http://2012.igem.org/wiki/index.php?title=Team:Carnegie_Mellon/Met-Challenges&feed=atom&action=historyTeam:Carnegie Mellon/Met-Challenges - Revision history2024-03-28T09:00:11ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:Carnegie_Mellon/Met-Challenges&diff=297656&oldid=prevYchoo at 03:31, 27 October 20122012-10-27T03:31:50Z<p></p>
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</table>Ychoohttp://2012.igem.org/wiki/index.php?title=Team:Carnegie_Mellon/Met-Challenges&diff=292469&oldid=prevEnpederson at 23:54, 26 October 20122012-10-26T23:54:31Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. Using our previous cloning protocols, the cloning using the plasmid vector worked in the first trial. Unfortunately, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. <br \></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. Using our previous cloning protocols, the cloning using the plasmid vector worked in the first trial. Unfortunately, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. <br \></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The key difference between our plasmid vector and the pSB1C3 vector from the registry was the fact that the submission vector was linearized. We have not been able to pinpoint the exact cause for this, so again, budget sufficient time for cloning! </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The key difference between our plasmid vector and the pSB1C3 vector from the registry was the fact that the submission vector was linearized. We have not been able to pinpoint the exact cause for this, so again, budget sufficient time for cloning! </p></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">We attempted to utilize flow cytometry to analyze expression data of our fluorogen-activating biosensors. In order to save time and prevent a queue from forming, we had to fix our cells with 5% formaldehyde before running it in the cytometer. This had negative effects on our results and were unable to see significant signal.</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Carnegie_Mellon/Templates/Footer}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Carnegie_Mellon/Templates/Footer}}</div></td></tr>
</table>Enpedersonhttp://2012.igem.org/wiki/index.php?title=Team:Carnegie_Mellon/Met-Challenges&diff=263916&oldid=prevYchoo at 01:31, 4 October 20122012-10-04T01:31:59Z<p></p>
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</table>Ychoohttp://2012.igem.org/wiki/index.php?title=Team:Carnegie_Mellon/Met-Challenges&diff=262223&oldid=prevYchoo at 00:12, 4 October 20122012-10-04T00:12:04Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A major help in diagnosing our failures was using the gel consistently to check the length of our inserts, and to sequence periodically to ensure the insert and vector are as expected. <br \></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A major help in diagnosing our failures was using the gel consistently to check the length of our inserts, and to sequence periodically to ensure the insert and vector are as expected. <br \></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Please refer to our protocols for our final cloning protocol, and remember to reserve some amount of time to allow for experimental failures!</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Please refer to our protocols for our final cloning protocol, and remember to reserve some amount of time to allow for experimental failures!</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><br \> <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><br \<ins class="diffchange diffchange-inline">> <br</ins>><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. Using our previous cloning protocols, the cloning using the plasmid vector worked in the first trial. Unfortunately, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. <br \></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. Using our previous cloning protocols, the cloning using the plasmid vector worked in the first trial. Unfortunately, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. <br \></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The key difference between our plasmid vector and the pSB1C3 vector from the registry was the fact that the submission vector was linearized. We have not been able to pinpoint the exact cause for this, so again, budget sufficient time for cloning! </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The key difference between our plasmid vector and the pSB1C3 vector from the registry was the fact that the submission vector was linearized. We have not been able to pinpoint the exact cause for this, so again, budget sufficient time for cloning! </p></div></td></tr>
</table>Ychoohttp://2012.igem.org/wiki/index.php?title=Team:Carnegie_Mellon/Met-Challenges&diff=259106&oldid=prevYchoo at 18:52, 3 October 20122012-10-03T18:52:45Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A major help in diagnosing our failures was using the gel consistently to check the length of our inserts, and to sequence periodically to ensure the insert and vector are as expected. <br \></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>A major help in diagnosing our failures was using the gel consistently to check the length of our inserts, and to sequence periodically to ensure the insert and vector are as expected. <br \></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Please refer to our protocols for our final cloning protocol, and remember to reserve some amount of time to allow for experimental failures!</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Please refer to our protocols for our final cloning protocol, and remember to reserve some amount of time to allow for experimental failures!</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. Using our previous cloning protocols, the cloning using the plasmid vector worked in the first trial. Unfortunately, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. <br \></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. Using our previous cloning protocols, the cloning using the plasmid vector worked in the first trial. Unfortunately, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. <br \></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The key difference between our plasmid vector and the pSB1C3 vector from the registry was the fact that the submission vector was linearized. We have not been able to pinpoint the exact cause for this, so again, budget sufficient time for cloning! </p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>The key difference between our plasmid vector and the pSB1C3 vector from the registry was the fact that the submission vector was linearized. We have not been able to pinpoint the exact cause for this, so again, budget sufficient time for cloning! </p></div></td></tr>
</table>Ychoohttp://2012.igem.org/wiki/index.php?title=Team:Carnegie_Mellon/Met-Challenges&diff=259104&oldid=prevYchoo at 18:52, 3 October 20122012-10-03T18:52:29Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Challenges with Molecular Cloning</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Challenges with Molecular Cloning</h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> Our cloning procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions. However, these issues were eventually ironed out and we managed to get the FAP and Spinach into a single construct. <br \><br \></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> <ins class="diffchange diffchange-inline">Cloning was a major challenge for us. It often took us multiple rounds of digestion/ligation/cloning to get our construct into the cells. </ins>Our cloning procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions. However, these issues were eventually ironed out and we managed to get the FAP and Spinach into a single construct. <br \></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We <del class="diffchange diffchange-inline">have </del>attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. <del class="diffchange diffchange-inline">The </del>cloning using the plasmid vector worked in the first trial. <del class="diffchange diffchange-inline">However</del>, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. We have not been able to pinpoint the cause for this<del class="diffchange diffchange-inline">. </del></p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">A major help in diagnosing our failures was using the gel consistently to check the length of our inserts, and to sequence periodically to ensure the insert and vector are as expected. <br \></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">Please refer to our protocols for our final cloning protocol, and remember to reserve some amount of time to allow for experimental failures!</ins></div></td></tr>
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<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. <ins class="diffchange diffchange-inline">Using our previous cloning protocols, the </ins>cloning using the plasmid vector worked in the first trial. <ins class="diffchange diffchange-inline">Unfortunately</ins>, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols<ins class="diffchange diffchange-inline">. <br \></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">The key difference between our plasmid vector and the pSB1C3 vector from the registry was the fact that the submission vector was linearized</ins>. We have not been able to pinpoint the <ins class="diffchange diffchange-inline">exact </ins>cause for this<ins class="diffchange diffchange-inline">, so again, budget sufficient time for cloning! </ins></p></div></td></tr>
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</table>Ychoohttp://2012.igem.org/wiki/index.php?title=Team:Carnegie_Mellon/Met-Challenges&diff=254940&oldid=prevYchoo at 05:48, 3 October 20122012-10-03T05:48:51Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> Our cloning procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions. However, these issues were eventually ironed out and we managed to get the FAP and Spinach into a single construct. <br \><br \></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p> Our cloning procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions. However, these issues were eventually ironed out and we managed to get the FAP and Spinach into a single construct. <br \><br \></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We have attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. The cloning using the plasmid vector worked in the first trial. However, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. We have not been able to pinpoint the <del class="diffchange diffchange-inline">problems</del>. </p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We have attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. The cloning using the plasmid vector worked in the first trial. However, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. We have not been able to pinpoint the <ins class="diffchange diffchange-inline">cause for this</ins>. </p></div></td></tr>
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</table>Ychoohttp://2012.igem.org/wiki/index.php?title=Team:Carnegie_Mellon/Met-Challenges&diff=254335&oldid=prevYchoo at 04:40, 3 October 20122012-10-03T04:40:20Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Challenges with Molecular Cloning</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Challenges with Molecular Cloning</h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p> Our cloning procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions. <br \></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> Our cloning procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions. <ins class="diffchange diffchange-inline">However, these issues were eventually ironed out and we managed to get the FAP and Spinach into a single construct. <br \></ins><br \></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>We have attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. The cloning using the plasmid vector worked in the first trial. However, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. We have not been able to pinpoint the problems. </<del class="diffchange diffchange-inline">li</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We have attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. The cloning using the plasmid vector worked in the first trial. However, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. We have not been able to pinpoint the problems. </<ins class="diffchange diffchange-inline">p</ins>></div></td></tr>
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</table>Ychoohttp://2012.igem.org/wiki/index.php?title=Team:Carnegie_Mellon/Met-Challenges&diff=254320&oldid=prevYchoo at 04:38, 3 October 20122012-10-03T04:38:35Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><li> </del><p> <del class="diffchange diffchange-inline">Cloning </del>procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions. <<del class="diffchange diffchange-inline">/p> </li</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p> <ins class="diffchange diffchange-inline">Our cloning </ins>procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions. <<ins class="diffchange diffchange-inline">br \</ins>></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><li> <p> </del>We have attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. The cloning using the plasmid vector worked in the first trial. However, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. We have not been able to pinpoint the problems. <del class="diffchange diffchange-inline"></p></del></li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>We have attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. The cloning using the plasmid vector worked in the first trial. However, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. We have not been able to pinpoint the problems. </li></div></td></tr>
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</table>Ychoohttp://2012.igem.org/wiki/index.php?title=Team:Carnegie_Mellon/Met-Challenges&diff=253683&oldid=prevYchoo at 03:26, 3 October 20122012-10-03T03:26:29Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Challenges with Molecular Cloning</h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1>Challenges with Molecular Cloning</h1></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline"><p></del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li> Cloning procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions.</li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li<ins class="diffchange diffchange-inline">> <p</ins>> Cloning procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions. <ins class="diffchange diffchange-inline"></p> </ins></li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><li> We have attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. The cloning using the plasmid vector worked in the first trial. However, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. We have not been able to pinpoint the problems.</<del class="diffchange diffchange-inline">li</del>></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><li<ins class="diffchange diffchange-inline">> <p</ins>> We have attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. The cloning using the plasmid vector worked in the first trial. However, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. We have not been able to pinpoint the problems. </<ins class="diffchange diffchange-inline">p</ins>></<ins class="diffchange diffchange-inline">li</ins>></div></td></tr>
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</table>Ychoo