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

Line 25: Line 25:
 
</h2>
 
</h2>
 
</header>
 
</header>
−
<p>Holi</p>
+
<h3 style="color:green">5 June 2015</h3>
−
<p>Holi</p>
+
 
−
<p>Holi</p>
+
<p>We had 2 cultures from the last day, corresponding to other 2 colonies of ligation. </p>
−
<p>Holi</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; &Omega;1</td><td>Etr8 CMV+Bxb1_T35S; &alpha;1</td></tr>
 +
 
 +
<tr><td>1µL (GB892) PIF; &alpha;1</td><td>1µL 1097 (Etr8 CMV) pUPD2</td></tr>
 +
 
 +
<tr><td>1µL (GB88E) PhyB; &alpha;2</td><td>1µL Bxb1; pUPD2</td></tr>
 +
 
 +
<tr><td>1µL &Omega;1 </td><td>1µL Tnos PuPD</td></tr>
 +
 
 +
<tr><td>1.2µL Buffer ligase</td><td>1µL &alpha;1</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>
 +
 
 +
<tr><td>FOTO</td></tr>
 +
 
 +
<tr><td></td></tr>
 +
 
 +
</br><h3 style="color:green">7 June 2015</h3>
 +
 
 +
<tr><td></td></tr>
 +
 
 +
<tr><td>We?ve got white colonies from PIF+Phy and Bxb1!</td></tr>
 +
 
 +
<tr><td>Pick two colonies from each construction.</td></tr>
 +
 
 +
<tr><td></td></tr>
 +
 
 +
<tr><td></td></tr>
 +
 
 +
</br><h3 style="color:green">8 June 2015</h3>
 +
 
 +
<tr><td></td></tr>
 +
 
 +
<tr><td>Minipreps of the 4 liquid cultures and digestion to see the band patterns.</td></tr>
 +
 
 +
<tr><td>Digestion:</td></tr>
 +
 
 +
<tr><td></td></tr>
 +
 
 +
<div class="table-wrapper"><table class="alt">
 +
 
 +
<tr><td>Etr8(CMV):Bxb1:Tnos; &alpha;1</td><td>EcoRI</td><td>6345, 238</td></tr>
 +
 
 +
<tr><td>EPIF6 + PhyB-PV16; &Omega;1</td><td>BamHI</td><td>6686, 1439, 2685, 2237</td></tr>
 +
 
 +
</div></table>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<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>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<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>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
</br><h3 style="color:green">10 June 2015</h3>
 +
 
 +
<ul><li>Check the primers and order LexA, Gal4, PIF6, LacI, Dronpa.</li>
 +
 
 +
<li>Check linker VP16 (88E) and make a primer for it.</li>
 +
 
 +
<li>Take out glycerinate of &Omega;2.</li>
 +
 
 +
</ul>
 +
 
 +
<p>Alfredo?s part is not working.</p>
 +
 
 +
<ul><li>Make liquid culture of E:PIF6:PhyB:VP16:luc:ren (C1-C3).</li>
 +
 
 +
</ul>
 +
 
 +
<ul><li>Minipreps of liquid culture (PIF + Phy), colonies C3, C4, C5, C6</li>
 +
 
 +
<li>Digestion:</li>
 +
 
 +
</ul></ul>
 +
 
 +
<div class="table-wrapper"><table class="alt">
 +
 
 +
<tr><td>PIF+Phy:VP16</td><td>PvuII (buffer green 10x)</td><td>3663, 9472</td></tr>
 +
 
 +
<tr><td>PIF+Phy:VP16</td><td>BamHI</td><td>1939, 2685, 2337, 6674</td></tr>
 +
 
 +
</div></table>
 +
 
 +
 
 +
 
 +
<ul><li>Agarose gel 1%:</li>
 +
 
 +
</ul>
 +
 
 +
<div class="table-wrapper"><table class="alt">
 +
 
 +
<tr><td>PIF + Phy (PvuII) C3</td><td>PIF + Phy (PvuII) C4</td><td>PIF + Phy (PvuII) C5</td><td>PIF + Phy (PvuII) C6</td></tr>
 +
 
 +
<tr><td>no</td><td>ok</td><td>no</td><td>No</td></tr>
 +
 
 +
<tr><td>PIF + Phy (BamHI) C3</td><td>PIF + Phy (BamHI) C4</td><td>PIF + Phy (BamHI) C5</td><td>PIF + Phy (BamHI) C6</td></tr>
 +
 
 +
<tr><td>no</td><td>ok</td><td>no</td><td>No</td></tr>
 +
 
 +
</div></table>
 +
 
 +
 
 +
 
 +
<ul><li>Transformation into <i>Agrobacterium</i>EPIF6-PhyB-VP16 + luciferase (GB896) and make petri dish culture. We are not going to have the positive control (renilla+P19) and we won?t be able to quantify and make ratios.</li>
 +
 
 +
</ul>
 +
 
 +
</br><h3 style="color:green">11 June 2015</h3>
 +
 
 +
<ul><li>Minipreps of the culture:</li>
 +
 
 +
</ul>
 +
 
 +
<ul><li>Digestion:</li>
 +
 
 +
</ul>
 +
 
 +
<div class="table-wrapper"><table class="alt">
 +
 
 +
<tr><td>E:PIF6:PhyB:VP16:luc:ren</td><td>BamHI</td><td>4209, 3756, 6100, 6674</td></tr>
 +
 
 +
<tr><td></td><td>EcoRV</td><td>3942, 2989, 2475, 381, 10952</td></tr>
 +
 
 +
</div></table>
 +
 
 +
 
 +
 
 +
 
 +
 
 +
<p>Gel:</p>
 +
 
 +
 
 +
 
 +
<div class="table-wrapper"><table class="alt">
 +
 
 +
<tr><td>PIF6:PhyB:VP16:luc:ren C1 (BamHI)</td><td>PIF6:PhyB:VP16:luc:ren C3 (BamHI)</td><td>PIF6:PhyB:VP16:luc:ren C1 (EcoRV)</td><td>PIF6:PhyB:VP16:luc:ren C3 (EcoRV)</td></tr>
 +
 
 +
<tr><td>no</td><td>no</td><td>no</td><td>no</td><td></td><td></td></tr>
 +
 
 +
</div></table>
 +
 
 +
 
 +
 
 +
<p>FOTO</p>
 +
 
 +
<p>Transformation into <i>Agrobacterium</i>of Renilla (GB160) because we could not join this construction with PIF:PhyB and so we will do a cotransfection of both plasmids.Make petri dish culture.</p>
 +
 
 +
 
 
</section>
 
</section>
 
</section>
 
</section>

Revision as of 12:39, 10 September 2015

Valencia UPV iGEM 2015

Notebook

5 June 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?

  • Select the sequence to amplify and save in FASTA format.
  • gbCloning, go to Tools-Domesticator-1º Category
  • Add FASTA and select parts.
  • On the protocol we have the primers
  • The oligos they give us:
    • 4 first nucleotides: so the enzyme can recognize without problems
    • 6 following bingind sites.
    • 1 extra nucleotide.
    • 4 overhangs.

Meeting with Daniel Ramón (Biopolis).

Ligation with part 2 and 24 of task sheet.

PIF6 + PhyB; Ω1Etr8 CMV+Bxb1_T35S; α1
1µL (GB892) PIF; α11µL 1097 (Etr8 CMV) pUPD2
1µL (GB88E) PhyB; α21µL Bxb1; pUPD2
1µL Ω1 1µL Tnos PuPD
1.2µL Buffer ligase1µL α1
1µL Bsmb15.8µL H2O
6.8µL H2O

Digestions:

(GB160) 35S:Renilla:tNOS-35S:P19:tNOSEcoRV2475, 381, 4601
(GB896) Luc:PIF6:PhyBEcoRV11608, 3942

6 June 2015

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.


7 June 2015


8 June 2015

GB160289PIF+PhyBBxbI
okno??
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:
Etr8(CMV):Bxb1:Tnos; α1EcoRI6345, 238
EPIF6 + PhyB-PV16; Ω1BamHI6686, 1439, 2685, 2237

Agarose gel was made:

Bxb1 (C1)Bxb1 (C2)EPIF6 + PhyB-PV16 (C1)EPIF6 + PhyB-PV16 (C2)
okok

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:

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

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.

  • Problem: domesticator is introduced in an old pUPD2. The new one has different bases.
  • Change manually the pUPD2 bases in the program (Benchling).

9 June 2015

Digestion of the ligation of yesterday containing: EPIF6-PhyB-VP16 (C1 y C2)

EPIF6-PhyB-VP16PvuII (green buffer)3663, 9472pb

Agarose gel 1%:

EPIF6-PhyB-VP16 (C1)EPIF6-PhyB-VP16 (C2)
nono

FOTO

We see three bands: 7000, 4000, 1900pb

Transform optimized ligation PIF-Phy-Luc-Renilla-P19 and make petri dish cultures.


10 June 2015

  • Check the primers and order LexA, Gal4, PIF6, LacI, Dronpa.
  • Check linker VP16 (88E) and make a primer for it.
  • Take out glycerinate of Ω2.

Alfredo?s part is not working.

  • Make liquid culture of E:PIF6:PhyB:VP16:luc:ren (C1-C3).
  • Minipreps of liquid culture (PIF + Phy), colonies C3, C4, C5, C6
  • Digestion:
PIF+Phy:VP16PvuII (buffer green 10x)3663, 9472
PIF+Phy:VP16BamHI1939, 2685, 2337, 6674
  • Agarose gel 1%:
PIF + Phy (PvuII) C3PIF + Phy (PvuII) C4PIF + Phy (PvuII) C5PIF + Phy (PvuII) C6
nooknoNo
PIF + Phy (BamHI) C3PIF + Phy (BamHI) C4PIF + Phy (BamHI) C5PIF + Phy (BamHI) C6
nooknoNo
  • Transformation into AgrobacteriumEPIF6-PhyB-VP16 + luciferase (GB896) and make petri dish culture. We are not going to have the positive control (renilla+P19) and we won?t be able to quantify and make ratios.

11 June 2015

  • Minipreps of the culture:
  • Digestion:
E:PIF6:PhyB:VP16:luc:renBamHI4209, 3756, 6100, 6674
EcoRV3942, 2989, 2475, 381, 10952

Gel:

PIF6:PhyB:VP16:luc:ren C1 (BamHI)PIF6:PhyB:VP16:luc:ren C3 (BamHI)PIF6:PhyB:VP16:luc:ren C1 (EcoRV)PIF6:PhyB:VP16:luc:ren C3 (EcoRV)
nononono

FOTO

Transformation into Agrobacteriumof Renilla (GB160) because we could not join this construction with PIF:PhyB and so we will do a cotransfection of both plasmids.Make petri dish culture.