http://2011.igem.org/wiki/index.php?title=Team:Calgary/Notebook/Calendar/OriT&feed=atom&action=historyTeam:Calgary/Notebook/Calendar/OriT - Revision history2024-03-28T21:04:17ZRevision history for this page on the wikiMediaWiki 1.16.0http://2011.igem.org/wiki/index.php?title=Team:Calgary/Notebook/Calendar/OriT&diff=203770&oldid=prevEmily Hicks at 04:44, 29 September 20112011-09-29T04:44:41Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>TITLE = Genomic Tools for Pseudomonas |</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>TITLE = Genomic Tools for <ins class="diffchange diffchange-inline"><i></ins>Pseudomonas<ins class="diffchange diffchange-inline"></i> </ins>|</div></td></tr>
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</table>Emily Hickshttp://2011.igem.org/wiki/index.php?title=Team:Calgary/Notebook/Calendar/OriT&diff=200039&oldid=prevMyarcell at 02:25, 29 September 20112011-09-29T02:25:43Z<p></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>TITLE = Genomic <del class="diffchange diffchange-inline">Library Expression </del>|</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>TITLE = Genomic <ins class="diffchange diffchange-inline">Tools for Pseudomonas </ins>|</div></td></tr>
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</table>Myarcellhttp://2011.igem.org/wiki/index.php?title=Team:Calgary/Notebook/Calendar/OriT&diff=189928&oldid=prevSaeedq at 07:31, 28 September 20112011-09-28T07:31:36Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>The digested parts were then modified using T4 Polymerase to create “blunt ends”, followed by another purification to isolate the DNA.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>The digested parts were then modified using T4 Polymerase to create “blunt ends”, followed by another purification to isolate the DNA.</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></ol></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div></ol></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Also, I had Emily check up on the parts sent for sequencing and his preliminary analysis indicated that the parts did not actually contain he promoter region, so there was an error in the procedure somewhere. However there is no time to go back and try to repeat this and this portion of the project must be abandoned.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>Also, I had Emily check up on the parts sent for sequencing and his preliminary analysis indicated that the parts did not actually contain he promoter region, so there was an error in the procedure somewhere. However there is no time to go back and try to repeat this and this portion of the project must be abandoned.</p></div></td></tr>
</table>Saeedqhttp://2011.igem.org/wiki/index.php?title=Team:Calgary/Notebook/Calendar/OriT&diff=181560&oldid=prevSj.dixon at 02:37, 27 September 20112011-09-27T02:37:33Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>I transformed TOP10 cells using the part I732019 which is a LacZ reporter fused to RBS.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>I transformed TOP10 cells using the part I732019 which is a LacZ reporter fused to RBS.</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>The transformed bacteria were then cultured and miniprepped and the identity confirmed by restriction digests (Figure 2).</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><li>The transformed bacteria were then cultured and miniprepped and the identity confirmed by restriction digests (Figure 2).</li></div></td></tr>
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</table>Sj.dixonhttp://2011.igem.org/wiki/index.php?title=Team:Calgary/Notebook/Calendar/OriT&diff=181557&oldid=prevSj.dixon at 02:36, 27 September 20112011-09-27T02:36:59Z<p></p>
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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 style="color: red; font-weight: bold; text-decoration: none;"><h3>Monday August 29, 2011 – Friday September 2, 2011</h3></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 style="color: red; font-weight: bold; text-decoration: none;"><h4> Author: Saeed</h4></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 style="color: red; font-weight: bold; text-decoration: none;"><h4>OBJECTIVE</h4></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 style="color: red; font-weight: bold; text-decoration: none;"><p> Designing a plasmid which contains ORI-T/R, gentamicin resistance and a double terminator flowed by site where genomic DNA from Pseudomonas Fluorescence and Putida can be inserted, followed by a specific LacZ reporter.</p></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 style="color: red; font-weight: bold; text-decoration: none;"><p><b>REFERENCE:</b> See the previous journal for the week of August 22-26.</p></ins></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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><h4> Summary of Actions Taken</h4></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 style="color: red; font-weight: bold; text-decoration: none;"><ol></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 style="color: red; font-weight: bold; text-decoration: none;"><li>Ran restriction digests of the cultured I732901 transformed bacteria as well as the bacteria transformed with the constructs, and ran them on a gel (Figure 1).</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>Designed a new approach to the main plasmid construction technique and ordered primers to accomplish this. </li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>Basically this new technique would involve adding new cut sites into amplified fragments, thereby making the procedure more efficient and shorter as previous attempts had failed.</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>Cleaned the lab.</li></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><ins style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"><h4>Additional Activities Performed</h4></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 style="color: red; font-weight: bold; text-decoration: none;"><ol></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 style="color: red; font-weight: bold; text-decoration: none;"><li>I transformed TOP10 cells using the part I732019 which is a LacZ reporter fused to RBS.</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>The transformed bacteria were then cultured and miniprepped and the identity confirmed by restriction digests (Figure 2).</li></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"><h4>RESULTS</h4></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"><img src="https://static.igem.org/mediawiki/2011/9/9c/Calgary2011_Saeed_Gel_%28Sept_2%29_fig1.JPG" width="500px"></img></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 style="color: red; font-weight: bold; text-decoration: none;"><p><b>Figure 1.</b> The gel is of poor quality due to breakage, two faint barely separable bands can be observed for lanes 2-11. Two distinct bands observed at lanes 12 and 13.</p></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"><img src="https://static.igem.org/mediawiki/2011/6/60/Calgary2011_Saeed_Gel_%28Sept_2%29_fig2.JPG" width="500px"></img> </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 style="color: red; font-weight: bold; text-decoration: none;"><p><b>Figure 2.</b> All lanes show either the digested or undigested I732019 part. *Note the legend should say should say I732019 not I732109.</p></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"><h4>INTERPRETATION</h4></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"><p>Figure 1 illustrates that the identity of the pUC plasmids cannot be ascertained as it is very unclear and some lanes show two bands while others only show one. However since three bands cannot be seen the construction/ligation/transformation was not successful and must be repeated. But the identity of the I732901 samples has been confirmed.</p></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"><p>Figure 2 illustrates that the I732019 was successfully digested and therefore the identity has been confirmed. Both the insert and the vector are of similar lengths so they overlap.</p></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The primers of the gene found in objective one needs to have primers designed to be included in RT-PCR experiment. The rpoD primer needs to be ordered.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The primers of the gene found in objective one needs to have primers designed to be included in RT-PCR experiment. The rpoD primer needs to be ordered.</p></div></td></tr>
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</table>Sj.dixonhttp://2011.igem.org/wiki/index.php?title=Team:Calgary/Notebook/Calendar/OriT&diff=181513&oldid=prevSj.dixon at 02:27, 27 September 20112011-09-27T02:27:31Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 1.</b>No bands can be observed for the PCR reactions. Very faint bands can be observed for the plasmid samples 301-308.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 1.</b>No bands can be observed for the PCR reactions. Very faint bands can be observed for the plasmid samples 301-308.</p></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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><h4>INTERPRETATION</h4></ins></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><del class="diffchange diffchange-inline">INTERPRETATION</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p>Observations from Figure 1 suggest that the process is going well but that significant quantity of the insert may have been lost along the way (which is expected) given the number of PCR clean ups that have taken place.</p></ins></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><del class="diffchange diffchange-inline">Observations from </del>Figure <del class="diffchange diffchange-inline">1 suggest </del>that the <del class="diffchange diffchange-inline">process is going well but that significant quantity of </del>the <del class="diffchange diffchange-inline">insert may have been lost along the way (which is expected) given the number of PCR clean ups </del>that <del class="diffchange diffchange-inline">have taken place</del>.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p></ins>Figure <ins class="diffchange diffchange-inline">2 suggests </ins>that the <ins class="diffchange diffchange-inline">both 151 and 152 were successfully amplified and </ins>the <ins class="diffchange diffchange-inline">single distinct band observed for lanes 6 and 8 suggest </ins>that <ins class="diffchange diffchange-inline">samples 303 and 304 were successfully restriction digested, though not much more can be determined</ins>. <ins class="diffchange diffchange-inline"></p></ins></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><del class="diffchange diffchange-inline">Figure 2 suggests that the both 151 and 152 were successfully amplified and the single distinct band observed for lanes 6 and 8 suggest that samples 303 and 304 were successfully restriction digested, though not much more can be determined. </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p></ins>Figure 3 indicates that the PCR was not successful perhaps because the buffer concentration was wrong, also a different Taq was used than normal. This will have to be looked into. And faint bands can be seen for the plasmid DNA tubes but only a single band can be seen suggesting that they are pure.<ins class="diffchange diffchange-inline"></p></ins></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </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>Figure 3 indicates that the PCR was not successful perhaps because the buffer concentration was wrong, also a different Taq was used than normal. This will have to be looked into. And faint bands can be seen for the plasmid DNA tubes but only a single band can be seen suggesting that they are pure.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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</table>Sj.dixonhttp://2011.igem.org/wiki/index.php?title=Team:Calgary/Notebook/Calendar/OriT&diff=181508&oldid=prevSj.dixon at 02:26, 27 September 20112011-09-27T02:26:48Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The competent cells from Maggie’s lab likely do not have the right genotype (i.e. they have a lacZ gene, rather than the M15 genotype, which does not have a lacZ), which explains why most colonies are blue. The colonies on the border are not white because the IPTG and XGal was not spread evenly on the border. White colonies in the repeated transformations using Top10 can be cultured overnight to test for the right genotype (by restriction digest). Saeed checked the colonies, but none seemed to have the right genotype. A new strategy will be developed by Saeed with Dr. Mayi and Maggie to ensure faster and more accurate cloning steps for the next week.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p>The competent cells from Maggie’s lab likely do not have the right genotype (i.e. they have a lacZ gene, rather than the M15 genotype, which does not have a lacZ), which explains why most colonies are blue. The colonies on the border are not white because the IPTG and XGal was not spread evenly on the border. White colonies in the repeated transformations using Top10 can be cultured overnight to test for the right genotype (by restriction digest). Saeed checked the colonies, but none seemed to have the right genotype. A new strategy will be developed by Saeed with Dr. Mayi and Maggie to ensure faster and more accurate cloning steps for the next week.</p></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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><br></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 style="color: red; font-weight: bold; text-decoration: none;"><h3>August 22nd-26th, 2011</h3></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 style="color: red; font-weight: bold; text-decoration: none;"><h4> Author: Saeed Qureshi</h4></ins></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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><h4>OBJECTIVE</h4></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 style="color: red; font-weight: bold; text-decoration: none;"><p> Designing a plasmid which contains ORI-T/R, gentamicin resistance and a double terminator flowed by site where genomic DNA from Pseudomonas Fluorescence and Putida can be inserted, followed by a specific LacZ reporter.</p></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 style="color: red; font-weight: bold; text-decoration: none;"><p><b>REFERENCE:</b> See Saeed's previous journal for the week of August 15-19.</p></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 style="color: red; font-weight: bold; text-decoration: none;"><h4> Summary of Actions Taken</h4></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 style="color: red; font-weight: bold; text-decoration: none;"><ol></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 style="color: red; font-weight: bold; text-decoration: none;"><li>A Poly A tail was added to the insert fragment.</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>This was then cut with Pst1 and run on a gel (Figure 1).</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>The Vector (plasmid was also prepared for insertion of the plasmid by cutting with SfiI</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>It was then blunt ended using T4 polymerase followed by a PCR cleanup and running a sample on a gel (Figure 1)</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>A poly T tail was added followed by a PCR cleanup.</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>It was then cut with Pst1 and run on a gel (Figure 2).</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>The vector tubes were then incubated with Antarctic phosphate.</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>The vector and Insert were ligated together for either an hour or overnight, and then this sample was used to transform DH5 alpha and TOP10 competent cells.</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>I tried selecting for white colonies (successfully transformed) and culturing them, as they would lack the LacZ gene necessary for blue color on an IPTG/X-Gal plate.</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>These cultures were then miniprepped with the intention of performing restriction digests to ascertain the identity of the plasmid used to transform the bacteria.</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>In preparation for the next step of the process strains carrying the specific LacZ gene (mini CTX 2) were cultured and then miniprepped.</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>In case the process had to be repeated some of the insert was again amplified by PCR and then purified (Figure 2).</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>To see whether any of the samples of the plasmid (vector) were impure, samples of all of these were run on a gel (Figure 3)</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 style="color: red; font-weight: bold; text-decoration: none;"></ol></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"><h4>Additional Activities Performed</h4></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 style="color: red; font-weight: bold; text-decoration: none;"><ol></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 style="color: red; font-weight: bold; text-decoration: none;"><li>I transformed TOP10 cells using the part I732901 which was an IPTG inducible LacZ reporter.</li></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 style="color: red; font-weight: bold; text-decoration: none;"><li>The transformed bacteria was then cultured and miniprepped.</li></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 style="color: red; font-weight: bold; text-decoration: none;"></ol></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"><h4>RESULTS</h4></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"> </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 style="color: red; font-weight: bold; text-decoration: none;"><img src="https://static.igem.org/mediawiki/2011/6/6c/Saeed_Gel_%28Aug_26%29_fig1.JPG" width="500px"></img></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 style="color: red; font-weight: bold; text-decoration: none;"><p><b>Figure 1.</b> Faint bands/smears can be seen in the 100bp range for the 151 and 152 working product (possibly due to the low concentration and small size). There appears to be two distinct bands for the 303 and 304 samples cut with SfiI but these haven’t been able separate sufficiently.</p></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"> </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 style="color: red; font-weight: bold; text-decoration: none;"><img src="https://static.igem.org/mediawiki/2011/9/9c/Saeed_Gel_%28Aug26%29_Fig2.JPG" width="500px"></img></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 style="color: red; font-weight: bold; text-decoration: none;"><p><b>Figure 2.</b>The PCR amplified products 151 and 152 show up in the 400-500 bp range. The 303 and 304 working product clearly displays distinct bands as opposed to smears proving that they have been digested.</p></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"> </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 style="color: red; font-weight: bold; text-decoration: none;"><img src="https://static.igem.org/mediawiki/2011/c/c1/Saeed_Gel_%28Aug_26%29_fig3.JPG" width="500px"></img></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 style="color: red; font-weight: bold; text-decoration: none;"><p><b>Figure 1.</b>No bands can be observed for the PCR reactions. Very faint bands can be observed for the plasmid samples 301-308.</p></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;">INTERPRETATION</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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;">Observations from Figure 1 suggest that the process is going well but that significant quantity of the insert may have been lost along the way (which is expected) given the number of PCR clean ups that have taken place.</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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;">Figure 2 suggests that the both 151 and 152 were successfully amplified and the single distinct band observed for lanes 6 and 8 suggest that samples 303 and 304 were successfully restriction digested, though not much more can be determined. </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 style="color: red; font-weight: bold; text-decoration: none;"></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 style="color: red; font-weight: bold; text-decoration: none;">Figure 3 indicates that the PCR was not successful perhaps because the buffer concentration was wrong, also a different Taq was used than normal. This will have to be looked into. And faint bands can be seen for the plasmid DNA tubes but only a single band can be seen suggesting that they are pure.</ins></div></td></tr>
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</table>Sj.dixonhttp://2011.igem.org/wiki/index.php?title=Team:Calgary/Notebook/Calendar/OriT&diff=181477&oldid=prevSj.dixon at 02:19, 27 September 20112011-09-27T02:19:20Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h4>INTERPRETATION</h4></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h4>INTERPRETATION</h4></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><p>The competent cells from Maggie’s lab likely do not have the right genotype (i.e. they have a lacZ gene, rather than the <del class="diffchange diffchange-inline">?</del>M15 genotype, which does not have a lacZ), which explains why most colonies are blue. The colonies on the border are not white because the IPTG and XGal was not spread evenly on the border. White colonies in the repeated transformations using Top10 can be cultured overnight to test for the right genotype (by restriction digest). Saeed checked the colonies, but none seemed to have the right genotype. A new strategy will be developed by Saeed with Dr. Mayi and Maggie to ensure faster and more accurate cloning steps for the next week.</p></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><p>The competent cells from Maggie’s lab likely do not have the right genotype (i.e. they have a lacZ gene, rather than the M15 genotype, which does not have a lacZ), which explains why most colonies are blue. The colonies on the border are not white because the IPTG and XGal was not spread evenly on the border. White colonies in the repeated transformations using Top10 can be cultured overnight to test for the right genotype (by restriction digest). Saeed checked the colonies, but none seemed to have the right genotype. A new strategy will be developed by Saeed with Dr. Mayi and Maggie to ensure faster and more accurate cloning steps for the next week.</p></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li>Choose additional candidate genes for RT-PCR experiments (with NA treatment) from Pseudomonas.com searches.</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li>Choose additional candidate genes for RT-PCR experiments (with NA treatment) from Pseudomonas.com searches.</li></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> <li>Help Saeed with assays of E. coli strains with IPTG-LacZ inserts with <del class="diffchange diffchange-inline">chlorophenolred</del>-ß-D-galactopyranoside.</li></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> <li>Help Saeed with assays of E. coli strains with IPTG-LacZ inserts with <ins class="diffchange diffchange-inline">chlorophenol red</ins>-ß-D-galactopyranoside.</li></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li>Design RT-PCR experiments to check validity of primers, and design a new primer for the housekeeping gene (rpoD)</li></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> <li>Design RT-PCR experiments to check validity of primers, and design a new primer for the housekeeping gene (rpoD)</li></div></td></tr>
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</table>Sj.dixonhttp://2011.igem.org/wiki/index.php?title=Team:Calgary/Notebook/Calendar/OriT&diff=181475&oldid=prevSj.dixon at 02:18, 27 September 20112011-09-27T02:18:22Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 02:18, 27 September 2011</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h4>RESULTS:</h4></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h4>RESULTS:</h4></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><img src="https://static.igem.org/mediawiki/2011/9/9a/Calgary2011_Gel_%28Saeed_for_ORiT_Aug_19%29_fig1.JPG" width="500px"></img></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 1.</b> B0015 (151B) and B0015 (152B) both show up in the 300-400 bp range. The positive control does not show up in the gel.</p></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><p><b>Figure 1.</b> B0015 (151B) and B0015 (152B) both show up in the 300-400 bp range. The positive control does not show up in the gel.</p></div></td></tr>
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</table>Sj.dixonhttp://2011.igem.org/wiki/index.php?title=Team:Calgary/Notebook/Calendar/OriT&diff=181469&oldid=prevSj.dixon at 02:16, 27 September 20112011-09-27T02:16:41Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h4>RESULTS:</h4></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h4>RESULTS:</h4></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 colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"><p><b>Figure 1.</b> B0015 (151B) and B0015 (152B) both show up in the 300-400 bp range. The positive control does not show up in the gel.</p></ins></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><del class="diffchange diffchange-inline">Figure 1. B0015 (151B) and B0015 (152B) both show up in the 300-400 bp range. The positive control does not show up in the gel.</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><h4>INTERPRETATION</h4></ins></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> </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><p></ins>Figure 1 shows bands for B0015 which confirm that the part was amplified and showed up in the appropriate size range. The failure of the (+) control to show up on the gel is likely because the during the PCR the annealing time was too short for the 800bp part, this was because times were reduced in order to obtain the PCR products faster. Though there was enough time for the actual desired product to be amplified.<ins class="diffchange diffchange-inline"></p></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">INTERPRETATION</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> </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>Figure 1 shows bands for B0015 which confirm that the part was amplified and showed up in the appropriate size range. The failure of the (+) control to show up on the gel is likely because the during the PCR the annealing time was too short for the 800bp part, this was because times were reduced in order to obtain the PCR products faster. Though there was enough time for the actual desired product to be amplified.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div></div></td></tr>
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</table>Sj.dixon