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| Line 3: |
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| | <body> | | <body> |
| − | <section>
| |
| − | <div class="container">
| |
| − | <div class="timeline">
| |
| − | <div class="timeline-hline"></div>
| |
| − | <div class="blog-post-item">
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| − | <div class="timeline-entry rounded">
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| − | 18<span>May</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">May 18, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Take glycerinated cultures of C58 <i>Agrobacterium</i>
| |
| − | with dsRED from GoldenBraid Collection. Prepare broth
| |
| − | culture (5 mL LB + 5 μL kanamycin + 5 μL Rifampin) 1:1000
| |
| − | and incubate overnight at 28^C.<br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 19<span>May</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">May 19, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Refresh previously made culture by inoculating 10 μL in
| |
| − | a new culture medium.<br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 20<span>May</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">May 20, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Agroinfiltration in <i>Nicotiana benthamiana</i> of C58
| |
| − | with dsRED. href<br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 06<span>Jun</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">June 06, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Take from the glycerinates of Goldenbraid
| |
| − | Collection:</li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Plasmid</th>
| |
| − | <th>GB Code</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>pD6B3 α1</td>
| |
| − | <td>GB0015</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>pD6B3 α2</td>
| |
| − | <td>GB0017</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>pD6B3 Ω1</td>
| |
| − | <td>GB0019</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>pD6B3 Ω2</td>
| |
| − | <td>GB0021</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>pUPD2</td>
| |
| − | <td>GB0307</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p>Prepare liquid culture (3 mL LB + 3 μL antibiotic)
| |
| − | 1:1000. Incubate at 37^C overnight.<br></p>
| |
| − | <ul>
| |
| − | <li>Experiment with snails:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>Two experiments: one with infiltrated
| |
| − | <i>Nicotiana benthamiana</i> and another with
| |
| − | not infiltrated N.benthamiana (negative
| |
| − | control). Lettuce leafs haven’t been correctly
| |
| − | infiltrated and it seems that the expression
| |
| − | level is low.</li>
| |
| − | <li>Let both <i>N. benthamiana</i> leafs with
| |
| − | snails overnight at room temperature in
| |
| − | separated boxes. We will observe if snails eat
| |
| − | the leafs and if appears fluorescence.</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 15<span>Jun</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">June 15, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Experiment is over due to snails haven’t eaten leafs
| |
| − | enough so we have not been able to see
| |
| − | fluorescence.<br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 30<span>Jun</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">June 30, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Orange Clemenules DNA Genome Extraction <a href=
| |
| − | "https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol">protocol</a><br>
| |
| − | </p>
| |
| − | <ul>
| |
| − | <li>Take from the glycerinates of GoldenBraid
| |
| − | Collection:</li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Plasmid</th>
| |
| − | <th>GB Code</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>35s:Cas9:nopaline synthase terminator
| |
| − | (Tnos)</td>
| |
| − | <td>GB0639</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Luciferase (Luc) in pUPD2</td>
| |
| − | <td>GB0096</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Tnos in pUPD2</td>
| |
| − | <td>GB0037</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 01<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 01, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Miniprep of 35s:Cas9:Tnos with E.Z.N.A ®. Plasmid Mini
| |
| − | Kit I, Q(capless) Spin.<br>
| |
| − | Luciferase and nopaline synthase terminator cultures
| |
| − | haven’t succeed. Repeat Luc and Tnos cultures.<br>
| |
| − | Primers IG16JUN01 and IG16JUN02 have arrived.<br>
| |
| − | Finish orange DNA Genome Extraction and check DNA
| |
| − | concentration with NanoDrop. Concentration is very low so
| |
| − | extraction will be done again.<br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 02<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 02, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Miniprep with E.Z.N.A ®. Plasmid Mini Kit I,
| |
| − | Q(capless) Spin of:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>Luc in pUPD2</li>
| |
| − | <li>Tnos in pUPD2</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Check orange DNA genome concentration with
| |
| − | NanoDrop.<br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Sample</th>
| |
| − | <th>DNA concentration (ng / μL)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>TFL 1</td>
| |
| − | <td>3153.8</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>TFL 2</td>
| |
| − | <td>4527.9</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p><br>
| |
| − | Perform a PCR to bind linker SAGTI with luciferase:<br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Reagent</th>
| |
| − | <th>Volume(μL)</th>
| |
| − | <th>Program</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>LuciferasepUPD</td>
| |
| − | <td>1</td>
| |
| − | <td>Temperature</td>
| |
| − | <td>Time</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Buffer HF</td>
| |
| − | <td>10</td>
| |
| − | <td>98^C</td>
| |
| − | <td>5 minutes</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>dNTPs</td>
| |
| − | <td>2</td>
| |
| − | <td>98^C</td>
| |
| − | <td>35x</td>
| |
| − | <td>30 seconds</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>IG16JUN01</td>
| |
| − | <td>2.5</td>
| |
| − | <td>70^C</td>
| |
| − | <td>35x</td>
| |
| − | <td>30 seconds</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>IG16JUN02</td>
| |
| − | <td>2.5</td>
| |
| − | <td>72^C</td>
| |
| − | <td>35x</td>
| |
| − | <td>1 minute 30 seconds</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Taq phusion</td>
| |
| − | <td>0.5</td>
| |
| − | <td>72^C</td>
| |
| − | <td>10 minutes</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>H<sub>2</sub>O milli-Q</td>
| |
| − | <td>31.5</td>
| |
| − | <td>16^C</td>
| |
| − | <td>∞</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 04<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 04, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Run electrophoresis gel of Clemenules DNA (agarose 1%).
| |
| − | 45 mL agarose gel at 1% 0.45 g of agarose with 45 mL of TAE
| |
| − | 1X. Voltage used is 100 V.<br>
| |
| − | Gleva Rice DNA Genome Extraction <a href=
| |
| − | "https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol">Protocol</a><br>
| |
| | | | |
| − | Run electrophoresis gel of Luciferase PCR product. 45 mL
| + | < section>< div class="container">< div class="timeline"><div class="timeline-hline"></ div>< div class="blog-post-item">< div class="timeline-entry rounded"> 18<span>May</ span><div class=" timeline- vline">< /div></div><ul class=" blog- post- info list-inline">< /ul>< p> Take glycerinated cultures of C58 < i> Agrobacterium< /i> with dsRED from GoldenBraid Collection. Prepare broth culture ( 5 mL LB + 5 μL kanamycin + 5 μL Rifampin) 1:1000 and incubate overnight at 28^C. <br></ p></ div>< div class="blog-post-item">< div class="timeline-entry rounded"> 19<span>May</ span>< div class="timeline-vline"></ div></ div>< ul class="blog-post-info list-inline"></ ul>< p> Refresh previously made culture by inoculating 10 μL in a new culture medium. <br></ p></ div> <div class="blog-post-item"><div class="timeline-entry rounded">20<span>May</ span><div class="timeline-vline"></div> </div><ul class="blog-post-info list-inline"></ul><p> Agroinfiltration in <i>Nicotiana benthamiana</i> of C58 with dsRED. href<br></p></div><div class="blog-post-item"><div class="timeline-entry rounded"> 06<span> Jun</span><div class="timeline-vline"></div></div><ul class="blog-post-info list-inline"> </ul><p></p><ul><li> Take from the glycerinates of Goldenbraid Collection:< /li></ul><p></p><div class=" table- responsive" style="width:55%;overflow:inherit"> <table class="table table-bordered table-striped"><tr><th>Plasmid</ th> <th>GB Code</ th></ tr><tr><td>pD6B3 α1</td><td>GB0015</td></tr><tr><td>pD6B3 α2</td><td>GB0017</td></tr><tr><td>pD6B3 Ω1</td><td>GB0019</td></tr><tr><td>pD6B3 Ω2</td><td>GB0021</td></tr><tr><td>pUPD2</td><td>GB0307</td></tr></table></div><p> Prepare liquid culture (3 mL LB + 3 μL antibiotic) 1:1000. Incubate at 37^C overnight. <br>< /p><ul><li>Experiment with snails: </ li></ p><ul><li>Two experiments: one with infiltrated <i>Nicotiana benthamiana</i> and another with not infiltrated N. benthamiana (negative control). Lettuce leafs haven’t been correctly infiltrated and it seems that the expression level is low.</ li><li> Let both <i>N. benthamiana</ i> leafs with snails overnight at room temperature in separated boxes. We will observe if snails eat the leafs and if appears fluorescence. </ li></ ul></ul><p><br ></p></div></div></div></section> |
| − | agarose gel at 1% 0.45 g of agarose with 45 mL of TAE 1X.
| + | |
| − | Voltage used is 100 V.<br>
| + | |
| − | Ligation Reaction of SAGTI:Luciferase into a pUPD2.<br>
| + | |
| − | Transform <i>E. coli</i> DH5α with it. The method that is
| + | |
| − | necessary to carry out this procedure is explained in
| + | |
| − | protocols href<br>
| + | |
| − | Check DNA concentration with NanoDrop.<br></p>
| + | |
| − | <div class=" table- responsive" style= | + | |
| − | "width:55%;overflow:inherit">
| + | |
| − | <table class="table table-bordered table-striped">
| + | |
| − | <tr>
| + | |
| − | <th>SAMPLE</th>
| + | |
| − | <th>DNA Concentration(ng / μL)</th>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Rice Gleva 1</td>
| + | |
| − | <td>22.8</td>
| + | |
| − | </tr>
| + | |
| − | <tr>
| + | |
| − | <td>Rice Gleva 2</td>
| + | |
| − | <td>17.3</td>
| + | |
| − | </tr>
| + | |
| − | </table>
| + | |
| − | </div>
| + | |
| − | | + | |
| − | </div>
| + | |
| − | <div class="blog-post-item"> | + | |
| − | <div class="timeline-entry rounded"> | + | |
| − | 05<span>Jul</span>
| + | |
| − | <div class="timeline-vline"></div> | + | |
| − | </div>
| + | |
| − | <ul class="blog-post-info list-inline"> | + | |
| − | <li><span class="font-lato">July 05, 2016</span></li>
| + | |
| − | </ul>
| + | |
| − | <p> Run electrophoresis gel of Gleva rice DNA. We have | + | |
| − | checked that there isn’t DNA.<br>
| + | |
| − | Repeat: Rice DNA Genome Extraction <a href=
| + | |
| − | "https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol">Protocol</a><br>
| + | |
| | | | |
| − | Check DNA concentration with NanoDrop.<br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>SAMPLE</th>
| |
| − | <th>DNA Concentration(ng / μL)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Rice Gleva 1</td>
| |
| − | <td>294.9</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Rice Gleva 2</td>
| |
| − | <td>193.7</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p>Run electrophoresis gel of Gleva rice DNA. It observed
| |
| − | that genome extraction is correctly done.<br>
| |
| − | <br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 06<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 06, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Take glycerinated cultures from Goldenbraid
| |
| − | Collection:<br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>GB part</th>
| |
| − | <th>Plasmid</th>
| |
| − | <th>Antibiotic</th>
| |
| − | <th>Number GB</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>psgRNA</td>
| |
| − | <td>pUPD</td>
| |
| − | <td>Ampicillin</td>
| |
| − | <td>0645</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>U6-26</td>
| |
| − | <td>pUPD</td>
| |
| − | <td>Ampicillin</td>
| |
| − | <td>1001</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p><br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 07<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 07, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Miniprep with E.Z.N.A ®. Plasmid Mini Kit I,
| |
| − | Q(capless) Spin of:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>psgRNA in pUPD2</li>
| |
| − | <li>U6-26 in pUPD2</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Primers IG16JUL01, IG16JUL02, IG16JUL03, IG16JUL04,
| |
| − | IG16JUL05, IG16JUL06, IG16JUL07 and IG16JUL08 arrived.<br>
| |
| − | gBlocks of - 35s:5’ region - have arrived.<br>
| |
| − | We perform a PCR of Clemenules Orange and Gleva Rice Genome
| |
| − | Extraction following the <a href=
| |
| − | "https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol">protocol</a>.
| |
| − | We want to obtain a fragment of the gene TFL of the orange
| |
| − | DNA extraction and the gene Ga20ox of the rice DNA
| |
| − | extraction.<br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Sample</th>
| |
| − | <th>Initial concentration(ng/μL)</th>
| |
| − | <th>Final concentration(ng/μL)</th>
| |
| − | <th>Initial volume(μL)</th>
| |
| − | <th>Final volume (μL)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>TFL 1</td>
| |
| − | <td>3153.8</td>
| |
| − | <td>150</td>
| |
| − | <td>4.756</td>
| |
| − | <td>100</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>TFL 2</td>
| |
| − | <td>4527.9</td>
| |
| − | <td>150</td>
| |
| − | <td>3.31</td>
| |
| − | <td>100</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Ga20ox 1</td>
| |
| − | <td>294.9</td>
| |
| − | <td>150</td>
| |
| − | <td>50.8647</td>
| |
| − | <td>100</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Ga20ox 2</td>
| |
| − | <td>193.7</td>
| |
| − | <td>150</td>
| |
| − | <td>77.44</td>
| |
| − | <td>100</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p><br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>REAGENT</th>
| |
| − | <th>VOLUME(μL)</th>
| |
| − | <th>PROGRAM</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>TFL DNA 1</td>
| |
| − | <td>TEMPERATURE</td>
| |
| − | <td>TIME</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Buffer HF</td>
| |
| − | <td>10</td>
| |
| − | <td>98^C</td>
| |
| − | <td>5 minutes</td>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>dNTPs</td>
| |
| − | <td>2</td>
| |
| − | <td>98^C</td>
| |
| − | <td>35x</td>
| |
| − | <td>30 seconds</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>IG16JUL01 (TFL_For)</td>
| |
| − | <td>2.5</td>
| |
| − | <td>64^C</td>
| |
| − | <td>35x</td>
| |
| − | <td>30 seconds</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>IG16JUL02 (TFL_Rev)</td>
| |
| − | <td>2.5</td>
| |
| − | <td>72^C</td>
| |
| − | <td>35x</td>
| |
| − | <td>30 seconds</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Taq phusion</td>
| |
| − | <td>0.5</td>
| |
| − | <td>72^C</td>
| |
| − | <td>10 minutes</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>H<sub>2</sub>O milli-Q</td>
| |
| − | <td>31.5</td>
| |
| − | <td>16^C</td>
| |
| − | <td>∞</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p><br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>REAGENT</th>
| |
| − | <th>VOLUME(μL)</th>
| |
| − | <th>PROGRAM</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Ga20ox DNA</td>
| |
| − | <td>1</td>
| |
| − | <td>TEMPERATURE</td>
| |
| − | <td>TIME</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Buffer HF</td>
| |
| − | <td>10</td>
| |
| − | <td>98^C</td>
| |
| − | <td>5 minutes</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>dNTPs</td>
| |
| − | <td>2</td>
| |
| − | <td>98^C</td>
| |
| − | <td>35x</td>
| |
| − | <td>30 seconds</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>IG16JUL03 (Ga20_for)</td>
| |
| − | <td>2.5</td>
| |
| − | <td>72^C</td>
| |
| − | <td>35x</td>
| |
| − | <td>30 seconds</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>IG16JUL02 (Ga20_rev)</td>
| |
| − | <td>2.5</td>
| |
| − | <td>72^C</td>
| |
| − | <td>35x</td>
| |
| − | <td>30 seconds</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Taq phusion</td>
| |
| − | <td>0.5</td>
| |
| − | <td>72^C</td>
| |
| − | <td>10 minutes</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>H<sub>2</sub>O milli-Q</td>
| |
| − | <td>31.5</td>
| |
| − | <td>16^C</td>
| |
| − | <td>∞</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p>Ligate reaction of 35s:5’ region in pUPD2. Following
| |
| − | <a href=
| |
| − | "https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol">ligation
| |
| − | protocol</a>, BsmbI enzyme is used in this reaction.<br>
| |
| − | <br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 08<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 08, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Run electrophoresis gel of TFL and Ga20ox PCR products.
| |
| − | 45 mL agarose gel at 1 % 0.45 g of agarose with 45 mL of
| |
| − | TAE 1X. Voltage used is 120 V.<br>
| |
| − | Transform <i>E. coli</i> with the next part: 35s:5’ region
| |
| − | (electroporation 2.5KV). Plating it and incubate overnight
| |
| − | at 37^C.<br>
| |
| − | Take glycerinated culture for Georgia collaboration. The
| |
| − | part is 35s:GFP:Tnos (α1 and kanamycin).<br>
| |
| − | <br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 09<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 09, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless)
| |
| − | Spin of 35s:GFP:Tnos<br>
| |
| − | No colonies have grown in the 35s:5’ region petri dishes.
| |
| − | Repeat transformation procedure, plating again and incubate
| |
| − | overnight at 37^C.<br>
| |
| − | <br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 11<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 11, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Pick a single E. coli DH5α (35s:5’ region in pUPD2)
| |
| − | colony from the plate that has been incubated overnight.
| |
| − | Inoculate a starter culture of 4 ml of LB medium with 4 μL
| |
| − | of chloramphenicol in a 50 ml tube with the colony and
| |
| − | incubate it overnight at 37^C with shaking.<br>
| |
| − | Check Georgia miniprep concentration with NanoDrop
| |
| − | (35s:GFP:Tnos)<br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Sample</th>
| |
| − | <th>DNA Concentration(ng / μL)</th>
| |
| − | <th>DNA Concentration(ng)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>1</td>
| |
| − | <td>105.2</td>
| |
| − | <td>5035.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>2</td>
| |
| − | <td>104.6</td>
| |
| − | <td>5035.2</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p>Targets ligations in pUPD2 (Orange TFL and Rice Ga20ox).
| |
| − | Following <a href=
| |
| − | "https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol">ligation
| |
| − | protocol</a>, BsmbI enzyme is used in this reaction.<br>
| |
| − | <br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 12<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 12, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Pick a single E. coli DH5α (target Ga20ox in pUPD2 and
| |
| − | target TFL in pUPD2) colony from the plate that has been
| |
| − | incubated overnight. Inoculate a starter culture of 4 ml of
| |
| − | LB medium with 4 μL of chloramphenicol in a 50 ml tube with
| |
| − | the colony and incubate it 16 hours at 37^C.<br>
| |
| − | Miniprep with E.Z.N.A ®. Plasmid Mini Kit I, Q(capless)
| |
| − | Spin of 35s:5’region in pUPD2<br>
| |
| − | Digestion of minipreps with NotI. Incubate 1 hour at
| |
| − | 37^C<br>
| |
| − | Run electrophoresis gel of the following part: 35s:5’
| |
| − | region in pUPD2. We remain the samples 1 and 3.<br></p>
| |
| − | <ul>
| |
| − | <li>Miniprep with E.Z.N.A ®. Plasmid Mini Kit I,
| |
| − | Q(capless) Spin of:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>Ga20ox PCR in pUPD2</li>
| |
| − | <li>TFL PCR in pUPD2</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Digestion of minipreps with NotI. Incubate 1 hour at
| |
| − | 37^C.<br></p>
| |
| − | <ul>
| |
| − | <li>Run electrophoresis gel of the same part:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>Ga20ox PCR in pUPD2</li>
| |
| − | <li>TFL PCR in pUPD2</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Ligation using Golden Braid assembly of the device
| |
| − | 35s:5’region:TFL PCR:Luc:Tnos and
| |
| − | 35s:5’region:Ga20oxPCR:Luc:Tnos in α1 plasmid.<br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 13<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 13, 2016</span></li>
| |
| − | </ul>
| |
| − | <p><i>E. coli</i> Transformation with the device
| |
| − | 35s:5’region:TFL PCR:Luc:Tnos and
| |
| − | 35s:5’region:Ga20oxPCR:Luc:Tnos in α1 plasmid.<br>
| |
| − | E. coli plating in plates with Kanamycin (antibiotic).
| |
| − | Incubate overnight at 37^C<br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 14<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 14, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Pick a single E. coli DH5α (35s:5’ region) colony from
| |
| − | the plate that has been incubated overnight. Inoculate a
| |
| − | starter culture of 4 ml of LB medium with 4 μL of
| |
| − | chloramphenicol in a 50 ml tube with the colony and
| |
| − | incubate it 16 hours at 37^C.<br>
| |
| − | Primers IG16JUL09, IG16JUL10, IG16JUL11, IG16JUL12,
| |
| − | IG16JUL13, IG16JUL14, IG16JUL15 and IG16JUL16
| |
| − | arrived.<br></p>
| |
| − | <ul>
| |
| − | <li>Ligation using Golden Braid assembly of the next
| |
| − | devices:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:5’ region:TFL PCR control
| |
| − | positive:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20oxPCR control
| |
| − | positive:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:TFL PCR
| |
| − | consensus:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox PCR
| |
| − | consensus:Luc:Tnos</li>
| |
| − | <li>U6-26:sgRNA TFL: psgRNA (scaffold)</li>
| |
| − | <li>U6-26:sgRNA Ga20ox: psgRNA (scaffold)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Step 1: Annealing of oligonucleotides. Received
| |
| − | primers are at 1uM. It is necessary taking to 10
| |
| − | uM.</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>Mix in an Eppendorf:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>8 μL of H<sub>2</sub>O milli-Q</li>
| |
| − | <li>1 μL forward primer</li>
| |
| − | <li>1 μL reverse primer</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | <li>Step 2: Ligation reaction</li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Reagent</th>
| |
| − | <th>Volume (μL)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>TFL target control + / Ga20ox target
| |
| − | control + / TFL target consensus / Ga20ox
| |
| − | target consensus</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>35s:5’ region</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Luciferase</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Tnos</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>α1 plasmid</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>BSA10X</td>
| |
| − | <td>1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Ligase Buffer</td>
| |
| − | <td>1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>BsaI</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>T4 ligase</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>H<sub>2</sub>O milli-Q</td>
| |
| − | <td>2.6</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p><br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Reagent</th>
| |
| − | <th>Volume (μL)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>sgRNA TFL / sgRNA Ga20ox</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>U6 -26</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>psgRNA (scaffold)</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>α1 plasmid</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>BSA10X</td>
| |
| − | <td>1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Ligase Buffer</td>
| |
| − | <td>1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>BsaI</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>T4 ligase</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>H<sub>2</sub>O milli-Q</td>
| |
| − | <td>3.6</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p><br>
| |
| − | <i>E. coli</i> Transformation with the devices previously
| |
| − | explained.<br>
| |
| − | E. coli plating in 6 plates (6 ligation reactions) with
| |
| − | Kanamycin (antibiotic). Incubate overnight at 37^C.<br></p>
| |
| − | <ul>
| |
| − | <li>Miniprep with E.Z.N.A ®. Plasmid Mini Kit I,
| |
| − | Q(capless) Spin of:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:5’ region : TFL PCR : Luc : Tnos
| |
| − | (3α1)</li>
| |
| − | <li>35s:5’ region : Ga20ox PCR : Luc : Tnos
| |
| − | (3α1)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Digestion of minipreps with EcoRI. Incubate 1 hour at
| |
| − | 37^C<br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 15<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 15, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Ligation using Golden Braid assembly of the next
| |
| − | devices:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:Cas9:Tnos – 35s:5’ region:TFL
| |
| − | PCR:Luc:Tnos</li>
| |
| − | <li>35s:Cas9:Tnos – 35s:5’ region:Ga20ox
| |
| − | PCR:Luc:Tnos</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>E. coli Transformation with the devices previously
| |
| − | explained.<br></p>
| |
| − | <ul>
| |
| − | <li>Run an electrophoresis gel of the products from the
| |
| − | digestion of minipreps. The devices are:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:5’ region:TFL PCR:Luc:Tnos (3α1)</li>
| |
| − | <li>35s:5’ region :Ga20ox PCR:Luc:Tnos
| |
| − | (3α1)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Sequencing 35s:5’region in pUPD2 → correct<br>
| |
| − | <br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 16<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 16, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Transformations in <i>E. coli</i> DH5α with:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:5’ region:TFL PCR:Luc:Tnos (3α1)</li>
| |
| − | <li>35s:5’ region:Ga20ox PCR:Luc:Tnos
| |
| − | (3α1)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Plating the last devices in 2 plates with
| |
| − | <i>Agrobacterium</i> C58. Incubate 2 days at 28^C.<br></p>
| |
| − | <ul>
| |
| − | <li>Minipreps (2 samples for each device) with E.Z.N.A
| |
| − | ®. Plasmid Mini Kit I, Q(capless) Spin of:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:5’ region:TFL target control
| |
| − | positive:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target control
| |
| − | positive:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:TFL target
| |
| − | consensus:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target
| |
| − | consensus:Luc:Tnos</li>
| |
| − | <li>U6-26:sgRNA TFL:psgRNA (scaffold)</li>
| |
| − | <li>U6-26:sgRNA Ga20ox:psgRNA (scaffold)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Ligation using Golden Braid assembly of the next
| |
| − | devices:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:Cas9:Tnos – U6:TFL sgRNA:psgRNA
| |
| − | (scaffold) (Ω1)</li>
| |
| − | <li>35s:Cas9:Tnos – U6: Ga20ox sgRNA:psgRNA
| |
| − | (scaffold) (Ω1)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Reagent</th>
| |
| − | <th>Volume (μL)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Promotor 35s:Cas9 : Tnos</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>U6-26:TFL sgRNA:psgRNA / U6-26:Ga20ox
| |
| − | sgRNA:psgRNA</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>3Ω1</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>BSA10X</td>
| |
| − | <td>1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Ligase Buffer</td>
| |
| − | <td>1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>BsmbI</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>T4 ligase</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>H<sub>2</sub>O milli-Q</td>
| |
| − | <td>4.6</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p>Digestion of the minipreps with EcoRI which have been
| |
| − | done before. Incubate 1 hour at 37^C. There is a total of
| |
| − | twelve minipreps. It has been followed the Miniprep
| |
| − | {digestion protocol
| |
| − | path:https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol].<br>
| |
| − |
| |
| − | Run electrophoresis gel of the digestion products.<br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Lanes</th>
| |
| − | <th>Samples</th>
| |
| − | <th>Verification</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>1</td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>2</td>
| |
| − | <td>gRNA Ga20ox 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>3</td>
| |
| − | <td>gRNA Ga20ox 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>4</td>
| |
| − | <td>Ga20ox consensus 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>5</td>
| |
| − | <td>Ga20ox consensus 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>6</td>
| |
| − | <td>Ga20ox knock-out 1</td>
| |
| − | <td>-</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>7</td>
| |
| − | <td>Ga20ox knock-out 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>8</td>
| |
| − | <td>TFL consensus 1</td>
| |
| − | <td>-</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>9</td>
| |
| − | <td>TFL consensus 2</td>
| |
| − | <td>-</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>10</td>
| |
| − | <td>TFL knock-out 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>11</td>
| |
| − | <td>TFL knock-out 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>12</td>
| |
| − | <td>TFL gRNA 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>13</td>
| |
| − | <td>TFL gRNA 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>14</td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Pick a single E. coli DH5α colony from the plates
| |
| − | that have been incubated overnight. The devices
| |
| − | are:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:TFL Knock-out:Luc:Tnos (4 samples)</li>
| |
| − | <li>U6-26:Ga20ox sgRNA:psgRNA (2 samples)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Inoculate a starter culture of 4 ml of LB medium with 4
| |
| − | μL of kanamycin in a 50 ml tube with the colony and
| |
| − | incubate it 16 hours at 37^C.<br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 17<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 17, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Transformation in DH5α <i>E. coli</i> with:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:Cas9:Tnos – U6:TFL sgRNA:psgRNA
| |
| − | (scaffold) (Ω1)</li>
| |
| − | <li>35s:Cas9:Tnos – U6:Ga20ox sgRNA:psgRNA
| |
| − | (scaffold) (Ω1)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Plating <i>E. coli</i> transformations in plates with LB
| |
| − | + agar + IPTG + XGal and incubate it overnight at
| |
| − | 37^C.<br></p>
| |
| − | <ul>
| |
| − | <li>Miniprep with E.Z.N.A ®. Plasmid Mini Kit I,
| |
| − | Q(capless) Spin of:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>U6-26:Ga20ox sgRNA:psgRNA</li>
| |
| − | <li>35s:5’ region:TFL Knock-out:Luc:Tnos
| |
| − | (3α1)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Digestion of the minipreps with EcoRI which have been
| |
| − | done before. Incubate 1 hour at 37^C. There is a total of
| |
| − | six minipreps. It has been followed the Miniprep digestion
| |
| − | protocol href.<br>
| |
| − | Run an electrophoresis gel of the digestion
| |
| − | products.<br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Lanes</th>
| |
| − | <th>Samples</th>
| |
| − | <th>Verification</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>1</td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>2</td>
| |
| − | <td>gRNA Ga20ox 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>3</td>
| |
| − | <td>gRNA Ga20ox 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>4</td>
| |
| − | <td>gRNA Ga20ox 3</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>5</td>
| |
| − | <td>gRNA Ga20ox 4</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>6</td>
| |
| − | <td>TFL knock-out 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>7</td>
| |
| − | <td>TFL knock-out 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>8</td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p>However, despite the fact that electrophoresis gel have
| |
| − | correctly run, we have decided to repeat the ligation
| |
| − | reactions. We have not get the expected results for a
| |
| − | gRNA.<br></p>
| |
| − | <ul>
| |
| − | <li>Ligation using Golden Braid assembly of the next
| |
| − | devices:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:5’ region:TFL target control
| |
| − | positive:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target control
| |
| − | positive:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:TFL target
| |
| − | consensus:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target
| |
| − | consensus:Luc:Tnos</li>
| |
| − | <li>U6-26:sgRNA TFL:psgRNA (scaffold)</li>
| |
| − | <li>U6-26:sgRNA Ga20ox:psgRNA (scaffold)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Reagent</th>
| |
| − | <th>Volume(μL)</th>
| |
| − | <th>Reagent</th>
| |
| − | <th>Volume(μL)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>TFL/Ga20ox gRNA</td>
| |
| − | <td>1</td>
| |
| − | <td>35s:5’region</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>U6-26</td>
| |
| − | <td>1</td>
| |
| − | <td>TFL/Ga20ox consensus TFL/Ga20ox
| |
| − | knock-out</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>psgRNA</td>
| |
| − | <td>1</td>
| |
| − | <td>luciferase</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>3α1</td>
| |
| − | <td>1</td>
| |
| − | <td>Tnos</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>BSA10X</td>
| |
| − | <td>1.2</td>
| |
| − | <td>3α1</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Ligase Buffer</td>
| |
| − | <td>1.2</td>
| |
| − | <td>BSA10X</td>
| |
| − | <td>1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>BsaI</td>
| |
| − | <td>1</td>
| |
| − | <td>Ligase Buffer</td>
| |
| − | <td>1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>T4 ligase</td>
| |
| − | <td>1</td>
| |
| − | <td>BsmbI</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>H<sub>2</sub>O milli-Q</td>
| |
| − | <td>3.6</td>
| |
| − | <td>T4 ligase</td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>H<sub>2</sub>O milli-Q</td>
| |
| − | <td>2.6</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p><br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 18<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 18, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Transformation in DH5α <i>E. coli</i> with ligation
| |
| − | products (consensus targets and knock-out targets of TFL
| |
| − | and Ga20ox as well as their gRNAs)<br>
| |
| − | Plating <i>E. coli</i> transformations in plates with LB +
| |
| − | agar + IPTG + XGal and incubate it overnight at
| |
| − | 37^C.<br></p>
| |
| − | <ul>
| |
| − | <li>Pick a single <i>Agrobacterium</i> C58 colony from
| |
| − | the plates that have been incubating since 16/06/2016.
| |
| − | The devices are:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:5’ region:TFL PCR:Luc:Tnos (3α1)</li>
| |
| − | <li>35s:5’ region:Ga20ox PCR:Luc:Tnos
| |
| − | (3α1)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Incubate it 48 hours at 28^C.<br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 19<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 19, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Pick transformed <i>E. coli</i> colony from the
| |
| − | incubated plates. The devices are:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:5’ region:TFL target control
| |
| − | positive:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target control
| |
| − | positive:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:TFL target
| |
| − | consensus:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target
| |
| − | consensus:Luc:Tnos</li>
| |
| − | <li>U6-26: sgRNA TFL:psgRNA (scaffold)</li>
| |
| − | <li>U6-26: sgRNA Ga20ox:psgRNA (scaffold)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Incubate it overnight at 37^C.<br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 20<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 20, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Miniprep with E.Z.N.A ®. Plasmid Mini Kit I,
| |
| − | Q(capless) Spin of:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:5’ region:TFL target
| |
| − | knock-out:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target control
| |
| − | knock-out:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:TFL target
| |
| − | consensus:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target
| |
| − | consensus:Luc:Tnos</li>
| |
| − | <li>U6-26:sgRNA TFL:psgRNA (scaffold)</li>
| |
| − | <li>U6-26:sgRNA Ga20ox:psgRNA (scaffold)</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Digestion of the minipreps with EcoRI which have been
| |
| − | done before. Incubate 1 hour at 37^C. There is a total of
| |
| − | six minipreps. It has been followed the Miniprep {digestion
| |
| − | protocol
| |
| − | path:https://2016.igem.org/Team:Valencia_UPV/Notebook/Protocol].<br>
| |
| − |
| |
| − | Run an electrophoresis gel of the digestion products. We
| |
| − | will keep the minipreps with “1”.<br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Lanes</th>
| |
| − | <th>Samples</th>
| |
| − | <th>Verification</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>1</td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>2</td>
| |
| − | <td>gRNA TFL 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>3</td>
| |
| − | <td>gRNA TFL 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>4</td>
| |
| − | <td>TFL consensus 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>5</td>
| |
| − | <td>TFL consensus 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>6</td>
| |
| − | <td>TFL knock-out 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>7</td>
| |
| − | <td>TFL knock-out 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>8</td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>9</td>
| |
| − | <td>gRNA Ga20ox 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>10</td>
| |
| − | <td>gRNA Ga20ox 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>11</td>
| |
| − | <td>Ga20ox consensus 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>12</td>
| |
| − | <td>Ga20ox consensus 2</td>
| |
| − | <td>-</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>13</td>
| |
| − | <td>Ga20ox knock-out 1</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>14</td>
| |
| − | <td>Ga20ox knock-out 2</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>15</td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Ligation using Golden Braid assembly of the next
| |
| − | devices. Is used the restriction enzyme BsmbI.</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:Cas 9:Tnos – U6-26:TFL
| |
| − | sgRNA:psgRNA</li>
| |
| − | <li>35s:Cas 9:Tnos – U6-26:Ga20ox
| |
| − | sgRNA:psgRNA</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 21<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 21, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Transformations in <i>E. coli</i> DH5α with:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>35s:Cas 9:Tnos – U6-26:TFL
| |
| − | sgRNA:psgRNA</li>
| |
| − | <li>35s:Cas 9:Tnos – U6-26:Ga20ox
| |
| − | sgRNA:psgRNA</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p>Incubate 2 hours at 37^C.<br>
| |
| − | Plating <i>E. coli</i> transformation explained before and
| |
| − | incubate it at 37^C overnight.<br>
| |
| − | #<i>Agrobacterium</i> C58 transformation with:<br></p>
| |
| − | <ul>
| |
| − | <li>#35s:5’ region:TFL target knock-out:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target control
| |
| − | knock-out:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:TFL target consensus:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target consensus:Luc:Tnos</li>
| |
| − | </ul>
| |
| − | <p>Incubate 48 hours at 28^C.<br>
| |
| − | <br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 22<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 22, 2016</span></li>
| |
| − | </ul>
| |
| − | <p>Sequencing reaction:<br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Reagent</th>
| |
| − | <th>Volume (μL)</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Primer in order to sequence</td>
| |
| − | <td>3</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Miniprep reaction</td>
| |
| − | <td>5</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>H<sub>2</sub>O milli-Q</td>
| |
| − | <td>6</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p><br></p>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>Sequence</th>
| |
| − | <th>Order</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>TFL gRNA</td>
| |
| − | <td>210.13.201</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>Ga20oxgRNA</td>
| |
| − | <td>210.13.202</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p>Ordered the necessary primers to sequence: TFL
| |
| − | consensus, TFL knockout, Ga20ox consensus and Ga20ox
| |
| − | knockout.<br>
| |
| − | <i>E. coli</i> transformations with 35s:Cas 9:Tnos –
| |
| − | U6-26:TFL sgRNA: psgRNA<br>
| |
| − | Plating the last device in plates with <i>Agrobacterium</i>
| |
| − | C58. Plates contain spec + IPTG + X-Gal. Incubate 2 days at
| |
| − | 28^C.<br>
| |
| − | Pick a single <i>E. coli</i> DH5α (35s:Cas 9:Tnos –
| |
| − | U6-26:TFL sgRNA:psgRNA and 35s:Cas 9:Tnos – U6-26:Ga20ox
| |
| − | sgRNA:psgRNA) colony from the plates that has been
| |
| − | incubated overnight. Inoculate a starter culture of 4 ml of
| |
| − | LB medium with 4 μL of spectinomycin in a 50 ml tube with
| |
| − | the colony and incubate it overnight at 37^C with
| |
| − | shaking.<br>
| |
| − | <br></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 23<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 23, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>#Minipreps (4 samples for each device) with E.Z.N.A
| |
| − | ®. Plasmid Mini Kit I, Q(capless) Spin of:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>#35s:Cas 9:Tnos – U6-26:TFL
| |
| − | sgRNA:psgRNA</li>
| |
| − | <li>35s:Cas 9:Tnos – U6-26:Ga20ox
| |
| − | sgRNA:psgRNA</li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </li>
| |
| − | </ul>Digestion of minipreps with BamHI following digestion
| |
| − | protocol href. Incubate 1 hour at 37^C.<br>
| |
| − | Run an electrophoresis gel of the digestion products. We
| |
| − | will keep the minipreps with the sample number 4 for TFL
| |
| − | sgRNA and the sample number 2 for Ga20ox sgRNA.<br>
| |
| − | Pick a single <i>Agrobacterium</i> C58 (35s:Cas 9:Tnos –
| |
| − | U6-26:TFL sgRNA:psgRNA and 35s:Cas 9:Tnos – U6-26:Ga20ox
| |
| − | sgRNA:psgRNA ) colony from the plates that has been
| |
| − | incubated overnight. Inoculate a starter culture of 5 ml of
| |
| − | LB medium with 5 μL of spectinomycin and kanamycin in a
| |
| − | falcon tube with the colony and incubate it overnight at
| |
| − | 28^C with shaking.<br>
| |
| − | #<i>Agrobacterium</i> C58 transformations with:<br>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>#35s:Cas 9:Tnos – U6-26:TFL sgRNA:psgRNA</li>
| |
| − | <li style="list-style: none">35s:Cas 9:Tnos –
| |
| − | U6-26:Ga20ox sgRNA:psgRNA<br></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 24<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 24, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>#Store the next cultures at -80^C:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>#35s:5’ region in pUPD2 (DH5α) number
| |
| − | 1</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>#SAGTI: luciferase in pUPD2 (DH5α)
| |
| − | number 2</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 25<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 25, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>Pick a single <i>Agrobacterium</i> C58 (35s:Cas
| |
| − | 9:Tnos – U6-26:TFL sgRNA:psgRNA in 3Ω1 and 35s:Cas
| |
| − | 9:Tnos – U6-26:Ga20ox sgRNA:psgRNA in 3Ω1 ) colony from
| |
| − | the plates that has been incubated overnight. Inoculate
| |
| − | a starter culture of 5 ml of LB medium with 5 μL of
| |
| − | spectinomycin and kanamycin in a falcon tube with the
| |
| − | colony and incubate it overnight at 28^C with
| |
| − | shaking.</li>
| |
| − | <li>Incubate it 48 hours at 28^C</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>inipreps of <i>Agrobacterium</i> with
| |
| − | E.Z.N.A ®. Plasmid Mini Kit I, Q(capless) Spin
| |
| − | of:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>5s:5’ region:TFL target control
| |
| − | positive:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target control
| |
| − | positive:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:TFL target
| |
| − | consensus:Luc:Tnos</li>
| |
| − | <li>35s:5’ region:Ga20ox target
| |
| − | consensus:Luc:Tnos</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | <li>Digestion of minipreps with the restriction enzyme
| |
| − | EcoRI. Incubate 1 hour at 37^C.</li>
| |
| − | <li>Run an electrophoresis gel of the digestion
| |
| − | products.</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class=
| |
| − | "table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>
| |
| − | <ul>
| |
| − | <li>Lanes</li>
| |
| − | </ul>
| |
| − | </th>
| |
| − | <th>Samples</th>
| |
| − | <th>Verification</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>1</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>2</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Target Ga20ox consensus</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>3</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Target Ga20ox Knock-out</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>4</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Target TFL consensus</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>5</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Target TFL knock-out</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>6</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p></p>
| |
| − | </li>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 26<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 26, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>inipreps with E.Z.N.A ®. Plasmid Mini Kit I,
| |
| − | Q(capless) Spin of:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>5s:5’ region:TFL PCR:Luc:Tnos in 3α1
| |
| − | plasmid from C58 <i>Agrobacterium</i></li>
| |
| − | <li>35s:5’ region:Ga20ox PCR:Luc:Tnos in 3α1
| |
| − | plasmid from C58 <i>Agrobacterium</i></li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li>Digestion of minipreps with the restriction enzyme
| |
| − | EcoRI. Incubate 1 hour at 37^C.</li>
| |
| − | <li>Run an electrophoresis gel of the digestion
| |
| − | products:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <table class=
| |
| − | "table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>
| |
| − | <ul>
| |
| − | <li>Lanes</li>
| |
| − | </ul>
| |
| − | </th>
| |
| − | <th>Samples</th>
| |
| − | <th>Verification</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>1</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | <td>correct</td>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>2</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35s:5’ region:PCR Ga20ox
| |
| − | :Luc:Tnos</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>3</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35s:5’ region:PCR TFL
| |
| − | Clemunules:Luc:Tnos</td>
| |
| − | <td>correct</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>6</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | <td>correct</td>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <p></p>
| |
| − | </li>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 26<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July 26, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>equencing products have arrived:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>6-26:Ga20ox gRNA:psgRNA in α1 -
| |
| − | correct</li>
| |
| − | <li>U6-26:TFL gRNA:psgRNA in α1 - correct</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>inipreps with E.Z.N.A ®.® Plasmid Mini Kit I,
| |
| − | Q(capless) Spin of:</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <ul>
| |
| − | <li>5s:5’ region: Ga20ox PCR : Luc : Tnos in
| |
| − | α1</li>
| |
| − | <li>35s:5’ region: TFL PCR : Luc : Tnos in
| |
| − | α1</li>
| |
| − | </ul>
| |
| − | </li>
| |
| − | </ul>
| |
| − | <ul>
| |
| − | <li>Digestion of minipreps with the restriction enzyme
| |
| − | EcoRI. Incubate 1 hour at 37^.</li>
| |
| − | <li>Run electrophoresis gel of the digestion
| |
| − | products.</li>
| |
| − | <li style="list-style: none; display: inline">
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 27<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July
| |
| − | 27, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 28<span>Jul</span>
| |
| − | <div class="timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li><span class="font-lato">July
| |
| − | 28, 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <div class="table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <div class="blog-post-item">
| |
| − | <div class=
| |
| − | "timeline-entry rounded">
| |
| − | 29<span>Jul</span>
| |
| − | <div class=
| |
| − | "timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class=
| |
| − | "blog-post-info list-inline">
| |
| − | <li><span class=
| |
| − | "font-lato">July 29,
| |
| − | 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class=
| |
| − | "timeline-entry rounded">
| |
| − | 01<span>Aug</span>
| |
| − | <div class=
| |
| − | "timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class=
| |
| − | "blog-post-info list-inline">
| |
| − | <li><span class=
| |
| − | "font-lato">August 01,
| |
| − | 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class=
| |
| − | "timeline-entry rounded">
| |
| − | 02<span>Aug</span>
| |
| − | <div class=
| |
| − | "timeline-vline"></div>
| |
| − | </div>
| |
| − | <ul class=
| |
| − | "blog-post-info list-inline">
| |
| − | <li><span class=
| |
| − | "font-lato">August 02,
| |
| − | 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class="table-responsive"
| |
| − | style="width:55%;overflow:inherit">
| |
| − | <div class="table-responsive"
| |
| − | style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <div class=
| |
| − | "blog-post-item">
| |
| − | <div class=
| |
| − | "timeline-entry rounded">
| |
| − | 03<span>Aug</span>
| |
| − | <div class=
| |
| − | "timeline-vline">
| |
| − | </div>
| |
| − | </div>
| |
| − | <ul class=
| |
| − | "blog-post-info list-inline">
| |
| − | <li><span class=
| |
| − | "font-lato">August 03,
| |
| − | 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class=
| |
| − | "table-responsive" style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <div class=
| |
| − | "table-responsive"
| |
| − | style="width:55%;overflow:inherit">
| |
| − | <div class=
| |
| − | "table-responsive"
| |
| − | style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <div class=
| |
| − | "table-responsive"
| |
| − | style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <div class=
| |
| − | "blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 04<span>Aug</span>
| |
| − | <div class="timeline-vline">
| |
| − | </div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li>
| |
| − | <span class="font-lato">
| |
| − | August
| |
| − | 04,
| |
| − | 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class=
| |
| − | "blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 05<span>Aug</span>
| |
| − | <div class="timeline-vline">
| |
| − | </div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li>
| |
| − | <span class="font-lato">
| |
| − | August
| |
| − | 05,
| |
| − | 2016</span></li>
| |
| − | </ul>
| |
| − | <p></p>
| |
| − | </div>
| |
| − | <div class=
| |
| − | "table-responsive"
| |
| − | style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <div class="table-responsive"
| |
| − | style=
| |
| − | "width:55%;overflow:inherit">
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 08<span>Aug</span>
| |
| − | <div class="timeline-vline">
| |
| − | </div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li>
| |
| − | <span class="font-lato">
| |
| − | August
| |
| − | 08,
| |
| − | 2016</span></li>
| |
| − | </ul>
| |
| − | <p>
| |
| − | </p>
| |
| − | </div>
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 09<span>Aug</span>
| |
| − | <div class="timeline-vline">
| |
| − | </div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li>
| |
| − | <span class="font-lato">
| |
| − | August
| |
| − | 09,
| |
| − | 2016</span></li>
| |
| − | </ul>
| |
| − | <p>
| |
| − | </p>
| |
| − | </div>
| |
| − | <div class="table-responsive"
| |
| − | style="width:55%;overflow:inherit">
| |
| − | <div class="table-responsive"
| |
| − | style="width:55%;overflow:inherit">
| |
| − | <div class="table-responsive"
| |
| − | style="width:55%;overflow:inherit">
| |
| − | <div class="table-responsive"
| |
| − | style="width:55%;overflow:inherit">
| |
| − | <div class="table-responsive"
| |
| − | style="width:55%;overflow:inherit">
| |
| − | <div class="table-responsive"
| |
| − | style="width:55%;overflow:inherit">
| |
| − | <div class="blog-post-item">
| |
| − | <div class="timeline-entry rounded">
| |
| − | 10<span>Aug</span>
| |
| − | <div class="timeline-vline">
| |
| − | </div>
| |
| − | </div>
| |
| − | <ul class="blog-post-info list-inline">
| |
| − | <li>
| |
| − | <span class="font-lato">
| |
| − | August
| |
| − | 10,
| |
| − | 2016</span></li>
| |
| − | </ul>
| |
| − | <p>
| |
| − | </p>
| |
| − | </div>
| |
| − | <div class="table-responsive"
| |
| − | style="width:55%;overflow:inherit">
| |
| − | <div class="table-responsive"
| |
| − | style="width:55%;overflow:inherit">
| |
| − | <table class="table table-bordered table-striped">
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | devices</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | Volume
| |
| − | (μL)</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | gTS
| |
| − | TFL/
| |
| − | Ga20ox-
| |
| − | Renilla</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Cas9
| |
| − | -
| |
| − | gRNA</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 3
| |
| − | α
| |
| − | 1
| |
| − | plasmid</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Bsa
| |
| − | 10x</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Buffer
| |
| − | ligase</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Bsa
| |
| − | I</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | T4
| |
| − | ligase</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | H20
| |
| − | milliQ</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 4.6</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Run
| |
| − | electrophoresis
| |
| − | gel
| |
| − | of:
| |
| − | 35s:
| |
| − | 5’
| |
| − | region:
| |
| − | TFL/Ga20oxPCR:
| |
| − | Luc:
| |
| − | Tnos
| |
| − | –
| |
| − | 35s:
| |
| − | Renilla:
| |
| − | Tnos.
| |
| − | We
| |
| − | keep
| |
| − | the
| |
| − | Miniprep
| |
| − | number
| |
| − | 1.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Refresh
| |
| − | the
| |
| − | cultures
| |
| − | of
| |
| − | TFL
| |
| − | PCR
| |
| − | (pUPD2)
| |
| − | and
| |
| − | Ga20oxPCR
| |
| − | (pUPD2)
| |
| − | because
| |
| − | we
| |
| − | suspect
| |
| − | that
| |
| − | these
| |
| − | cultures
| |
| − | are
| |
| − | the
| |
| − | correct
| |
| − | ones
| |
| − | but
| |
| − | we
| |
| − | are
| |
| − | not
| |
| − | sure
| |
| − | so
| |
| − | we
| |
| − | want
| |
| − | to
| |
| − | check
| |
| − | them.
| |
| − | The
| |
| − | cultures
| |
| − | that
| |
| − | we
| |
| − | use
| |
| − | to
| |
| − | refresh
| |
| − | were
| |
| − | made
| |
| − | on
| |
| − | 12/07/2016.
| |
| − | It
| |
| − | is
| |
| − | important
| |
| − | to
| |
| − | remember
| |
| − | that
| |
| − | we
| |
| − | need
| |
| − | them
| |
| − | to
| |
| − | assemble
| |
| − | the
| |
| − | gTS
| |
| − | with
| |
| − | the
| |
| − | new
| |
| − | linkers.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Miniprep
| |
| − | with
| |
| − | E.Z.N.A
| |
| − | ®.®
| |
| − | Plasmid
| |
| − | Mini
| |
| − | Kit
| |
| − | I,
| |
| − | Q(capless)
| |
| − | Spin
| |
| − | of
| |
| − | TFL
| |
| − | /
| |
| − | Ga20oxPCR
| |
| − | in
| |
| − | pUPD2</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Digestion
| |
| − | of
| |
| − | minipreps
| |
| − | with
| |
| − | NotI.
| |
| − | Incubate
| |
| − | 1
| |
| − | hour
| |
| − | at
| |
| − | 37^</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Run
| |
| − | electrophoresis
| |
| − | gel
| |
| − | of
| |
| − | Miniprep
| |
| − | products.
| |
| − | We
| |
| − | have
| |
| − | made
| |
| − | a
| |
| − | small
| |
| − | gel
| |
| − | so
| |
| − | we
| |
| − | have
| |
| − | mix
| |
| − | 0.45
| |
| − | g
| |
| − | of
| |
| − | Agarose
| |
| − | with
| |
| − | 45
| |
| − | mL
| |
| − | of
| |
| − | TAE
| |
| − | 1X.
| |
| − | Voltage
| |
| − | used
| |
| − | is
| |
| − | 100V.
| |
| − | Both
| |
| − | samples
| |
| − | are
| |
| − | correct.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Pick
| |
| − | a
| |
| − | single
| |
| − | E.
| |
| − | coli
| |
| − | DH5α
| |
| − | (promoter35s:
| |
| − | 5’
| |
| − | region:
| |
| − | TFL/Ga20oxcons:
| |
| − | Tnos
| |
| − | -
| |
| − | promoter35s:
| |
| − | Renilla:
| |
| − | Tnos
| |
| − | -
| |
| − | 35s:
| |
| − | Cas9:
| |
| − | Tnos
| |
| − | -
| |
| − | U6:
| |
| − | TFL/Ga20oxgRNA:
| |
| − | psgRNA)
| |
| − | colony
| |
| − | from
| |
| − | the
| |
| − | plate
| |
| − | that
| |
| − | has
| |
| − | been
| |
| − | incubated
| |
| − | overnight.
| |
| − | Inoculate
| |
| − | a
| |
| − | starter
| |
| − | culture
| |
| − | of
| |
| − | 4
| |
| − | ml
| |
| − | of
| |
| − | LB
| |
| − | medium
| |
| − | with
| |
| − | 4
| |
| − | μL
| |
| − | of
| |
| − | chloramphenicol
| |
| − | in
| |
| − | a
| |
| − | 50
| |
| − | ml
| |
| − | tube
| |
| − | with
| |
| − | the
| |
| − | colony
| |
| − | and
| |
| − | incubate
| |
| − | it
| |
| − | overnight
| |
| − | at
| |
| − | 37^
| |
| − | with
| |
| − | shaking.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | We
| |
| − | throw
| |
| − | out
| |
| − | from
| |
| − | Golden
| |
| − | Braid
| |
| − | collection
| |
| − | the
| |
| − | glycerinate
| |
| − | number
| |
| − | 1107
| |
| − | (Cas9
| |
| − | -
| |
| − | XT1gRNA)
| |
| − | and
| |
| − | 0549
| |
| − | (35s:
| |
| − | TEV:
| |
| − | Tnos)</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Ligation
| |
| − | reaction
| |
| − | of
| |
| − | 35s:
| |
| − | 5’
| |
| − | region:
| |
| − | TFL/
| |
| − | Ga20:
| |
| − | Tnos
| |
| − | -
| |
| − | 35s:
| |
| − | Renilla:
| |
| − | Tnos
| |
| − | +
| |
| − | 35s:
| |
| − | Cas9:
| |
| − | Tnos
| |
| − | -
| |
| − | U6:
| |
| − | TFL/Ga20oxgRNA:
| |
| − | psgRNA</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 5</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:Ga20cons:Luc:Tnos</td>
| |
| − | <td>
| |
| − | 3α1</td>
| |
| − | <td>
| |
| − | KAN</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 6</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:Ga20KO:Luc:Tnos</td>
| |
| − | <td>
| |
| − | 3α1</td>
| |
| − | <td>
| |
| − | KAN</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 7</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:TFLcons:Luc:Tnos</td>
| |
| − | <td>
| |
| − | 3α1</td>
| |
| − | <td>
| |
| − | KAN</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 8</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:TFLKO:Luc:Tnos</td>
| |
| − | <td>
| |
| − | 3α1</td>
| |
| − | <td>
| |
| − | KAN</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 9</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:Ga20cons:Luc:Tnos-35s:Ren:Tnos</td>
| |
| − | <td>
| |
| − | 3Ω2</td>
| |
| − | <td>
| |
| − | SPEC</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 10</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:Ga20KO:Luc:Tnos-35s:Ren:Tnos</td>
| |
| − | <td>
| |
| − | 3Ω2</td>
| |
| − | <td>
| |
| − | SPEC</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 11</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:Ga20PCR:Luc:Tnos-35s:Ren:Tnos</td>
| |
| − | <td>
| |
| − | 3Ω2</td>
| |
| − | <td>
| |
| − | SPEC</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 12</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:TFLcons:Luc:Tnos-35s:Ren:Tnos</td>
| |
| − | <td>
| |
| − | 3Ω2</td>
| |
| − | <td>
| |
| − | SPEC</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 13</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:TFLKO:Luc:Tnos-35s:Ren:Tnos</td>
| |
| − | <td>
| |
| − | 3Ω2</td>
| |
| − | <td>
| |
| − | SPEC</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 14</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:TFLPCR:Luc:Tnos-35s:Ren:Tnos</td>
| |
| − | <td>
| |
| − | 3Ω2</td>
| |
| − | <td>
| |
| − | SPEC</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 15</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | U6:Ga20sgRNA:psgRNA</td>
| |
| − | <td>
| |
| − | 3α1</td>
| |
| − | <td>
| |
| − | KAN</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 16</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | U6:TFLsgRNA:psgRNA</td>
| |
| − | <td>
| |
| − | 3α1</td>
| |
| − | <td>
| |
| − | KAN</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 17</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:Cas9:Tnos-U6:Ga20gRNA:psgRNA</td>
| |
| − | <td>
| |
| − | 3Ω1</td>
| |
| − | <td>
| |
| − | SPEC</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 18</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:Cas9:Tnos-U6:TFLgRNA:psgRNA</td>
| |
| − | <td>
| |
| − | 3Ω1</td>
| |
| − | <td>
| |
| − | SPEC</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 19</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | Ga20PCR</td>
| |
| − | <td>
| |
| − | pUPD2</td>
| |
| − | <td>
| |
| − | CAM</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 20</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | TFLPCR</td>
| |
| − | <td>
| |
| − | pUPD2</td>
| |
| − | <td>
| |
| − | CAM</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Transform
| |
| − | the
| |
| − | products
| |
| − | of
| |
| − | ligation
| |
| − | in
| |
| − | DH5
| |
| − | α.
| |
| − | Incubate
| |
| − | it
| |
| − | at
| |
| − | 37^
| |
| − | during
| |
| − | 2
| |
| − | hours.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | We
| |
| − | store
| |
| − | at
| |
| − | -80^:</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Promoter35s</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | pNos</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | TFLKO</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | Ga20KO</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | TFLPCRXT1</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | Ga20PCRXT1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | TFLPCROK</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | Ga20PCROK</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | TFLconsOK</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | Ga20consOK</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | U6:Ga20oxgRNA:
| |
| − | psgRNA
| |
| − | -
| |
| − | 35s:
| |
| − | Cas9:
| |
| − | Tnos
| |
| − | -
| |
| − | Miniprep
| |
| − | number
| |
| − | 2
| |
| − | was
| |
| − | empty.
| |
| − | We
| |
| − | have
| |
| − | resuspended
| |
| − | it
| |
| − | with
| |
| − | 40
| |
| − | μL
| |
| − | of
| |
| − | H20
| |
| − | milliQ
| |
| − | and
| |
| − | we
| |
| − | have
| |
| − | checked
| |
| − | the
| |
| − | DNA
| |
| − | concentration
| |
| − | with
| |
| − | the
| |
| − | Nanodrop.
| |
| − | The
| |
| − | results
| |
| − | show
| |
| − | us
| |
| − | that
| |
| − | the
| |
| − | DNA
| |
| − | concentration
| |
| − | in
| |
| − | the
| |
| − | Eppendorf
| |
| − | was
| |
| − | 140
| |
| − | ng/
| |
| − | μL
| |
| − | so
| |
| − | we
| |
| − | have
| |
| − | used
| |
| − | it.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Miniprep
| |
| − | with
| |
| − | E.Z.N.A
| |
| − | ®.®
| |
| − | Plasmid
| |
| − | Mini
| |
| − | Kit
| |
| − | I,
| |
| − | Q(capless)
| |
| − | Spin
| |
| − | of
| |
| − | 35s:
| |
| − | 5’
| |
| − | region:
| |
| − | TFL/Ga20oxPCR:
| |
| − | Luc:
| |
| − | Tnos
| |
| − | –
| |
| − | 35s:
| |
| − | Renilla:
| |
| − | Tnos</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Transform
| |
| − | in
| |
| − | C58
| |
| − | <i>
| |
| − | Agrobacterium</i>
| |
| − | 35s:
| |
| − | Renilla
| |
| − | :
| |
| − | Tnos
| |
| − | (3
| |
| − | α2)</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Plating
| |
| − | promoter35s:
| |
| − | Renilla:
| |
| − | Tnos
| |
| − | (3α2)</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Luciferase
| |
| − | assay</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | devices</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | Volume
| |
| − | (μL)</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 35s
| |
| − | :
| |
| − | 5’
| |
| − | region:
| |
| − | Ga20/TFL
| |
| − | KO:
| |
| − | Luc:
| |
| − | Tnos
| |
| − | -
| |
| − | 35s:
| |
| − | Renilla:
| |
| − | Tnos
| |
| − | -
| |
| − | 35s:
| |
| − | hCas9:
| |
| − | Tnos:
| |
| − | U6:
| |
| − | Ga20/TFL
| |
| − | gRNA:
| |
| − | psgRNA</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 35s
| |
| − | :
| |
| − | 5’
| |
| − | region:
| |
| − | Ga20/TFL
| |
| − | cons:
| |
| − | Luc:
| |
| − | Tnos
| |
| − | -
| |
| − | 35s:
| |
| − | Renilla:
| |
| − | Tnos
| |
| − | -
| |
| − | 35s:
| |
| − | hCas9:
| |
| − | Tnos:
| |
| − | U6:
| |
| − | Ga20/TFL
| |
| − | gRNA:
| |
| − | psgRNA</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 3
| |
| − | α
| |
| − | 1
| |
| − | plasmid</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Bsa
| |
| − | 10X</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Buffer
| |
| − | ligase</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Bsa
| |
| − | I</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | T4
| |
| − | ligase</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | H20
| |
| − | milliQ</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 4.6</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>
| |
| − | nfiltration
| |
| − | cultures:</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 5s:
| |
| − | Luc:
| |
| − | Tnos</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | Laboratory
| |
| − | controls</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | Pnos:
| |
| − | Luc:
| |
| − | Tnos</td>
| |
| − | <td>
| |
| − | Laboratory
| |
| − | controls</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | gTS
| |
| − | TFL
| |
| − | KO
| |
| − | +
| |
| − | Cas9
| |
| − | -
| |
| − | XT1:XT2</td>
| |
| − | <td>
| |
| − | Positive
| |
| − | controls</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | gTSGa20oxKO
| |
| − | +
| |
| − | Cas9
| |
| − | -
| |
| − | XT1:XT2</td>
| |
| − | <td>
| |
| − | Positive
| |
| − | controls</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | gTS
| |
| − | TFL
| |
| − | PCR
| |
| − | +
| |
| − | Cas9
| |
| − | -
| |
| − | XT1:XT2</td>
| |
| − | <td>
| |
| − | Negative
| |
| − | controls</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | gTS
| |
| − | Ga20oxPCR
| |
| − | +
| |
| − | Cas9
| |
| − | -
| |
| − | XT1:XT2</td>
| |
| − | <td>
| |
| − | Negative
| |
| − | controls</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | gTS
| |
| − | TFL
| |
| − | PCR
| |
| − | +
| |
| − | Cas9
| |
| − | -
| |
| − | TFLgRNA</td>
| |
| − | <td>
| |
| − | Samples</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | gTS
| |
| − | TFL
| |
| − | cons
| |
| − | +
| |
| − | Cas9
| |
| − | -
| |
| − | TFLgRNA</td>
| |
| − | <td>
| |
| − | Samples</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | gTS
| |
| − | Ga20oxPCR
| |
| − | +
| |
| − | Cas9
| |
| − | -
| |
| − | Ga20gRNA</td>
| |
| − | <td>
| |
| − | Samples</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | gTS
| |
| − | Ga20oxcons
| |
| − | +
| |
| − | Cas9
| |
| − | -
| |
| − | Ga20gRNA</td>
| |
| − | <td>
| |
| − | Samples</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Transplant
| |
| − | agroinfiltrated
| |
| − | plants
| |
| − | (<i>Nicotiana
| |
| − | benthamiana</i>)</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Pick
| |
| − | up
| |
| − | 6
| |
| − | disks
| |
| − | per
| |
| − | each
| |
| − | plant
| |
| − | in
| |
| − | order
| |
| − | to
| |
| − | carry
| |
| − | out
| |
| − | the
| |
| − | luciferase
| |
| − | assay
| |
| − | on
| |
| − | 09/08</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Ligate
| |
| − | reaction
| |
| − | of:</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | device</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | Volume
| |
| − | of
| |
| − | culture
| |
| − | (mL)</td>
| |
| − | <td>
| |
| − | Volume
| |
| − | of
| |
| − | Agroinfiltration
| |
| − | solution
| |
| − | (mL)</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Cas9
| |
| − | -
| |
| − | TFL
| |
| − | gRNA</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 0.7</td>
| |
| − | <td>
| |
| − | 9.3</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Cas
| |
| − | 9
| |
| − | -
| |
| − | Ga20oxgRNA</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 0.7</td>
| |
| − | <td>
| |
| − | 9.3</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Cas
| |
| − | 9
| |
| − | -
| |
| − | XT1:
| |
| − | XT2</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 0.59</td>
| |
| − | <td>
| |
| − | 9.41</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | TFL
| |
| − | KO</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 0.67</td>
| |
| − | <td>
| |
| − | 9.33</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | TFL
| |
| − | PCR</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 0.73</td>
| |
| − | <td>
| |
| − | 9.27</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Ga20oxconsensus</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 0.71</td>
| |
| − | <td>
| |
| − | 9.28</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Pnos</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 0.68</td>
| |
| − | <td>
| |
| − | 9.32</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 35s
| |
| − | :
| |
| − | Luc</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 0.78</td>
| |
| − | <td>
| |
| − | 9.22</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Ga20oxPCR</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 0.74</td>
| |
| − | <td>
| |
| − | 9.26</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | TFL
| |
| − | consensus</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 0.71</td>
| |
| − | <td>
| |
| − | 9.29</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Ga20ox-
| |
| − | KO</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 0.73</td>
| |
| − | <td>
| |
| − | 9.27</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class="table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Miniprep
| |
| − | with
| |
| − | E.Z.N.A
| |
| − | ®.®
| |
| − | Plasmid
| |
| − | Mini
| |
| − | Kit
| |
| − | I,
| |
| − | Q(capless)
| |
| − | Spin
| |
| − | of
| |
| − | TMV
| |
| − | CDS</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Transform
| |
| − | DH5
| |
| − | α
| |
| − | <i>
| |
| − | E.
| |
| − | coli</i>
| |
| − | with
| |
| − | the
| |
| − | device
| |
| − | 35s
| |
| − | :
| |
| − | 5’
| |
| − | region:
| |
| − | PCR
| |
| − | PCR:
| |
| − | Luc:
| |
| − | Tnos
| |
| − | -
| |
| − | 35s:
| |
| − | Renilla:
| |
| − | Tnos.
| |
| − | After
| |
| − | incubating
| |
| − | 2
| |
| − | hours,
| |
| − | it
| |
| − | must
| |
| − | be
| |
| − | plated.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Refresh
| |
| − | C58
| |
| − | <i>
| |
| − | <i>
| |
| − | Agrobacterium</i>
| |
| − | tumefaciens</i>
| |
| − | cultures
| |
| − | with
| |
| − | the
| |
| − | aim
| |
| − | of
| |
| − | infiltrating
| |
| − | in
| |
| − | N.benthamiana
| |
| − | on
| |
| − | 05/08.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Pick
| |
| − | a
| |
| − | single
| |
| − | E.
| |
| − | coli
| |
| − | DH5α
| |
| − | (35S
| |
| − | :
| |
| − | 5’
| |
| − | region:
| |
| − | KO/cons
| |
| − | target:
| |
| − | Luc:Tnos
| |
| − | -
| |
| − | promoter35s:
| |
| − | renilla:
| |
| − | Tnos)
| |
| − | colony
| |
| − | from
| |
| − | the
| |
| − | plate
| |
| − | that
| |
| − | has
| |
| − | been
| |
| − | incubated
| |
| − | overnight.
| |
| − | Inoculate
| |
| − | a
| |
| − | starter
| |
| − | culture
| |
| − | of
| |
| − | 4
| |
| − | ml
| |
| − | of
| |
| − | LB
| |
| − | medium
| |
| − | with
| |
| − | 4
| |
| − | μL
| |
| − | of
| |
| − | spectinomycin
| |
| − | in
| |
| − | a
| |
| − | 50
| |
| − | ml
| |
| − | tube
| |
| − | with
| |
| − | the
| |
| − | colony
| |
| − | and
| |
| − | incubate
| |
| − | it
| |
| − | overnight
| |
| − | at
| |
| − | 37^
| |
| − | with
| |
| − | shaking.Sequencing
| |
| − | TMV
| |
| − | empty
| |
| − | vector
| |
| − | with
| |
| − | the
| |
| − | primer
| |
| − | D09OCT01
| |
| − | (10
| |
| − | uM).
| |
| − | 5μL
| |
| − | of
| |
| − | Miniprep
| |
| − | and
| |
| − | 9μL
| |
| − | of
| |
| − | primer
| |
| − | (dilution
| |
| − | 1:3).
| |
| − | Sequencing
| |
| − | code
| |
| − | of
| |
| − | 210.13.300
| |
| − | and
| |
| − | 210.13.301.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Pick
| |
| − | a
| |
| − | single
| |
| − | E.
| |
| − | coli
| |
| − | DH5α
| |
| − | (promoter35s:
| |
| − | 5’
| |
| − | region:
| |
| − | TFL/Ga20oxPCR:
| |
| − | Luc:
| |
| − | Tnos
| |
| − | -
| |
| − | promoter35s:
| |
| − | Renilla:
| |
| − | Tnos
| |
| − | )
| |
| − | colony
| |
| − | from
| |
| − | the
| |
| − | plate
| |
| − | that
| |
| − | has
| |
| − | been
| |
| − | incubated
| |
| − | overnight.
| |
| − | Inoculate
| |
| − | a
| |
| − | starter
| |
| − | culture
| |
| − | of
| |
| − | 4
| |
| − | ml
| |
| − | of
| |
| − | LB
| |
| − | medium
| |
| − | with
| |
| − | 4
| |
| − | μL
| |
| − | of
| |
| − | kanamycin
| |
| − | in
| |
| − | a
| |
| − | 50
| |
| − | ml
| |
| − | tube
| |
| − | with
| |
| − | the
| |
| − | colony
| |
| − | and
| |
| − | incubate
| |
| − | it
| |
| − | overnight
| |
| − | at
| |
| − | 37^
| |
| − | with
| |
| − | shaking.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>
| |
| − | iniprep
| |
| − | with
| |
| − | E.Z.N.A
| |
| − | ®.®
| |
| − | Plasmid
| |
| − | Mini
| |
| − | Kit
| |
| − | I,
| |
| − | Q(capless)
| |
| − | Spin
| |
| − | of:</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 5s:
| |
| − | 5’
| |
| − | region:
| |
| − | TFL
| |
| − | PCR:
| |
| − | Luc:
| |
| − | Tnos
| |
| − | -
| |
| − | promoter35s:
| |
| − | Renilla:
| |
| − | Tnos</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:
| |
| − | 5’
| |
| − | region:
| |
| − | Ga20oxPCR:
| |
| − | Luc:
| |
| − | Tnos
| |
| − | -
| |
| − | promoter35s:
| |
| − | Renilla:
| |
| − | Tnos</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:
| |
| − | 5’
| |
| − | region:
| |
| − | Ga20oxconsensus:
| |
| − | Luc:
| |
| − | Tnos
| |
| − | -
| |
| − | promoter35s:
| |
| − | Renilla:
| |
| − | Tnos</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:
| |
| − | 5’
| |
| − | region:
| |
| − | Ga20oxKO:
| |
| − | Luc:
| |
| − | Tnos
| |
| − | -
| |
| − | promoter35s:
| |
| − | Renilla:
| |
| − | Tnos</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Digestion
| |
| − | of
| |
| − | minipreps
| |
| − | with
| |
| − | EcoRV.
| |
| − | Incubate
| |
| − | 1
| |
| − | hour
| |
| − | at
| |
| − | 37^</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Run
| |
| − | electrophoresis
| |
| − | gel
| |
| − | of
| |
| − | the
| |
| − | digestion
| |
| − | products:
| |
| − | TFLK01,
| |
| − | TFLK02,
| |
| − | TFLcons01,
| |
| − | TFLcons02,
| |
| − | GAK01,
| |
| − | GAK02,
| |
| − | GAcons01,
| |
| − | GAcons02.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Prepare
| |
| − | the
| |
| − | Agroinfiltration
| |
| − | with
| |
| − | the
| |
| − | correct
| |
| − | protocol.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Centrifuge
| |
| − | the
| |
| − | cultures
| |
| − | at
| |
| − | 3000
| |
| − | rpm
| |
| − | during
| |
| − | 15
| |
| − | minutes.
| |
| − | We
| |
| − | discard
| |
| − | the
| |
| − | supernatant
| |
| − | and
| |
| − | it
| |
| − | is
| |
| − | necessary
| |
| − | to
| |
| − | resuspend
| |
| − | in
| |
| − | 5mL
| |
| − | of
| |
| − | Agroinfiltration
| |
| − | solution.
| |
| − | Let
| |
| − | shaking
| |
| − | it
| |
| − | a
| |
| − | RT
| |
| − | during
| |
| − | 2
| |
| − | hours.
| |
| − | OD’s
| |
| − | measurement</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class=
| |
| − | "table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Reagent</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | Volume(μL)</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 35s:5’region:
| |
| − | TFL/Ga20oxPCR:
| |
| − | Luc:
| |
| − | Tnos</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Promoter35s:
| |
| − | Renilla:
| |
| − | Tnos</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | pUPD2</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | BSA10X</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Ligase
| |
| − | Buffer</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | BsmbI</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | T4
| |
| − | ligase</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | H<sub>2</sub>O
| |
| − | milli-Q</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 4.6</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class=
| |
| − | "table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Ligate
| |
| − | reactions
| |
| − | of
| |
| − | TFL
| |
| − | PCR
| |
| − | and
| |
| − | Ga20ox
| |
| − | PCR
| |
| − | with
| |
| − | Renilla</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class=
| |
| − | "table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 1</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35S:5’</td>
| |
| − | <td>pUPD2</td>
| |
| − | <td>CAM</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 2</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | SAGTI:Luc</td>
| |
| − | <td>pUPD2</td>
| |
| − | <td>CAM</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 3</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:TFLPCR:Luc:Tnos</td>
| |
| − | <td>3α1</td>
| |
| − | <td>KAN</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 4</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’:Ga20PCR:Luc:Tnos</td>
| |
| − | <td>3α1</td>
| |
| − | <td>KAN</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class=
| |
| − | "table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>H5α
| |
| − | <i>E.
| |
| − | coli</i>
| |
| − | transformation
| |
| − | with the
| |
| − | devices:</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>5S : 5’
| |
| − | region :
| |
| − | TFL KO :
| |
| − | Luc : Tnos
| |
| − | -
| |
| − | promoter35s:
| |
| − | Renilla:Tnos
| |
| − | in Ω2</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35S : 5’ region
| |
| − | : TFL consensus :
| |
| − | Luc : Tnos -
| |
| − | promoter35s:
| |
| − | Renilla:Tnos in
| |
| − | Ω2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35S : 5’ region
| |
| − | : Ga20ox KO : Luc :
| |
| − | Tnos - promoter35s:
| |
| − | Renilla:Tnos in
| |
| − | Ω2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35S : 5’ region
| |
| − | : Ga20ox consensus
| |
| − | : Luc : Tnos -
| |
| − | promoter35s:
| |
| − | Renilla:Tnos in
| |
| − | Ω2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>E. coli
| |
| − | plating in
| |
| − | plates with
| |
| − | Kanamycin
| |
| − | (antibiotic).
| |
| − | Incubate 16
| |
| − | hours at
| |
| − | 37^</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>
| |
| − | inipreps
| |
| − | with
| |
| − | E.Z.N.A ®.®
| |
| − | Plasmid
| |
| − | Mini Kit I,
| |
| − | Q(capless)
| |
| − | Spin
| |
| − | of:</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>
| |
| − | 5s:5’region:TFL
| |
| − | PCR:Luc:Tnos
| |
| − | in α1</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>
| |
| − | 35s:5’region:Ga20oxPCR:
| |
| − | Luc: Tnos in
| |
| − | α1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Pick a
| |
| − | single E.
| |
| − | coli DH5α
| |
| − | (TMV CDS)
| |
| − | colony from
| |
| − | the plate
| |
| − | that has
| |
| − | been
| |
| − | incubated
| |
| − | overnight.
| |
| − | Inoculate a
| |
| − | starter
| |
| − | culture of
| |
| − | 4 ml of LB
| |
| − | medium with
| |
| − | 4 μL of
| |
| − | kanamycin
| |
| − | in a 50 ml
| |
| − | tube with
| |
| − | the colony
| |
| − | and
| |
| − | incubate it
| |
| − | overnight
| |
| − | at 37^ with
| |
| − | shaking.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Digestion
| |
| − | of
| |
| − | minipreps
| |
| − | with EcoRI.
| |
| − | Incubate 1
| |
| − | hour at
| |
| − | 37^</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Run
| |
| − | electrophoresis
| |
| − | gel of the
| |
| − | devices. We
| |
| − | remain the
| |
| − | samples
| |
| − | 1.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Store
| |
| − | at -80^ the
| |
| − | devices of
| |
| − | gTS
| |
| − | PCR</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class=
| |
| − | "table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Reagent</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Volume(μL)</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>TFL
| |
| − | KO/Ga20KO/TFL
| |
| − | cons /
| |
| − | Ga20oxcons</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Promoter35s:
| |
| − | Renilla:
| |
| − | Tnos</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>pUPD2</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>BSA10X</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Ligase
| |
| − | Buffer</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1.2</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>BsmbI</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>T4
| |
| − | ligase</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | H<sub>2</sub>O
| |
| − | milli-Q</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>4.6</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class=
| |
| − | "table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>iniprep with
| |
| − | E.Z.N.A ®.® Plasmid
| |
| − | Mini Kit I,
| |
| − | Q(capless) Spin
| |
| − | of:</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>5s: TFL PCR :
| |
| − | Luc : Tnos</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35s : Ga20ox PCR : Luc
| |
| − | : Tnos</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Digestion of
| |
| − | minipreps with
| |
| − | EcoRI following
| |
| − | digestion protocol.
| |
| − | Incubate 1 hour at
| |
| − | 37^.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Run
| |
| − | electrophoresis gel
| |
| − | of the digestion
| |
| − | products. We will
| |
| − | discard this
| |
| − | culture because the
| |
| − | gel result doesn’t
| |
| − | correspond with
| |
| − | what we
| |
| − | expect.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>
| |
| − | Agroinfiltration
| |
| − | procedure with 9
| |
| − | plants of
| |
| − | N.benthamiana
| |
| − | following the
| |
| − | Agroinfiltration
| |
| − | protocol.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Pick up the
| |
| − | samples of
| |
| − | infiltrated plants.
| |
| − | We keep 3 disks per
| |
| − | plant in a
| |
| − | Eppendorf tube and
| |
| − | we take 2 samples
| |
| − | of each plant.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Luciferase
| |
| − | assay is made
| |
| − | following the
| |
| − | correct protocol.
| |
| − | After analyzing all
| |
| − | the data obtained,
| |
| − | it seems that the
| |
| − | system works but we
| |
| − | must optimized it
| |
| − | to increase the
| |
| − | signal range. In
| |
| − | this way, we will
| |
| − | be able to
| |
| − | distinguish signal
| |
| − | from noise.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>onclusions
| |
| − | luciferase
| |
| − | assay:</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>liminate 5’
| |
| − | region of the
| |
| − | device</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Change linker
| |
| − | sequence</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Add renilla in
| |
| − | luciferase assay</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Change reporter to
| |
| − | +1</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Use pLess as
| |
| − | control</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Use a Wild Type as
| |
| − | control</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Insert devices in
| |
| − | cis</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Design a consensus
| |
| − | target as longer as the
| |
| − | amplified.</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Culture refresh
| |
| − | of C58
| |
| − | <i>Agrobacterium</i>
| |
| − | to infiltrate on
| |
| − | Friday.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Pick a single
| |
| − | E. coli DH5α
| |
| − | (35s:5’region:TFL
| |
| − | PCR:Luc:Tnos in α1
| |
| − | and
| |
| − | 35s:5’region:Ga20oxPCR:
| |
| − | Luc: Tnos in α1)
| |
| − | colony from the
| |
| − | plate that has been
| |
| − | incubated
| |
| − | overnight.
| |
| − | Inoculate a starter
| |
| − | culture of 4 ml of
| |
| − | LB medium with 4 μL
| |
| − | of kanamycin in a
| |
| − | 50 ml tube with the
| |
| − | colony and incubate
| |
| − | it overnight at 37^
| |
| − | with shaking.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Targets
| |
| − | ligations with
| |
| − | Renilla
| |
| − | reporters.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class=
| |
| − | "table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Device</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Order</td>
| |
| − | <td>Sequencing</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>TFL PCR</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>210.13.250</td>
| |
| − | <td>SNP in the position 652 of
| |
| − | the target. Position 1 of the
| |
| − | gRNA. GA</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Ga20ox PCR</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>210.13.253</td>
| |
| − | <td>Same sequence</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>TFL consensus</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>210.13.251</td>
| |
| − | <td>Same sequence</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>TFL KO</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>210.13.252</td>
| |
| − | <td>Same sequence</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Ga20ox
| |
| − | Consensus</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>210.13.254</td>
| |
| − | <td>Same sequence</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Ga20ox KO</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>210.13.255</td>
| |
| − | <td>Same sequence</td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class=
| |
| − | "table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Received the necessary
| |
| − | primers to sequence: TFL
| |
| − | consensus, TFL knockout,
| |
| − | Ga20ox consensus and Ga20ox
| |
| − | knockout.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>repare
| |
| − | <i>Agrobacterium</i>
| |
| − | cultures: Inoculate a
| |
| − | starter culture of 5 ml of
| |
| − | LB medium with 5 μL of
| |
| − | Kanamycin and 5 μL of
| |
| − | rifampin in a 50 ml tube
| |
| − | with the colony and
| |
| − | incubate it 48 hours at 28^
| |
| − | with shaking. The devices
| |
| − | are:</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <p></p>
| |
| − | <ul>
| |
| − | <li>5S : 5’Region : TFL
| |
| − | consensus : Luc : Tnos</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35S : 5’Region : TFL knockout :
| |
| − | Luc : Tnos</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35S : 5’Region :TFL PCR: Luc :
| |
| − | Tnos</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35S : 5’Region : Ga20ox
| |
| − | consensus : Luc : Tnos</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35S : 5’Region : Ga20ox
| |
| − | knockout : Luc : Tnos</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>35S : 5’Region :Ga20ox PCR: Luc
| |
| − | : Tnos</td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Sequencing the last
| |
| − | devices to check if these
| |
| − | are correct.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li></li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Refresh
| |
| − | <i><i>Agrobacterium</i>
| |
| − | tumefaciens</i> cultures
| |
| − | with the aim of
| |
| − | infiltrating in
| |
| − | N.benthamiana on 29/07</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Refresh cultures of the
| |
| − | devices 35s: 5’ region: TFL
| |
| − | PCR : Luc : Tnos and 35s :
| |
| − | 5’ region: Ga20ox PCR : Luc
| |
| − | : Tnos in order to prepare
| |
| − | the more Miniprep
| |
| − | reaction.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>Sequencing results have
| |
| − | arrived.</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | <table class=
| |
| − | "table table-bordered table-striped">
| |
| − | <tr>
| |
| − | <th>
| |
| − | <ul>
| |
| − | <li>Lanes</li>
| |
| − | </ul>
| |
| − | </th>
| |
| − | <th>Samples</th>
| |
| − | <th>Verification</th>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>1</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>1 Kb molecular weight marker</td>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>2</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>TFL PCR</td>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | <tr>
| |
| − | <td>
| |
| − | <ul>
| |
| − | <li>3</li>
| |
| − | </ul>
| |
| − | </td>
| |
| − | <td>Ga20ox PCR</td>
| |
| − | <td></td>
| |
| − | </tr>
| |
| − | </table>
| |
| − | </div>
| |
| − | </li>
| |
| − | </ul>
| |
| − | </div>
| |
| − | </div>
| |
| − | </div>
| |
| − | </section>
| |
| | </body> | | </body> |
| | | | |
