ||
+ | |||
+ | <p class="p_notebook"><i>Agrobacterium</i> culture of promoter less: Luciferase + Renilla </p> | ||
+ | |||
+ | |||
+ | |||
+ | <p class="p_notebook">Minipreps</p> | ||
+ | |||
+ | <p class="p_notebook">Digestion with BamHI and EcoRV</p> | ||
+ | |||
+ | <p class="p_notebook">Agarose gel 1%</p> | ||
+ | |||
+ | <table class="table_notebook"> | ||
+ | |||
+ | <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> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | <p class="p_notebook">How to ask and make primers?</p> | ||
+ | |||
+ | <ul class="ul_notebook"><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_ul_notebook"><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 class="p_notebook">Meeting with Daniel Ramón (Biopolis). </p> | ||
+ | |||
+ | <p class="p_notebook">Ligation with part 2 and 24 of task sheet.</p> | ||
+ | |||
+ | <table class="table_notebook"> | ||
+ | |||
+ | <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> | ||
+ | |||
+ | <tr class="tr_notebook"><td class="td_notebook">1µL 88E (Phy α2)</td><td class="td_notebook">1µL Bxb1 (PuPD)</td></tr> | ||
+ | |||
+ | <tr class="tr_notebook"><td class="td_notebook">1µL ?1 </td><td class="td_notebook">1µL Tnos PuPD</td></tr> | ||
+ | |||
+ | <tr class="tr_notebook"><td class="td_notebook">1.2µL Buffer ligase</td><td class="td_notebook">1µL α1</td></tr> | ||
+ | |||
+ | <tr class="tr_notebook"><td class="td_notebook">1µL Bsmb1</td><td class="td_notebook">5.8µL H2O</td></tr> | ||
+ | |||
+ | <tr class="tr_notebook"><td class="td_notebook">6.8µL H2O</td><td class="td_notebook"></td></tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | |||
+ | |||
+ | <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> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h4 class="date_notebook">June 2015</h4> | ||
+ | |||
+ | |||
+ | |||
+ | <p class="p_notebook">Transform to E.coli from Pif+Phy and Bxb1</p> | ||
+ | |||
+ | <ul class="ul_notebook"><li>1.5µL of ligation</li> | ||
+ | |||
+ | <ul class="ul_ul_notebook"><li>Cuvette on ice</li> | ||
+ | |||
+ | <li>Competent cells + 1.5µL of ligation</li> | ||
+ | |||
+ | <li>Pulse (electroporator) at 1500V</li> | ||
+ | |||
+ | <li>Add 300µL shock medium and put Eppendorf 1h at 37ºC</li> | ||
+ | |||
+ | </ul></ul> | ||
+ | |||
+ | <p class="p_notebook">Culture on petri dishes the ligations.</p> | ||
+ | |||
+ | <p class="p_notebook">Digest of 160, 289 and the two ligations, pif+phy and Etr8+BxbI. </p> | ||
+ | |||
+ | <p class="p_notebook">Agarose gel.</p> | ||
+ | |||
+ | <table class="table_notebook"> | ||
+ | |||
+ | <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> | ||
+ | |||
+ | <tr class="tr_notebook"><td class="td_notebook">1Kb</td></tr> | ||
+ | |||
+ | </table> | ||
+ | |||
+ | <p class="p_notebook">Storage of gel on: Basura en Arabidopsis – Igem – 2015 – 150606_Digestion_ToggleRojo</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h4 class="date_notebook">7 June 2015</h4> | ||
+ | |||
+ | |||
+ | |||
+ | <p class="p_notebook">We’ve got white colonies! (from Pif+Phy and Bxb1)</p> | ||
+ | |||
+ | <p class="p_notebook">Pick two colonies from each construction.</p> | ||
+ | |||
+ | <p class="p_notebook">4 tubes</p> | ||
+ | |||
+ | <ul class="ul_notebook"><li>3.5µL LB each tube</li> | ||
+ | |||
+ | </ul> | ||
+ | |||
+ | <p class="p_notebook">2) 2 tubes + 3.5µL Kanamycin (K)</p> | ||
+ | |||
+ | |||
+ | |||
+ | </br><h4 class="date_notebook">8 June 2015</h4> | ||
+ | |||
+ | |||
+ | |||
+ | <p class="p_notebook">Minipreps of the 4 liquid cultures and digestion to see the band patterns.</p> | ||
+ | |||
+ | <p class="p_notebook">Digestion:</p> | ||
+ | |||
+ | <table class="table_notebook"> | ||
+ | |||
+ | <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> | ||
+ | |||
+ | </div> | ||
+ | <div id="space-margin"></div> | ||
+ | </html></p> | ||
</header> | </header> | ||
<span class="image featured"><img src="images/pic01.jpg" alt="" /></span> | <span class="image featured"><img src="images/pic01.jpg" alt="" /></span> |
Revision as of 11:23, 28 August 2015
{{:Team:Valencia_UPV/header}}
CONSTRUCTIONS 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% How to ask and make primers? Meeting with Daniel Ramón (Biopolis). Ligation with part 2 and 24 of task sheet. If we make a digestion of 160 (35S:Renilla:tNOS-35S:P19:tNOS) with EcoRV, we obtain: 2475, 381, 4601 pb. If we make a digestion of 896 (Luc:Pif6:PhyB)with EcoRV, we obtain: 11608, 3942 pb. Transform to E.coli from Pif+Phy and Bxb1 Culture on petri dishes the ligations. Digest of 160, 289 and the two ligations, pif+phy and Etr8+BxbI. Agarose gel. Storage of gel on: Basura en Arabidopsis – Igem – 2015 – 150606_Digestion_ToggleRojo We’ve got white colonies! (from Pif+Phy and Bxb1) Pick two colonies from each construction. 4 tubes 2) 2 tubes + 3.5µL Kanamycin (K) Minipreps of the 4 liquid cultures and digestion to see the band patterns. Digestion: </div>
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The project
Notebook
Constructions
5 June 2015
Mw Ples 4E 4B 3E 3B 1kb
Pif6 + PhyB; ?1 Etr8 CMV_Bxb1_T35S 1µL 892 (Pif α1) 1µL 1097 (Etr8 CMV) Pupd2 1µL 88E (Phy α2) 1µL Bxb1 (PuPD) 1µL ?1 1µL Tnos PuPD 1.2µL Buffer ligase 1µL α1 1µL Bsmb1 5.8µL H2O 6.8µL H2O June 2015
6µL ladder 160 289 ligation ligation Ladder 1Kb 7 June 2015
8 June 2015
Etr8(CMV):Bxb1:Tnos; Ω1 EcoRI 6345, 238 EPIF6 + PhyB-PV16; Ω1 BamHI 6686, 1439, 2685, 2237 Contact
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