Team:Gothenburg/Calendar

From 2014.igem.org

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                     <span class="hidden">
                     <span class="hidden">
<p>On the first day of laboration, we prepared LB, YPD and SC-Ura/His/Trp-Leu and YPD media.  
<p>On the first day of laboration, we prepared LB, YPD and SC-Ura/His/Trp-Leu and YPD media.  
-
<br>Click here to see the protocol.  [[PUT METHOD]]!
+
-
<br>[[File:20140623_103124.jpg]],[[File:20140623_115809.jpg|frameless|300px]]</span>
+
                 </td>
                 </td>
                  
                  
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                     <span class="hidden">
                     <span class="hidden">
<p> Succesfully PCR amplified five out of the seven parts from yeast genome via a Phusion High-Fidelity DNA Polymerase.  
<p> Succesfully PCR amplified five out of the seven parts from yeast genome via a Phusion High-Fidelity DNA Polymerase.  
-
<br>Click here to see the protocol. [[PUT METHOD]]!<br>
 
<p>The PCR program used was: <br>
<p>The PCR program used was: <br>
<ol>
<ol>
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<li>10 min at 72ºC. </li>
<li>10 min at 72ºC. </li>
</ol>
</ol>
-
<p>A diagnostic gel was performed and the result was as follows:
+
<p>A diagnostic gel was performed and the purification of the succesfully amplified parts was done using a PCR Purification Kit from GenJet following the instructions  
-
[[File: Bio-Rad 2014-06-25 15hr 34min.jpg|frameless|300px]] <br>
+
-
<p>The purification of the succesfully amplified parts was done using a PCR Purification Kit from GenJet following the instructions  
+
of the manufacture's manual but with no addition of isopropanol. <br>
of the manufacture's manual but with no addition of isopropanol. <br>
       </span>
       </span>
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<li>10 min at 72ºC. </li>
<li>10 min at 72ºC. </li>
</ol>
</ol>
-
<p>A diagnostic gel was performed and the result is as follows: <br>
+
<p>A diagnostic gel was performed and the purification of the succesfully amplified parts was done using a PCR Purification Kit from GenJet following the instructions
-
[[File: Bio-Rad 2014-06-26 17hr 53min.jpg|frameless|300px]] <br>
+
of the manufacture's manual but with no addition of isopropanol. <br>
</span>
</span>
                 </td>
                 </td>
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<h2>PCR amplification and merging</h2>
<h2>PCR amplification and merging</h2>
<p>Performed a PCR to fuse the pDSE4, pHO and dCas9 parts together.<br>
<p>Performed a PCR to fuse the pDSE4, pHO and dCas9 parts together.<br>
-
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase (see protocol here) and the program used was: <br>
+
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
<ol>
<ol>
<li>3 min at 98ºC </li>
<li>3 min at 98ºC </li>
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<li>10 min at 72ºC.</li>
<li>10 min at 72ºC.</li>
</ol>
</ol>
-
<p>A diagnostic gel was performed and the result is as follows: <br>
+
<p>A diagnostic gel was performed. The bands did not seem correct, so this PCR was repeated on the next day.<br>
-
[[File: Bio-Rad 2014-06-27 15hr 07min-2.jpg|frameless|300px]] <br>
+
-
<p>The bands did not seem correct, so this PCR was repeated on the next day.<br>
+
</span>
</span>
                 </td>
                 </td>
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<li>10 min at 72ºC.</li>
<li>10 min at 72ºC.</li>
</ol>
</ol>
-
<p>A diagnostic gel was performed and the result is as follows: <br>
+
<p>A diagnostic gel was performed and this did not seemed to work either.<br>
-
[File: Bio-Rad 2014-06-30 15hr 24min.jpg|frameless|300px]] <br>
+
-
<p>This did not seemed to work either.
+
<h2>Plasmid restriction</h2>
<h2>Plasmid restriction</h2>
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. <br>
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. <br>
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<li>10 min at 72ºC.</li>
<li>10 min at 72ºC.</li>
</ol>
</ol>
-
<p> A diagnostic gel was performed, however the sizes of the resulting fragments did not match the expected.
+
<p> A diagnostic gel was performed, however the sizes of the resulting fragments did not match the expected.<br>
<h2>Plasmid restriction</h2>
<h2>Plasmid restriction</h2>
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. <br>
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. <br>
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                         <li>Plasmid restriction</li>
                         <li>Plasmid restriction</li>
<li>Gel extraction</li>
<li>Gel extraction</li>
-
<li>Concentration determination</li>
+
                     </ul>
                     </ul>
                     <span class="hidden">
                     <span class="hidden">
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<h2>Gel extraction</h2>
<h2>Gel extraction</h2>
<p>Extracted the cleaved plasmids with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
<p>Extracted the cleaved plasmids with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
-
<h2>Concentration determination</h2>
+
-
<p>The concentration of the circular and linear plasmids obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 6.55 ng of DNA per UL to 543.4ng/UL.<br>
+
</span>
</span>
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                     <h1>5</h1>
                     <h1>5</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        
+
                       <li>Concentration determination</li>
                     </ul>
                     </ul>
-
                     <span class="hidden"></span>
+
                     <span class="hidden"><h2>Concentration determination</h2>
 +
<p>The concentration of the circular and linear plasmids obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 6.55 ng of DNA per UL to 543.4ng/UL.<br> </span>
                 </td>
                 </td>
                  
                  
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             <tr class="week">
             <tr class="week">
                 <td id="2014-06-30" class="date prev-month">
                 <td id="2014-06-30" class="date prev-month">
-
                     <h1>30</h1>
+
                     <li>PCR amplification and merging</li>
-
                    <ul class="titles">
+
-
                        <li>Title one</li>
+
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>PCR amplification and merging</h2>
 +
Repeated the PCR to fuse the pDSE4, pHO and dCas9 parts together.<br>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>50ºC for 30s</li>
 +
<li>58ºC for 30s</li>
 +
<li>2min 30s at 72ºC</li>
 +
<li>Go to step 2 (repeat 40 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p> A diagnostic gel was performed, however the sizes of the resulting fragments did not match the expected.
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. <br>
 +
<p>Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<p>Samples were loaded into a gelGreen, that showed p413, p415 and p416 approximately in the 5000bp size band, while p414 presented two bands, one around 4000bp and another at 2000bp.<br>
 +
<p> this indicates that the 5000bp band corresponds to the uncut plasmid, while the 4000bp and 2000bp are probably contaminations.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
-
                <td id="2014-07-01" class="date"> <!-- My birthday -->
+
                    <td id="2014-07-01" class="date">  
                     <h1>1</h1>
                     <h1>1</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                      <li>Plasmid restriction</li>
-
                         <li>Title two</li>
+
                         <li>PCR amplification</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. <br>
 +
<p>Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<h2>PCR amplification</h2>
 +
<p>Amplified the parts pDSE4, cas9 and pHO. The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>50ºC for 30s</li>
 +
<li>58ºC for 30s</li>
 +
<li>2min 15s at 72ºC</li>
 +
<li>Go to step 2 (repeat 40 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
<p> A diagnostic gel was performed, however the sizes of the resulting fragments did not match the expected.
 +
 +
</span>
                 </td>
                 </td>
                  
                  
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                     <h1>2</h1>
                     <h1>2</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>PCR amplification</li>
-
                         <li>Title two</li>
+
<li>Gel extraction</li>
 +
                         <li>Concentration determination</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>PCR amplification</h2>
 +
<p>Repeated the amplification of the parts pDSE4, cas9 and pHO. The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>50ºC for 30s</li>
 +
<li>58ºC for 30s</li>
 +
<li>2min 15s at 72ºC</li>
 +
<li>Go to step 2 (repeat 40 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
<p> A diagnostic gel was performed and the sizes of the fragments matched for the pDSE4 and pHO, but not for the Cas9.<br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the DNA fragments previously amplified with a GeneJet gel extraction kit.The manufacter's
 +
instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Concentration determination</h2>
 +
<p>The concentration of all the fragments obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 8.5 ng of DNA per UL to 42ng/UL.<br>
 +
</span>
                 </td>
                 </td>
                  
                  
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                     <h1>3</h1>
                     <h1>3</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                    </ul>
-
                        <li>Title two</li>
+
                     <span class="hidden"></span>
-
                    </ul>
+
-
                     <span class="hidden">This is where all the information goes!</span>
+
                 </td>
                 </td>
                  
                  
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                     <h1>4</h1>
                     <h1>4</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Plasmid Purification</li>
-
                         <li>Title two</li>
+
                         <li>Plasmid restriction</li>
-
                    </ul>
+
<li>Gel extraction</li>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                    </ul>
 +
                     <span class="hidden">
 +
<h2>Plasmid purification</h2>
 +
<p>Performed plasmid purification on E. coli cells containing plasmids p413, p414, p415, p416 and p2055 via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by
 +
water on the last step. Each plasmid was purified in duplicates.
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XbaI. <br>
 +
<p>Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<p> A diagnostic gelGreen was performed and showed that the cleavage of plasmids p413, p415 and p416 was successful.<br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the cleaved plasmids with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
 +
</span>
                 </td>
                 </td>
                  
                  
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                     <h1>5</h1>
                     <h1>5</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                      <li>Concentration determination</li>  
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"><h2>Concentration determination</h2>
 +
<p>The concentration of the circular and linear plasmids obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 6.55 ng of DNA per UL to 543.4ng/UL.<br> </span>
                 </td>
                 </td>
                  
                  
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                     <h1>6</h1>
                     <h1>6</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
 
-
                        <li>Title two</li>
 
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
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                     <h1>7</h1>
                     <h1>7</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Plasmid restriction</li>
-
                        <li>Title two</li>
+
               
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p414TEF with the enzymes SacI and XbaI. <br>
 +
<p>Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<p> A diagnostic gelGreen was performed and showed multiple size bands for the samples treated with XbaI. Consultation to the literature showed that this enzyme has not an unique restriction site for p414. <br>
 +
<p>Therefore the restriction was repeated with the enzyme SpeI, that has an unique cleavage site in p414 located 6bp away from the XbaI's desirable cleavage site. The same procedure described above was used, substituting XbaI for SpeI.<br>
 +
<p> The disgnostic gel performed on this second cleavage showed correct band sizes, but with too low lines. This indicates low concentration of the plasmid, therefore it was not extracted.<br>
 +
</span>
                 </td>
                 </td>
                  
                  
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                     <h1>8</h1>
                     <h1>8</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                        <li>Plasmid restriction</li>
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p414TEF with the enzymes SacI and SpeI. <br>
 +
<p>Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of SpeI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<p> A diagnostic gelGreen was performed and showed again too light bands or no bands at all. <br>
 +
</span>
                 </td>
                 </td>
                  
                  
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                     <h1>9</h1>
                     <h1>9</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Plasmid restriction</li>
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid restriction</h2>
 +
<p>Repeated the cleavage the plasmids p414TEF with the enzymes SacI and SpeI. <br>
 +
<p>Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of SpeI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<p> A diagnostic gelGreen was performed and SacI seemed to be leaving an excessive amount of uncut plasmid while SpeI apparently degrades the sample. <br>
 +
</span>
 +
                </td></span>
                 </td>
                 </td>
                  
                  
Line 646: Line 725:
                     <h1>10</h1>
                     <h1>10</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Plasmid restriction</li>
-
                        <li>Title two</li>
+
<li>Gel extraction</li>
                     </ul>
                     </ul>
-
                    <span class="hidden">This is where all the information goes!</span>
+
<span class="hidden">
 +
                    <h2>Plasmid restriction</h2>
 +
<p>Repeated the cleavage the plasmids p414TEF with the enzymes SacI with SpeI or BamHI. <br>
 +
<p>Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of SpeI or BamHI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each and without phosphatase were also prepared.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 20 minutes and then enzymatic inactivation was performed for 5min in a heating block at 75ºC for samples containing BamHI and at 65ºC for samples containing SpeI.<br>
 +
<p> A diagnostic gelGreen was performed and SacI again seemed to be leaving an excessive amount of uncut plasmid. However, samples treated with SacI and SpeI with and without phosphatase and one of the samples treated with SacI, BamHI and phosphatase seemed to work and were extracted. <br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the succesfully cleaved plasmids with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 655: Line 744:
                     <h1>11</h1>
                     <h1>11</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 664: Line 752:
                     <h1>12</h1>
                     <h1>12</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 673: Line 760:
                     <h1>13</h1>
                     <h1>13</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 684: Line 770:
                     <h1>14</h1>
                     <h1>14</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 693: Line 778:
                     <h1>15</h1>
                     <h1>15</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
 
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 702: Line 786:
                     <h1>16</h1>
                     <h1>16</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 711: Line 794:
                     <h1>17</h1>
                     <h1>17</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                    <li>PCR amplification and merging</li>
-
                        <li>Title two</li>
+
<li>Gel extraction</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>PCR amplification and merging</h2>
 +
<p>Performed a PCR to fuse the following fragments: <br>
 +
<li>pHO and Sic1 fragments for the Cas9 construct </li>
 +
<li>D-tag and YFP for the Daughter Resetter construct</li>
 +
<li>pCLN1 and Sic1 for the Csy4 construct</li>
 +
<li>D-tag and CFP for the Counter 2 construct </li>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>50ºC for 30s</li>
 +
<li>30s at 60ºC</li>
 +
<li>2min 15s at 72ºC</li>
 +
<li>Go to step 2 (repeat 39 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p>A diagnostic gelRed was performed and revealed the expected band sizes for the merging of D-tag with CFP and pHO with Sic1.<br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully merged fragments with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 720: Line 824:
                     <h1>18</h1>
                     <h1>18</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>PCR amplification and merging</li>
-
                        <li>Title two</li>
+
<li>Gel extraction</li>
 +
<li>Dissolution of synthetic parts</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>PCR amplification and merging</h2>
 +
<p>Performed a PCR to fuse the following fragments: <br>
 +
<li>D-tag and YFP for the Daughter Resetter construct</li>
 +
<li>pCLN1 and Sic1 for the Csy4 construct</li>
 +
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>Gradient from 47 to 55ºC for 30s</li>
 +
<li>30s at 60ºC</li>
 +
<li>2min 15s at 72ºC</li>
 +
<li>Go to step 2 (repeat 39 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p>A diagnostic gelRed was performed and revealed the expected band sizes for the merging of D-tag with YFP.<br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully merged fragments with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Dissolution of synthetic parts</h2>
 +
<p>The received synthetic parts (g-blocks) were centrifuged at 3000g for 3min and then dissolved with 10UL of sterile water. The solution was transferred to 10UL of TE buffer, resulting in a final concentration of 10ng/UL.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 729: Line 856:
                     <h1>19</h1>
                     <h1>19</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 738: Line 864:
                     <h1>20</h1>
                     <h1>20</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 749: Line 874:
                     <h1>21</h1>
                     <h1>21</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 758: Line 882:
                     <h1>22</h1>
                     <h1>22</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Amplification and purification of synthetic parts</li>
-
                         <li>Title two</li>
+
                         <li>Gel extraction</li>
 +
<li>Preparation of competent cells</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Amplification and purification of synthetic parts</h2>
 +
<p>Performed a PCR amplification of the synthetic parts previously diluted. Namely
 +
<li>pCYC1 m1</li>
 +
<li>Csy4</li>
 +
<li>Counter 1</li>
 +
<li>Counter 2</li>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC </li>
 +
<li>54ºC for 30s </li>
 +
<li>30s at 60ºC </li>
 +
<li>20s at 72ºC </li>
 +
<li>Go to step 2 (repeat 39 times) </li>
 +
<li>10 min at 72ºC. </li>
 +
</ol>
 +
<p>A diagnostic gel was performed and showed the expected band sizes for all the fragments except pCYC1.<br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully merged parts with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Preparation of competent cells</h2>
 +
<p>Following the instructions of the protocol "Frozen-EZ Yeast Transformation II" by ZymoResearch, Saccaromyces cerevisiae competent cells were prepared and stored at -80ºC.<br>
 +
</span>
                 </td>
                 </td>
                  
                  
Line 767: Line 914:
                     <h1>23</h1>
                     <h1>23</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Amplification and purification of synthetic parts</li>
-
                         <li>Title two</li>
+
                         <li>Gel extraction</li>
 +
<li>Yeast transformation</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Amplification and purification of synthetic parts</h2>
 +
<p>Repeated the PCR amplification of the synthetic part pCYC1 m1.<br>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC </li>
 +
<li>54ºC for 30s </li>
 +
<li>30s at 60ºC </li>
 +
<li>20s at 72ºC </li>
 +
<li>Go to step 2 (repeat 39 times) </li>
 +
<li>10 min at 72ºC. </li>
 +
</ol>
 +
<p>A diagnostic gel was performed and showed the expected band sizes.<br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully merged part with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Yeast Transformation</h2>
 +
<p> Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".<br>
 +
<p> Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:
 +
<li>Cas9 construct: pHO, Sic1 and Cas9 fragments into the plasmid p413 (Histidine marker)</li>
 +
<li>Csy4 construct: Sic1, pCLN1 and Csy4 fragments into p415 plasmid (Leucine marker)</li>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 776: Line 946:
                     <h1>24</h1>
                     <h1>24</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                      <li>Plasmid restriction</li>
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF and p416TEF with the enzymes SacI and XbaI. <br>
 +
<p>Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 30 minutes and then enzymatic inactivation was performed for 5min in a heating block at 65ºC.<br>
 +
<p> A diagnostic gelGreen was performed and showed fragments with the correct sizes. <br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully restricted plasmids with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 785: Line 964:
                     <h1>25</h1>
                     <h1>25</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Dissolution of synthetic parts</li>
-
                         <li>Title two</li>
+
                         <li>Amplification of synthetic parts</li>
 +
<li>PCR purification</li>
 +
<li>Concentration determination</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Dissolution of synthetic parts</h2>
 +
<p>The received synthetic part pCYC1 m2 was centrifuged at 3000g for 3min and then dissolved with 10UL of sterile water. The solution was transferred to 10UL of TE buffer, resulting in a final concentration of 10ng/UL.<br>
 +
<h2>Amplification of synthetic parts</h2>
 +
<p>Performed PCR amplification of the synthetic part pCYC1 m2.<br>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC </li>
 +
<li>54ºC for 30s </li>
 +
<li>30s at 60ºC </li>
 +
<li>20s at 72ºC </li>
 +
<li>Go to step 2 (repeat 39 times) </li>
 +
<li>10 min at 72ºC. </li>
 +
</ol>
 +
<p>A diagnostic gel was performed and showed the expected band sizes.<br>
 +
<h2>PCR purification</h2>
 +
<p>The purification of the succesfully amplified part was done using a PCR Purification Kit from GenJet following the instructions
 +
of the manufacture's manual but with no addition of isopropanol and elution was done with 30UL of sterile water. <br>
 +
<h2>Concentration determination</h2>
 +
<p>The concentration of the pCYC1 m2 samples obtained was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 15.1 ng of DNA per UL to 34.2ng/UL.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 794: Line 997:
                     <h1>26</h1>
                     <h1>26</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 803: Line 1,005:
                     <h1>27</h1>
                     <h1>27</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 814: Line 1,015:
                     <h1>28</h1>
                     <h1>28</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Yeast Transformation</li>
-
                         <li>Title two</li>
+
                         <li></li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Yeast Transformation</h2>
 +
<p> Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".<br>
 +
<p> Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:
 +
<li>Cas9 construct: pHO, Sic1 and Cas9 fragments into the plasmid p413 (Histidine marker)</li>
 +
<li>Daughter Resetter construct: D-tag, YFP, pDSE4 and Counter 1 synthetic part fragments into p414 plasmid</li>
 +
<li>Counter 2 construct: D-tag, CFP, pCYC1 and Counter 2 synthetic part fragments into p416 plasmid</li>
 +
</span>
                 </td>
                 </td>
                  
                  
Line 823: Line 1,031:
                     <h1>29</h1>
                     <h1>29</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                                            </ul>
-
                        <li>Title two</li>
+
                     <span class="hidden">/span>
-
                    </ul>
+
-
                     <span class="hidden">This is where all the information goes!</span>
+
                 </td>
                 </td>
                  
                  
Line 832: Line 1,038:
                     <h1>30</h1>
                     <h1>30</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Observation of the previously transformed cells</li>
-
                         <li>Title two</li>
+
                         <li>Plasmid restriction</li>
 +
<li>Preparation of Competent cells</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Observation of the previously transformed cells</h2>
 +
<p>One of the plates with cells transformed on the 28th showed growth, but with contamination by filamentous fungi, while the other presented no growth at all.<br>
 +
<p>A single yeast colony from the contaminated plate was replated on a YPD-agar plate.
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF, p414TEF and p416TEF with the enzymes SacI and XbaI. <br>
 +
<p>Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 30 minutes and then enzymatic inactivation was performed for 5min in a heating block at 65ºC.<br>
 +
<p> A diagnostic gelGreen was performed and showed fragments with the correct sizes. <br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully restricted plasmids with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Preparation of Competent cells</h2>
 +
<p> Competent E.coli cells were prepared according to the protocol described by Hanahan(1983) and stored at -80ºC.
 +
</span>
                 </td>
                 </td>
                  
                  
Line 841: Line 1,062:
                     <h1>31</h1>
                     <h1>31</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Inoculation of cells</li>
-
                        <li>Title two</li>
+
                    </ul>
-
                    </ul>
+
                     <span class="hidden">
-
                     <span class="hidden">This is where all the information goes!</span>
+
<h2>Inoculation of cells</h2>
 +
<p>The single colony replated on the day before was inoculated in 4mL YPD medium as an overnight culture for transformation.
 +
</span>
                 </td>
                 </td>
                  
                  
Line 850: Line 1,073:
                     <h1>1</h1>
                     <h1>1</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Yeast transformation</li>
-
                         <li>Title two</li>
+
                         <li>Inoculation of cells</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Yeast Transformation</h2>
 +
<p> Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".<br>
 +
<p> Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:
 +
<li>Cas9 construct: pHO, Sic1 and Cas9 fragments into the plasmid p413</li>
 +
<li>Daughter Resetter construct: D-tag, YFP, pDSE4 and Counter 1 synthetic part fragments into p414 plasmid</li>
 +
<li>Counter 2 construct: D-tag, CFP, pCYC1 and Counter 2 synthetic part fragments into p416 plasmid</li>
 +
 +
<h2>Inoculation of cells</h2>
 +
<p>Cells transformed on the 28th that showed growth after 3 days were plated on new adequate selective media.
 +
</span>
                 </td>
                 </td>
                  
                  
Line 859: Line 1,092:
                     <h1>2</h1>
                     <h1>2</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 868: Line 1,100:
                     <h1>3</h1>
                     <h1>3</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 882: Line 1,113:
                     <h1>28</h1>
                     <h1>28</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Yeast Transformation</li>
-
                         <li>Title two</li>
+
                         <li></li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Yeast Transformation</h2>
 +
<p> Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".<br>
 +
<p> Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:
 +
<li>Cas9 construct: pHO, Sic1 and Cas9 fragments into the plasmid p413 (Histidine marker)</li>
 +
<li>Daughter Resetter construct: D-tag, YFP, pDSE4 and Counter 1 synthetic part fragments into p414 plasmid</li>
 +
<li>Counter 2 construct: D-tag, CFP, pCYC1 and Counter 2 synthetic part fragments into p416 plasmid</li>
 +
</span>
                 </td>
                 </td>
                  
                  
Line 891: Line 1,129:
                     <h1>29</h1>
                     <h1>29</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 900: Line 1,137:
                     <h1>30</h1>
                     <h1>30</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Observation of the previously transformed cells</li>
-
                         <li>Title two</li>
+
                         <li>Plasmid restriction</li>
 +
<li>Preparation of Competent cells</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Observation of the previously transformed cells</h2>
 +
<p>One of the plates with cells transformed on the 28th showed growth, but with contamination by filamentous fungi, while the other presented no growth at all.<br>
 +
<p>A single yeast colony from the contaminated plate was replated on a YPD-agar plate.
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF, p414TEF and p416TEF with the enzymes SacI and XbaI. <br>
 +
<p>Approximately 500ng of the plasmid was mixed with o.5UL of SacI, 0.5UL of XbaI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water. Control tubes containing just one of the enzymes each were also prepared.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 30 minutes and then enzymatic inactivation was performed for 5min in a heating block at 65ºC.<br>
 +
<p> A diagnostic gelGreen was performed and showed fragments with the correct sizes. <br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully restricted plasmids with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Preparation of Competent cells</h2>
 +
<p> Competent E.coli cells were prepared according to the protocol described by Hanahan(1983) and stored at -80ºC.
 +
</span>
                 </td>
                 </td>
                  
                  
Line 909: Line 1,161:
                     <h1>31</h1>
                     <h1>31</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Inoculation of cells</li>
-
                         <li>Title two</li>
+
                          
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Inoculation of cells</h2>
 +
<p>The single colony replated on the day before was inoculated in 4mL YPD medium as an overnight culture for transformation.
 +
</span>
                 </td>
                 </td>
                  
                  
Line 918: Line 1,173:
                     <h1>1</h1>
                     <h1>1</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                        <li>Yeast transformation</li>
-
                         <li>Title two</li>
+
                         <li>Inoculation of cells</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Yeast Transformation</h2>
 +
<p> Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".<br>
 +
<p> Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:
 +
<li>Cas9 construct: pHO, Sic1 and Cas9 fragments into the plasmid p413</li>
 +
<li>Daughter Resetter construct: D-tag, YFP, pDSE4 and Counter 1 synthetic part fragments into p414 plasmid</li>
 +
<li>Counter 2 construct: D-tag, CFP, pCYC1 and Counter 2 synthetic part fragments into p416 plasmid</li>
 +
 +
<h2>Inoculation of cells</h2>
 +
<p>Cells transformed on the 28th that showed growth after 3 days were plated on new adequate selective media.
 +
</span>
                 </td>
                 </td>
                  
                  
Line 927: Line 1,192:
                     <h1>2</h1>
                     <h1>2</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 936: Line 1,200:
                     <h1>3</h1>
                     <h1>3</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 947: Line 1,210:
                     <h1>4</h1>
                     <h1>4</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>PCR amplification and merging</li>
-
                         <li>Title two</li>
+
                         <li>Gel extraction</li>
 +
<li>Plasmid restriction</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>PCR amplification and merging</h2>
 +
<p>Performed PCR to fuse the following fragments:<br>
 +
<li>D-tag and CFP </li>
 +
<li>pHO and Sic1 </li>
 +
<li>pDSE4 and YFP-D-tag(previously merged) </li>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>30s at 98ºC</li>
 +
<li>Gradient from 43 to 53ºC for 30s</li>
 +
<li>1min 30s at 72ºC</li>
 +
<li>Go to step 2 (repeat 40 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p> A diagnostic gelGreen was performed and the sizes of all the resulting fragments match the expected.<br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully merged parts with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Plasmid restriction</h2>
 +
<p>While reviewing primer design documents, the team realized we were using the wrong enzyme to cleave the plasmids: it should be SacI with XhoI instead of XbaI. Therefore, our plasmids did not have the correct overlap with the constructs.<br>
 +
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XhoI. <br>
 +
<p>Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XhoI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<p>A diagnostic gelGreen was performed and the cleavage showed to be unsuccessful.
 +
</span>
                 </td>
                 </td>
                  
                  
Line 956: Line 1,245:
                     <h1>5</h1>
                     <h1>5</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Plasmid restriction</li>
-
                         <li>Title two</li>
+
                         <li>Gel extraction</li>
 +
<li>Concentration determination</li>
 +
<li>PCR amplification and merging</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid restriction</h2>
 +
<p>Repeated the cleavage of the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XhoI. <br>
 +
<p>Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XhoI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<p>A diagnostic gelGreen was performed and the expected band sizes were observed. <br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully cleaved plasmids with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Concentration determination</h2>
 +
<p>Used a NanoDrop spectrometer to determinate the concentration of the previously extracted plasmids. The concentrations ranged from 5.95 to 7.75ng/UL.<br>
 +
<h2>PCR amplification and merging</h2>
 +
<p>Performed PCR to fuse the following fragments:<br>
 +
<li>D-tag-CFP (previously merged) with pCYC1 </li>
 +
<li>pHO and Sic1 (previously merged)with Cas9 </li>
 +
<li>pDSE4-YFP-D-tag(previously merged) and Counter 1 (synthetic part)</li>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>30s at 98ºC</li>
 +
<li>Gradient from 43 to 60ºC for 30s</li>
 +
<li>2min 30s at 72ºC</li>
 +
<li>Go to step 2 (repeat 40 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p> A diagnostic gelGreen was performed and the sizes of all the resulting fragments did not match the expected.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 965: Line 1,283:
                     <h1>6</h1>
                     <h1>6</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>PCR amplification and merging</li>
-
                         <li>Title two</li>
+
                         <li>Gibson Assembly</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>PCR amplification and merging</h2>
 +
<p>Repeated the PCR to fuse the following fragments:<br>
 +
<li>D-tag-CFP (previously merged) with pCYC1 </li>
 +
<li>pHO and Sic1 (previously merged)with Cas9 </li>
 +
<li>pDSE4-YFP-D-tag(previously merged) and Counter 1 (synthetic part)</li>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>Gradient from 45 to 62ºC for 30s</li>
 +
<li>2min 30s at 72ºC</li>
 +
<li>Go to step 2 (repeat 40 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p> A diagnostic gelGreen was performed and the sizes of all the resulting fragments were smaller than expected and the bands were difuse.<br>
 +
 +
<h2>Gibson Assembly</h2>
 +
<p> Attempted to transform the Daughter Resetter construct (pDSE4,YFP,D-tag and Counter 1) into E. coli following the Gibson Assembly protocol from New England Biolab.
 +
<p> The cells were inoculated in LB-Amp medium and let to grow overnight.
 +
</span>
                 </td>
                 </td>
                  
                  
Line 974: Line 1,312:
                     <h1>7</h1>
                     <h1>7</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Plasmid Purification</li>
-
                         <li>Title two</li>
+
                         <li>Gel extraction</li>
 +
<li>Gibson Assembly</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid Purification</h2>
 +
<p> Performed plasmid purification on E. coli cells containing plasmids p413, p414, p415, p416, p2055 and a plasmid containig our Cas9 sequence
 +
(pTPG1-dCas9-UPGP)via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by
 +
water on the last step. <br>
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF, p414TEF, p415TEF and p416TEF by using the enzymes SacI and XhoI. <br>
 +
<p>Approximately 500ng of each plasmid was mixed with o.5UL of SacI, 0.5UL of XhoI, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 10 minutes and then enzymatic inactivation was performed for 5min in a heating block at 60ºC.<br>
 +
<p>A diagnostic gelGreen was performed and the expected band sizes were observed. <br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully cleaved plasmids with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Gibson Assembly</h2>
 +
<p>Continued with the Gibson Assembly protocol (New England Biolab) and used p414 in the cells with the assembly poducts. A positive control with cells and the circular plasmid was also prepared.<br>
 +
<p>All the inoculated cultures were placed at 37ºC.
 +
</span>
                 </td>
                 </td>
                  
                  
Line 983: Line 1,338:
                     <h1>8</h1>
                     <h1>8</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Observed results from the Gibson Assembly</li>
-
                         <li>Title two</li>
+
                         <li>Yeast Transformation</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Observed results from the Gibson Assembly</h2>
 +
<p>The positive control showed several monoclonal colonies.<br>
 +
<p>However, the plate with the actual construct showed only one colony which is unlikely to contain the construct.<br>
 +
<h2>Yeast Transformation</h2>
 +
<p> Performed transformation on the competent cells previously prepared according to the protocol "Frozen-EZ Yeast Transformation II".<br>
 +
<p> Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:
 +
<li>Cas9 construct: pHO, Sic1 and Cas9 fragments into the plasmid p413</li>
 +
<li>Daughter Resetter construct: D-tag, YFP, pDSE4 and Counter 1 synthetic part fragments into p414 plasmid</li>
 +
<li>Counter 2 construct: D-tag, CFP, pCYC1 and Counter 2 synthetic part fragments into p416 plasmid</li>
 +
</span>
                 </td>
                 </td>
                  
                  
Line 992: Line 1,357:
                     <h1>9</h1>
                     <h1>9</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,001: Line 1,365:
                     <h1>10</h1>
                     <h1>10</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">!</span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 1,012: Line 1,375:
                     <h1>11</h1>
                     <h1>11</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>PCR amplification and merging</li>
-
                         <li>Title two</li>
+
                         <li>E. coli transformation</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>PCR amplification and merging</h2>
 +
<p>Performed PCR to fuse the following fragments:<br>
 +
<li>D-tag and CFP </li>
 +
<li>pHO and Sic1 </li>
 +
<li>pDSE4 and D-tag and YFP</li>
 +
<li>dCas9</li>
 +
<li>YFP and Counter 1</li>
 +
<li>D-tag and pCYC1</li>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>30s at 98ºC</li>
 +
<li>53ºC for 30s</li>
 +
<li>2min 30s at 72ºC</li>
 +
<li>Go to step 2 (repeat 40 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p> The samples were stored under refrigeration and analysed on the next day.<br>
 +
 +
<h2>E. coli transformation</h2>
 +
<p>Added 10UL of the registry Cas9 plasmid (after a 1:10 dilution) to competent E.coli cells. A control with no DNA addition was also prepared.<br>
 +
<p>The cells were kept on ice for 30min and then heat shocked at 42ºC for 60s and then kept on ice again for 3min.<br>
 +
<p>250mL of LB medium were then added and cells were incubated at 37ºC for 90min, under 200rpm shaking.<br>
 +
<p>After incubation, the cells were centrifuged at 9000rpm for 1min and the supernatant was discarded. The pellet was plated in LB+chlorophenicol plates and let to grow overnight at 37ºC.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,021: Line 1,410:
                     <h1>12</h1>
                     <h1>12</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Diagnostic gel and gel extraction</li>
-
                         <li>Title two</li>
+
                         <li>E. coli transformation</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Diagnostic gel and gel extraction</h2>
 +
<p>A gelRed was run to evaluate the PCR products from the day before. The fragments of dCas9, YFP-Counter 1, D-tag-pCYC1 and D-tag-CFP presented the expected band sizes.<br>
 +
<p>The successfully merged and amplified were extracted fragments with a GeneJet gel extraction kit.The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>E. coli transformation</h2>
 +
<p>The succesfully transformed colonies from the day before were replated on new LB medium. <br>
 +
<p>The transormation procedure was repeated for dCas9 and p413TEF.<br>
 +
<p>Added 10UL of the registry Cas9 and p413TEF plasmids (after a 1:10 dilution) to competent E.coli cells. A control with no DNA addition was also prepared.<br>
 +
<p>The cells were kept on ice for 30min and then heat shocked at 42ºC for 60s and then kept on ice again for 3min.<br>
 +
<p>250mL of LB medium were then added and cells were incubated at 37ºC for 90min, under 200rpm shaking.<br>
 +
<p>After incubation, the cells were centrifuged at 9000rpm for 1min and the supernatant was discarded. The pellet was plated in LB+chlorophenicol plates and let to grow overnight at 37ºC.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,030: Line 1,431:
                     <h1>13</h1>
                     <h1>13</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Concentration determination</li>
-
                         <li>Title two</li>
+
                         <li>Yeast Electroporation</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Concentration determination</h2>
 +
<p>The concentration of the fragments obtained on the day before was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 15.3 ng of DNA per UL to 101.6ng/UL.<br>
 +
<h2>Yeast Electroporation</h2>
 +
<p>Prepared an overnight culture according to the "Yeast electroporation" protocol.
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,039: Line 1,445:
                     <h1>14</h1>
                     <h1>14</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Yeast electroporation</li>
-
                         <li>Title two</li>
+
                          
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Yeast Electroporation</h2>
 +
<p>Following the "Yeast Electroporation" protocol, Saccaromyces cerevisiae were transformed with the Daughter Resetter construct (D-tag, YFP, pDSE4 and Counter 1 synthetic part fragments into p414 plasmid),
 +
the Cas9 construct (pHO, Sic1 and Cas9 fragments into the plasmid p413) and the Counter 2 construct (D-tag, CFP, pCYC1 and Counter 2 synthetic part fragments into p416 plasmid).<br>
 +
<p>Cells were plated in the adequate restrictive media.<br>
 +
 +
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,048: Line 1,462:
                     <h1>15</h1>
                     <h1>15</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Plasmid Purification</li>
-
                         <li>Title two</li>
+
                         <li>PCR amplification and merging</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid Purification</h2>
 +
<p> Performed plasmid purification on the overnight culture of E. coli cells containing the registry Cas9 via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by
 +
water on the last step. <br>
 +
<h2>PCR amplification and merging</h2>
 +
<p>Performed PCR to fuse and amplify the following fragments:<br>
 +
<li>Registry Cas9 plasmid </li>
 +
<li>p413TEF YFP </li>
 +
<li>Registry Cas9</li>
 +
<li>pDSE4</li>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>58ºC for 30s</li>
 +
<li>2min 30s at 72ºC</li>
 +
<li>Go to step 2 (repeat 40 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p> The samples were stored under refrigeration and analysed on August 18th.<br>
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,057: Line 1,491:
                     <h1>16</h1>
                     <h1>16</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,066: Line 1,499:
                     <h1>17</h1>
                     <h1>17</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 1,077: Line 1,509:
                     <h1>18</h1>
                     <h1>18</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Diagnostic gel</li>
-
                         <li>Title two</li>
+
                         <li>PCR purification</li>
 +
<li>Plasmid restriction</li>
 +
<li>Gel extraction</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Diagnostic gel</h2>
 +
<p>A gelGreen was run to evaluate the PCR products from the 15th. The fragments showed the expected band sizes.<br>
 +
<h2>PCR purification</h2>
 +
<p>The purification of the successfully amplified part was done using a PCR Purification Kit from GenJet following the instructions
 +
of the manufacture's manual but with no addition of isopropanol and elution was done with 30UL of sterile water. <br>
 +
<h2>Plasmid restriction</h2>
 +
<p>Cleaved the plasmids p413TEF, p414TEF, p416TEF and the registry cas9 plasmid by using the enzymes SpeI and XbaI (for the resgistry plasmid) and SacI and XhoI (for the others). <br>
 +
<p>Approximately 500ng of each plasmid was mixed with o.5UL of each enzyme, 2UL of FastDigest buffer, 1UL of FastAP
 +
(alkaline phosphatase) and up to 20 UL of deionized sterile water.<br>
 +
<p>This reaction mixture was incubated at in a water bath at 37ºC for 30 minutes and then enzymatic inactivation was performed for 5min in a heating block at 65ºC.<br>
 +
<p> A diagnostic gelGreen was performed and the sizes of all the resulting fragments match the expected except p415TEF.<br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully restricted plasmids with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,086: Line 1,535:
                     <h1>19</h1>
                     <h1>19</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Gibson Assembly</li>
-
                         <li>Title two</li>
+
                         <li>Yeast transformation</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Gibson Assembly</h2>
 +
<p> Attempted to transform the following constructs into E. coli following the Gibson Assembly protocol from New England Biolab:
 +
<li>Daughter Resetter construct (pDSE4,YFP,D-tag and Counter 1 synthetic part) </li>
 +
<li>Cas9 construct (pHO, Sic1 and dCas9)</li>
 +
<li>Counter 2 construct (D-tag, CFP, pCYC1 and Counter 2 synthetic part)</li>
 +
<p> The cells were inoculated in LB-Amp medium and let to grow overnight.<br>
 +
<h2>Yeast Transformation</h2>
 +
<p> Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".<br>
 +
<p> Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:
 +
<li>Cas9 construct: pHO, Sic1 and Cas9 fragments into the plasmid p413</li>
 +
<li>Csy4 construct: Sic1, pCLN1 and Csy4 fragments into p415 plasmid</li>
 +
<p> The cells were inoculated in LB-Amp medium and let to grow overnight at 30ºC.<br>
 +
<h2>E. coli transformation</h2>
 +
<p>Added 10UL of the registry Cas9 plasmid (after a 1:10 dilution) to competent E.coli cells. A control with no DNA addition was also prepared.<br>
 +
<p>The cells were kept on ice for 30min and then heat shocked at 42ºC for 60s and then kept on ice again for 3min.<br>
 +
<p>250mL of LB medium were then added and cells were incubated at 37ºC for 90min, under 200rpm shaking.<br>
 +
<p>After incubation, the cells were centrifuged at 9000rpm for 1min and the supernatant was discarded. The pellet was plated in LB+chlorophenicol plates and let to grow overnight at 37ºC.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,095: Line 1,563:
                     <h1>20</h1>
                     <h1>20</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Colony PCR</li>
-
                         <li>Title two</li>
+
                          
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Colony PCR</h2>
 +
<p>Ran colony PCR on the colonies obtained so far (from different transformation attempts and both in E.coli and yeast). A single colony from each plate was suspended in 100mL LiAc, 1% SD solution and incubated at 70ºC for 5min.
 +
Then 300mL of 96% ethanol were added and the solution was centrifuged at 15000g for 3min.<br>
 +
<p>The pellet obtained was washed with 70% ethanol and dissolved in 100mL of water and then centrifuged at 15000g for 15s. 1mL of the obtained supernatant was used for the PCR.<br>>
 +
<p>The PCR program used was: <br>
 +
<ol>
 +
<li>10 min at 98ºC </li>
 +
<li>10s at 98ºC </li>
 +
<li>Gradient from 50 to 59ºC for 30s </li>
 +
<li>2 min at 72ºC </li>
 +
<li>Go to step 2 (repeat 39 times) </li>
 +
<li>10 min at 72ºC. </li>
 +
</ol>
 +
<p>A diagnostic gel was performed and correct band sizes were observed for counter 1 and counter 2 transformations done in 18/8.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,104: Line 1,588:
                     <h1>21</h1>
                     <h1>21</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,113: Line 1,596:
                     <h1>22</h1>
                     <h1>22</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Plasmid Purification</li>
                         <li>Title two</li>
                         <li>Title two</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Plasmid Purification</h2>
 +
<p> Performed plasmid purification on E. coli cells containing the plasmid containing the Cas9 part improvement (from 19/8) via a GenJet Plasmid Purification Kit. The manufacter's instructions were followed, except the elution buffer was subtituted by
 +
water on the last step. <br>
 +
instructions were followed, except the elution buffer was substituted by water on the last step.
 +
<h2>Concentration determination</h2>
 +
<p>The concentration of the obtained plasmids was measured via a NanoDrop. The samples were measured in four replicates. The results ranged from 35.4 ng of DNA per UL to 289ng/UL.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,122: Line 1,613:
                     <h1>23</h1>
                     <h1>23</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Fluorescence microscopy</li>
-
                        <li>Title two</li>
+
<li>PCR amplification</li>
 +
<li>Gel extraction</li>
 +
                     
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Fluorescence microscopy</h2>
 +
<p> Checked cells transformed with the Daughter Resetter construct under a fluorescent microscope and fluorescent yellow cells were observed, as well as dead cells under the green and yellow filter.<br>
 +
<h2>PCR amplification</h2>
 +
<p><p>Performed a PCR to amplify Csy4 cconstruct, dCas9, pHO, Sic1 and pCLN1.<br>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>Gradient from 50 to 55ºC for 30s</li>
 +
<li>30s at 60ºC</li>
 +
<li>2min 15s at 72ºC</li>
 +
<li>Go to step 2 (repeat 39 times)</li>
 +
<li>10 min at 72ºC.</li>
 +
</ol>
 +
<p>A diagnostic gel was performed. The bands match the expected size for all samples except one of the replicates of dCas9.<br>
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully amplified constructs with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,131: Line 1,643:
                     <h1>24</h1>
                     <h1>24</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>PCR amplification</li>
-
                         <li>Title two</li>
+
                         <li>Concentration determination</li>
 +
<li>Yeast plasmid extraction</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>PCR amplification</h2>
 +
<p>Performed a PCR to amplify the D-tag of the intended part submission.<br>
 +
<p>The PCR was done using the Phusion High-Fidelity DNA Polymerase and the program used was: <br>
 +
<ol>
 +
<li>3 min at 98ºC </li>
 +
<li>10s at 98ºC</li>
 +
<li>Gradient from 50 to 60ºC for 10s</li>
 +
<li>7s at 72ºC</li>
 +
<li>Go to step 2 (repeat 39 times)</li>
 +
<li>5 min at 72ºC.</li>
 +
</ol>
 +
<p>A diagnostic gel was performed and the purification of the succesfully amplified parts was done using a PCR Purification Kit from GenJet following the instructions
 +
of the manufacture's manual but with no addition of isopropanol. <br>
 +
 +
<h2>Yeast plasmid extraction<h2>
 +
<p>Extracted plasmids from yeast cells transformed with the Daughter Resetter and Counter 2 constructs via a Zymoprep Yeast plasmid Miniprep II, following the instructions on the kit. <br>
 +
<h2>Concentration determination</h2>
 +
<p>The concentration of the D-tag and plasmid samples was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 17.1 ng of DNA per UL to 54.6ng/UL.<br>
 +
</span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 1,142: Line 1,674:
                     <h1>25</h1>
                     <h1>25</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Restriction digestion</li>
-
                        <li>Title two</li>
+
                     
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Restriction digestion</h2>
 +
<p>Using a FastDigest kit with enzymes XbaI and SpeI, attempted to cleave p413, p415, D-tag and D-tag-CFP and linearized psB1C3 backbone. <br>
 +
<p> A diagnostic gelGreen was performed and the obtained fragments did not match the expected sizes.<br>
 +
 +
 
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,151: Line 1,689:
                     <h1>26</h1>
                     <h1>26</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                        <li>Restriction digestion</li>
-
                         <li>Title two</li>
+
                         <li>Gel extraction</li>
 +
<li>Concentration determination</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Restriction digestion</h2>
 +
<p>Repeated the cleavage of p413, p415, D-tag and D-tag-CFP and linearized psB1C3 backbone, using a FastDigest kit with enzymes EcoRI and PstI.<br> <p> A diagnostic gelGreen was performed and the obtained fragments did not match the expected sizes.<br>
 +
<p> A diagnostic gelGreen was performed and the obtained fragments match the expected sizes.<br>
 +
 +
<h2>Gel extraction</h2>
 +
<p>Extracted the successfully restricted constructs with a GeneJet gel extraction kit. The manufacter's instructions were followed, except the elution buffer was subtituted by water on the last step.
 +
<h2>Concentration determination</h2>
 +
<p>The concentration of the restricted constructs was measured via a NanoDrop. The samples were measured in duplicates. The results ranged from 1.9 ng of DNA per UL to 39.15ng/UL.<br>
 +
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,160: Line 1,710:
                     <h1>27</h1>
                     <h1>27</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Yeast Transformation</li>
-
                         <li>Title two</li>
+
                         <li>E. coli transformation</li>
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Yeast Transformation</h2>
 +
<p> Performed transformation on the competent cells previously prepared according to the protocol "Yeast Transformation".<br>
 +
<p> Equimollar quantities of the fragments were added to plasmids with different markers. More specifically:
 +
<li>Cas9 construct: pHO, Sic1 and Cas9 fragments into the plasmid p413 (Histidine marker)</li>
 +
<li>Csy4 construct: Sic1, pCLN1 and Csy4 fragments into p415 plasmid (Leucine marker)</li>
 +
 +
<h2>E. coli transformation</h2>
 +
<p>Added 3UL of the linearize biobrick backbone and 0.5UL of D-tag to competent E.coli cells. A control with only the linearized backbone was also prepared.<br>
 +
<p>The cells were kept on ice for 30min and then heat shocked at 42ºC for 60s and then kept on ice again for 3min.<br>
 +
<p>250mL of LB medium were then added and cells were incubated at 37ºC for 90min, under 200rpm shaking.<br>
 +
<p>After incubation, the cells were centrifuged at 9000rpm for 1min and the supernatant was discarded. The pellet was plated in LB+chlorophenicol plates and let to grow overnight at 37ºC.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,169: Line 1,732:
                     <h1>28</h1>
                     <h1>28</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Inoculation of plates</li>
-
                        <li>Title two</li>
+
                     
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Inoculation of plates</h2>
 +
<p>Replated transformed yeast cells that showed growth (constructs Csy4 and dCas9 from August  8th and 14th, respectively), as well as E.coli cells transformed with the D-tag part submission.<br>
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,178: Line 1,745:
                     <h1>29</h1>
                     <h1>29</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Colony PCR</li>
-
                         <li>Title two</li>
+
                          
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Colony PCR</h2>
 +
<p>Ran colony PCR on the replated plates from the day before. A single colony from each plate was suspended in 100mL LiAc, 1% SD solution and incubated at 70ºC for 5min.
 +
Then 300mL of 96% ethanol were added and the solution was centrifuged at 15000g for 3min.<br>
 +
<p>The pellet obtained was washed with 70% ethanol and dissolved in 100mL of water and then centrifuged at 15000g for 15s. 1mL of the obtained supernatant was used for the PCR.<br>>
 +
<p>The PCR program used was: <br>
 +
<ol>
 +
<li>10 min at 98ºC </li>
 +
<li>10s at 98ºC </li>
 +
<li>Gradient from 50 to 59ºC for 30s </li>
 +
<li>2 min at 72ºC </li>
 +
<li>Go to step 2 (repeat 39 times) </li>
 +
<li>10 min at 72ºC. </li>
 +
</ol>
 +
<p>A diagnostic gel was performed and correct band sizes were not observed for Csy4 and dCas9. Degradadion and difused bands were observed for the D-tag.<br>
 +
 +
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,187: Line 1,771:
                     <h1>30</h1>
                     <h1>30</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,196: Line 1,779:
                     <h1>31</h1>
                     <h1>31</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 1,210: Line 1,792:
                     <h1>1</h1>
                     <h1>1</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                         <li>Restriction digestion</li>
-
                        <li>Title two</li>
+
                     
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden">
 +
<h2>Restriction digestion</h2>
 +
<p>Restriction of possible fully constructed Csy4 construct from the 22nd of August using a FastDigest kit with enzymes XhoI and SpeI.<br> <p> A diagnostic gelGreen was performed and the obtained fragments did not match the expected sizes.<br>
 +
<p> A diagnostic gelGreen was performed and the obtained fragments did not match the expected sizes.<br>
 +
</span>
                 </td>
                 </td>
                  
                  
Line 1,219: Line 1,805:
                     <h1>2</h1>
                     <h1>2</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,228: Line 1,813:
                     <h1>3</h1>
                     <h1>3</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,237: Line 1,821:
                     <h1>4</h1>
                     <h1>4</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,246: Line 1,829:
                     <h1>5</h1>
                     <h1>5</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,255: Line 1,837:
                     <h1>6</h1>
                     <h1>6</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                 
-
                        <li>Title two</li>
+
                   
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,264: Line 1,846:
                     <h1>7</h1>
                     <h1>7</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                   
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 1,275: Line 1,856:
                     <h1>8</h1>
                     <h1>8</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                 
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,284: Line 1,864:
                     <h1>9</h1>
                     <h1>9</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,293: Line 1,872:
                     <h1>10</h1>
                     <h1>10</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                   
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,302: Line 1,880:
                     <h1>11</h1>
                     <h1>11</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,311: Line 1,888:
                     <h1>12</h1>
                     <h1>12</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,320: Line 1,896:
                     <h1>13</h1>
                     <h1>13</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,329: Line 1,904:
                     <h1>14</h1>
                     <h1>14</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 1,340: Line 1,915:
                     <h1>15</h1>
                     <h1>15</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,349: Line 1,923:
                     <h1>16</h1>
                     <h1>16</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                   
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,358: Line 1,931:
                     <h1>17</h1>
                     <h1>17</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                   
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,367: Line 1,939:
                     <h1>18</h1>
                     <h1>18</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,376: Line 1,947:
                     <h1>19</h1>
                     <h1>19</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                   
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,385: Line 1,955:
                     <h1>20</h1>
                     <h1>20</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,394: Line 1,963:
                     <h1>21</h1>
                     <h1>21</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                   
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 1,405: Line 1,973:
                     <h1>22</h1>
                     <h1>22</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,414: Line 1,981:
                     <h1>23</h1>
                     <h1>23</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,423: Line 1,989:
                     <h1>24</h1>
                     <h1>24</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,432: Line 1,997:
                     <h1>25</h1>
                     <h1>25</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,441: Line 2,005:
                     <h1>26</h1>
                     <h1>26</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,450: Line 2,013:
                     <h1>27</h1>
                     <h1>27</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,459: Line 2,021:
                     <h1>28</h1>
                     <h1>28</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
             </tr>
             </tr>
Line 1,470: Line 2,031:
                     <h1>29</h1>
                     <h1>29</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,479: Line 2,039:
                     <h1>30</h1>
                     <h1>30</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                         <li>Title one</li>
+
                          
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,488: Line 2,047:
                     <h1>1</h1>
                     <h1>1</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     </ul>
                     </ul>
-
                     <span class="hidden">This is where all the information goes!</span>
+
                     <span class="hidden"></span>
                 </td>
                 </td>
                  
                  
Line 1,497: Line 2,055:
                     <h1>2</h1>
                     <h1>2</h1>
                     <ul class="titles">
                     <ul class="titles">
-
                        <li>Title one</li>
+
                     
-
                        <li>Title two</li>
+
                     <span class="hidden"></span>
-
                    </ul>
+
-
                     <span class="hidden">This is where all the information goes!</span>
+
                 </td>
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Latest revision as of 03:01, 14 October 2014

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June

26

27

28

29

30

31

1

2

3

4

5

6

7

8

9

10

  • Safety Instructions

11

12

13

14

15

16

17

  • Introduction to centrifuges and autoclaves

18

19

20

  • Finished mandatory training

21

22

23

  • Media Preparation

24

25

  • Amplification and Purification of non-synthetic parts

26

  • Plasmid Purification
  • Amplification and purification of parts

27

  • Gel extraction
  • PCR amplification and merging

28

  • PCR amplification and merging
  • Plasmid restriction

29

30

  • PCR amplification and merging

1

  • Plasmid restriction
  • PCR amplification

2

  • PCR amplification
  • Gel extraction
  • Concentration determination

3

4

  • Plasmid Purification
  • Plasmid restriction
  • Gel extraction

5

  • Concentration determination

6

July

  • PCR amplification and merging
    		•

    1

    • Plasmid restriction
    • PCR amplification

    2

    • PCR amplification
    • Gel extraction
    • Concentration determination

    3

    4

    • Plasmid Purification
    • Plasmid restriction
    • Gel extraction

    5

    • Concentration determination

    6

    7

    • Plasmid restriction

    8

    • Plasmid restriction

    9

    • Plasmid restriction

    10

    • Plasmid restriction
    • Gel extraction

    11

    12

    13

    14

    15

    16

    17

    • PCR amplification and merging
    • Gel extraction

    18

    • PCR amplification and merging
    • Gel extraction
    • Dissolution of synthetic parts

    19

    20

    21

    22

    • Amplification and purification of synthetic parts
    • Gel extraction
    • Preparation of competent cells

    23

    • Amplification and purification of synthetic parts
    • Gel extraction
    • Yeast transformation

    24

    • Plasmid restriction

    25

    • Dissolution of synthetic parts
    • Amplification of synthetic parts
    • PCR purification
    • Concentration determination

    26

    27

    28

    • Yeast Transformation

    29

    30

    • Observation of the previously transformed cells
    • Plasmid restriction
    • Preparation of Competent cells

    31

    • Inoculation of cells

    1

    • Yeast transformation
    • Inoculation of cells

    2

    3

    Augusti

    28

    • Yeast Transformation

    29

    30

    • Observation of the previously transformed cells
    • Plasmid restriction
    • Preparation of Competent cells

    31

    • Inoculation of cells

    1

    • Yeast transformation
    • Inoculation of cells

    2

    3

    4

    • PCR amplification and merging
    • Gel extraction
    • Plasmid restriction

    5

    • Plasmid restriction
    • Gel extraction
    • Concentration determination
    • PCR amplification and merging

    6

    • PCR amplification and merging
    • Gibson Assembly

    7

    • Plasmid Purification
    • Gel extraction
    • Gibson Assembly

    8

    • Observed results from the Gibson Assembly
    • Yeast Transformation

    9

    10

    11

    • PCR amplification and merging
    • E. coli transformation

    12

    • Diagnostic gel and gel extraction
    • E. coli transformation

    13

    • Concentration determination
    • Yeast Electroporation

    14

    • Yeast electroporation

    15

    • Plasmid Purification
    • PCR amplification and merging

    16

    17

    18

    • Diagnostic gel
    • PCR purification
    • Plasmid restriction
    • Gel extraction

    19

    • Gibson Assembly
    • Yeast transformation

    20

    • Colony PCR

    21

    22

    • Plasmid Purification
    • Title two

    23

    • Fluorescence microscopy
    • PCR amplification
    • Gel extraction

    24

    • PCR amplification
    • Concentration determination
    • Yeast plasmid extraction

    25

    • Restriction digestion

    26

    • Restriction digestion
    • Gel extraction
    • Concentration determination

    27

    • Yeast Transformation
    • E. coli transformation

    28

    • Inoculation of plates

    29

    • Colony PCR

    30

    31

    September

    1

    • Restriction digestion

    2

    3

    4

    5

    6

    7

    8

    9

    10

    11

    12

    13

    14

    15

    16

    17

    18

    19

    20

    21

    22

    23

    24

    25

    26

    27

    28

    29

    30

    1

    2

    3

    4

    5

    Retrieved from "http://2014.igem.org/Team:Gothenburg/Calendar"