http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/miRNA_Kit/October&feed=atom&action=historyTeam:Heidelberg/Notebook/miRNA Kit/October - Revision history2024-03-29T10:35:58ZRevision history for this page on the wikiMediaWiki 1.16.5http://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/miRNA_Kit/October&diff=178228&oldid=prevOla: /* 24/10/2010 */2010-10-27T09:58:01Z<p><span class="autocomment">24/10/2010</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Transfection Transfection]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Transfection Transfection]</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;">1) HeLa luc2-miR122BS and miR122</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;">2) for ELISA - pBS_U6 containing hAAT with miRsAg imperfect binding sites, and miRsAg </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;">ratios 1:1 ans 4:1</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===26/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===26/10/2010===</div></td></tr>
</table>Olahttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/miRNA_Kit/October&diff=178181&oldid=prevOla: /* 26/10/2010 */2010-10-27T09:54:30Z<p><span class="autocomment">26/10/2010</span></p>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#ELISA ELISA] was performed on media in which HeLa cells we grown</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#ELISA ELISA] was performed on media in which HeLa cells we grown<ins class="diffchange diffchange-inline">, as hAAT is secreted by cells.</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:Heidelberg/Single_Bottom}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/Single_Bottom}}</div></td></tr>
</table>Olahttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/miRNA_Kit/October&diff=178104&oldid=prevOla: /* 26/10/2010 */2010-10-27T09:49:35Z<p><span class="autocomment">26/10/2010</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===26/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===26/10/2010===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Dual_Luciferase_Assay Dual Luciferase Assay]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Dual_Luciferase_Assay Dual Luciferase Assay]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:PsiCheck.png|thumb|center|600px|'''Figure 3: Tuning of gene expression through different imperfect miR122 binding sites in psiCHECK-2.''' Construct was transfected into HeLa cells together with an plasmid expressing miR122. Control without binding site was used for normalization.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:PsiCheck.png|thumb|center|600px|'''Figure 3: Tuning of gene expression through different imperfect miR122 binding sites in psiCHECK-2.''' Construct was transfected into HeLa cells together with an plasmid expressing miR122. Control without binding site was used for normalization.]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del style="color: red; font-weight: bold; text-decoration: none;">* [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#ELISA ELISA]</del></div></td><td colspan="2"> </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: #eee; color:black; font-size: smaller;"><div>[[Image:Haat U6HD2010.jpg|thumb|center|600px|'''Figure 1: Tuning of gene expression through different imperfect shRNA miR binding sites in pBS_U6''' Gene expression quantified via dual luciferase assay for constructs containing different imperfect binding sites for shhAAT.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Haat U6HD2010.jpg|thumb|center|600px|'''Figure 1: Tuning of gene expression through different imperfect shRNA miR binding sites in pBS_U6''' Gene expression quantified via dual luciferase assay for constructs containing different imperfect binding sites for shhAAT.]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Haat_H1HD2010.jpg|thumb|center|600px|'''Figure 2: Tuning of gene expression through different imperfect shRNA miR binding sites in pBS_H1.''' Gene expression quantified via dual luciferase assay for constructs containing different imperfect binding sites for shhAAT.]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:Haat_H1HD2010.jpg|thumb|center|600px|'''Figure 2: Tuning of gene expression through different imperfect shRNA miR binding sites in pBS_H1.''' Gene expression quantified via dual luciferase assay for constructs containing different imperfect binding sites for shhAAT.]]</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;"><br><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;"></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;">* [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#ELISA ELISA] was performed on media in which HeLa cells we grown</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:Heidelberg/Single_Bottom}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/Single_Bottom}}</div></td></tr>
</table>Olahttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/miRNA_Kit/October&diff=177803&oldid=prevOla: /* 26/10/2010 */2010-10-27T09:32:45Z<p><span class="autocomment">26/10/2010</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Dual_Luciferase_Assay Dual Luciferase Assay]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Dual_Luciferase_Assay Dual Luciferase Assay]</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;">[[Image:PsiCheck.png|thumb|center|600px|'''Figure 3: Tuning of gene expression through different imperfect miR122 binding sites in psiCHECK-2.''' Construct was transfected into HeLa cells together with an plasmid expressing miR122. Control without binding site was used for normalization.]]</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;">* [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#ELISA ELISA]</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;">[[Image:Haat U6HD2010.jpg|thumb|center|600px|'''Figure 1: Tuning of gene expression through different imperfect shRNA miR binding sites in pBS_U6''' Gene expression quantified via dual luciferase assay for constructs containing different imperfect binding sites for shhAAT.]]</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;">[[Image:Haat_H1HD2010.jpg|thumb|center|600px|'''Figure 2: Tuning of gene expression through different imperfect shRNA miR binding sites in pBS_H1.''' Gene expression quantified via dual luciferase assay for constructs containing different imperfect binding sites for shhAAT.]]</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: #eee; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/Single_Bottom}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/Single_Bottom}}</div></td></tr>
</table>Olahttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/miRNA_Kit/October&diff=177738&oldid=prevOla at 09:29, 27 October 20102010-10-27T09:29:23Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* dilution and co-transfection with miRsAg expressing construct into HeLa cells for ELISA measurements</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* dilution and co-transfection with miRsAg expressing construct into HeLa cells for ELISA measurements</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;">===23/10/2010===</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;">seeding HeLa cells</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;">===24/10/2010===</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;">[https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Transfection Transfection]</ins></div></td></tr>
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<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">===26/10/2010===</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><ins class="diffchange diffchange-inline">* [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Dual_Luciferase_Assay Dual Luciferase Assay]</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:Heidelberg/Single_Bottom}}</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>{{:Team:Heidelberg/Single_Bottom}}</div></td></tr>
</table>Olahttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/miRNA_Kit/October&diff=175233&oldid=prevOla: /* 10/10/2010 */2010-10-27T06:29:26Z<p><span class="autocomment">10/10/2010</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In presence of Tet repressor and miR122 that is not targeting it, less of firefly luciferase is expressed, but when miR sAg is expressed is recovered to 60% of original activity.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In presence of Tet repressor and miR122 that is not targeting it, less of firefly luciferase is expressed, but when miR sAg is expressed is recovered to 60% of original activity.</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div><del class="diffchange diffchange-inline">[[Image:101010on system.jpg | thumb | 350px | center| ON system test]]</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline"><br /><br /></ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===11/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===11/10/2010===</div></td></tr>
</table>Olahttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/miRNA_Kit/October&diff=175215&oldid=prevOla: /* 10/10/2010 */2010-10-27T06:28:01Z<p><span class="autocomment">10/10/2010</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In presence of Tet repressor and miR122 that is not targeting it, less of firefly luciferase is expressed, but when miR sAg is expressed is recovered to 60% of original activity.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>In presence of Tet repressor and miR122 that is not targeting it, less of firefly luciferase is expressed, but when miR sAg is expressed is recovered to 60% of original activity.</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;">[[Image:101010on system.jpg | thumb | 350px | center| ON system test]]</ins></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===11/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===11/10/2010===</div></td></tr>
</table>Olahttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/miRNA_Kit/October&diff=175201&oldid=prevOla at 06:26, 27 October 20102010-10-27T06:26:58Z<p></p>
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 06:26, 27 October 2010</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===05/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===05/10/2010===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>repressor construct:<br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>repressor construct:<br/></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* <i>sequencing results</i> from <del class="diffchange diffchange-inline">[[Igem2010/Main/synthetic miR Kit/October#05/10/2010 | </del>previous day<del class="diffchange diffchange-inline">]]</del>:</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* <i>sequencing results</i> from previous day:</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** once or twice the binding site in each of the T1 constructs, respectively</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** once or twice the binding site in each of the T1 constructs, respectively</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** once the binding site for the T2 construct</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** once the binding site for the T2 construct</div></td></tr>
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<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: #eee; color:black; font-size: smaller;"><div>===06/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===06/10/2010===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* waiting for colonies from <del class="diffchange diffchange-inline">[[Igem2010/Main/synthetic miR Kit/October#05/10/2010 | </del>previous night<del class="diffchange diffchange-inline">]]</del>, which did not grow after all</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* waiting for colonies from previous night, which did not grow after all</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: #eee; color:black; font-size: smaller;"><div>===07/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===07/10/2010===</div></td></tr>
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<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: #eee; color:black; font-size: smaller;"><div>===09/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===09/10/2010===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* sequencing <del class="diffchange diffchange-inline">[[Igem2010/Main/synthetic miR Kit/October#08/10/2010 | yesterday]] </del>was fine but construct contains TetO<sub>2</sub>, thus: maybe Q2 and Q3 were confused</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* sequencing <ins class="diffchange diffchange-inline">the day before </ins>was fine but construct contains TetO<sub>2</sub>, thus: maybe Q2 and Q3 were confused</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** that would explain why measurements with TetR constructs are not working properly even though sequences were fine</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** that would explain why measurements with TetR constructs are not working properly even though sequences were fine</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* cloning of binding sites for shhAAT (perfect binding site generated via annealing, and randomized ones PCR amplified)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* cloning of binding sites for shhAAT (perfect binding site generated via annealing, and randomized ones PCR amplified)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** touch-down PCR setup: 17 µl ddH<sub>2</sub>O, 4 µl each oligo (i.e. respective forward primer + second strand oligo[[Igem2010/Main/Primer_database#Standart_Kit_Cloning_Primers | D121]]), 25 µl Phusion PCR MasterMix (2x)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** touch-down PCR setup: 17 µl ddH<sub>2</sub>O, 4 µl each oligo (i.e. respective forward primer + second strand oligo[[Igem2010/Main/Primer_database#Standart_Kit_Cloning_Primers | D121]]), 25 µl Phusion PCR MasterMix (2x)</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*** touch-down PCR protocol: [<del class="diffchange diffchange-inline">[Igem2010</del>/<del class="diffchange diffchange-inline">Main</del>/<del class="diffchange diffchange-inline">Protocols</del>/<del class="diffchange diffchange-inline">Phusion_PCR</del>#shRNA-<del class="diffchange diffchange-inline">PCR Stefan M. protocol| </del>like this<del class="diffchange diffchange-inline">]</del>] but with increase of annealing temperature from 70°C to 62°C (i. e. 0.5°C per cycle, 16x) and four additional cycles with 62°C</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*** touch-down PCR protocol: [<ins class="diffchange diffchange-inline">https:</ins>//<ins class="diffchange diffchange-inline">2010.igem.org</ins>/<ins class="diffchange diffchange-inline">Team:Heidelberg/Notebook/Methods</ins>#shRNA-<ins class="diffchange diffchange-inline">PCR_protocol </ins>like this] but with increase of annealing temperature from 70°C to 62°C (i. e. 0.5°C per cycle, 16x) and four additional cycles with 62°C</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** annealing of perfect binding against shhAAT sites with an oligo-based strategy following the protocol from the [[Igem2010/Main/synthetic miR Kit/October#03/10/2010 | 3<sup>rd</sup> October]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** annealing of perfect binding against shhAAT sites with an oligo-based strategy following the protocol from the [[Igem2010/Main/synthetic miR Kit/October#03/10/2010 | 3<sup>rd</sup> October]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** digestion of PCR amplified binding sites with AgeI and XhoI, digestion of Q2 backbone containing the shhAAT with XhoI and XmaI, afterwards heat inactivation, nucleotide removal and ligation following [https://2010.igem.org/3A_Assembly standard protocol recommendations]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** digestion of PCR amplified binding sites with AgeI and XhoI, digestion of Q2 backbone containing the shhAAT with XhoI and XmaI, afterwards heat inactivation, nucleotide removal and ligation following [https://2010.igem.org/3A_Assembly standard protocol recommendations]</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===10/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===10/10/2010===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:20101010 colonypcr shhAAT p 12 22 bs LOLA.png|thumb|350px|right|Gel 101010-1. Colony PCR for construct containing shhAAT and binding sites. Expected is an amplified fragment a bit smaller than 300 bp which can be seen on lane 3, 4, 6, 7, 8 (perfect binding sites), 12, 13, 14, 16 and 17 (randomized binding sites). Lane 1 and Lane 26: 1kb Plus Ladder (Invitrogen)]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Image:20101010 colonypcr shhAAT p 12 22 bs LOLA.png|thumb|350px|right|Gel 101010-1. Colony PCR for construct containing shhAAT and binding sites. Expected is an amplified fragment a bit smaller than 300 bp which can be seen on lane 3, 4, 6, 7, 8 (perfect binding sites), 12, 13, 14, 16 and 17 (randomized binding sites). Lane 1 and Lane 26: 1kb Plus Ladder (Invitrogen)]]</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* colony pcr reveals some positive clones (see gel 101010-1) from <del class="diffchange diffchange-inline">[[Igem2010/Main/synthetic miR Kit/October#09/10/2010 | </del>previous day<del class="diffchange diffchange-inline">]]</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* colony pcr reveals some positive clones (see gel 101010-1) from previous day</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** inoculation of 5 ml LB Amp cultures for mini-prep over night</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** inoculation of 5 ml LB Amp cultures for mini-prep over night</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>
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<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: #eee; color:black; font-size: smaller;"><div>===11/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===11/10/2010===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* mini-prep of ten cultures from <del class="diffchange diffchange-inline">[[Igem2010/Main/synthetic miR Kit/October#10/10/2010 | yesterday]] </del>and direct transfection to test the system</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* mini-prep of ten cultures from <ins class="diffchange diffchange-inline">previous day </ins>and direct transfection to test the system</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** amount: each 100 µl (c=2.5 ng/µl)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** amount: each 100 µl (c=2.5 ng/µl)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><br/></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** 25 µl Phusion PCR MasterMix (2x)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** 25 µl Phusion PCR MasterMix (2x)</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** touch-down PCR protocol: </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** touch-down PCR protocol: </div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*** [<del class="diffchange diffchange-inline">[Igem2010</del>/<del class="diffchange diffchange-inline">Main</del>/<del class="diffchange diffchange-inline">Protocols</del>/<del class="diffchange diffchange-inline">Phusion_PCR</del>#shRNA-<del class="diffchange diffchange-inline">PCR Stefan M. protocol| </del>like this<del class="diffchange diffchange-inline">]</del>] but with increase of annealing temperature from 70°C to 62°C (i. e. 0.5°C per cycle, 16x) </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*** [<ins class="diffchange diffchange-inline">https:</ins>//<ins class="diffchange diffchange-inline">2010.igem.org</ins>/<ins class="diffchange diffchange-inline">Team:Heidelberg/Notebook/Methods</ins>#shRNA-<ins class="diffchange diffchange-inline">PCR_protocol </ins>like this] but with increase of annealing temperature from 70°C to 62°C (i. e. 0.5°C per cycle, 16x) </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** and four additional cycles with 62°C, elongation time only 10 seconds</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** and four additional cycles with 62°C, elongation time only 10 seconds</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* digestion of generated binding sites and the pSMB_miMeasure backbone with EcoRI and PstI, then nucleotide removal</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* digestion of generated binding sites and the pSMB_miMeasure backbone with EcoRI and PstI, then nucleotide removal</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** elongation time: 90<nowiki>''</nowiki></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*** elongation time: 90<nowiki>''</nowiki></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** <u>no positive</u> results, maybe due to insufficient screening primers (one for BBb standard, the other one for CMV resulting in an 1300 bp fragment)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** <u>no positive</u> results, maybe due to insufficient screening primers (one for BBb standard, the other one for CMV resulting in an 1300 bp fragment)</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>*** as a consequence: repetition of entire cloning with [http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_ef0511.pdf SAP] treated backbone <del class="diffchange diffchange-inline">[[Igem2010/Main/synthetic miR Kit/October#12/10/2010| tomorrow]]</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>*** as a consequence: repetition of entire cloning with [http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_ef0511.pdf SAP] treated backbone <ins class="diffchange diffchange-inline">following day</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: #eee; color:black; font-size: smaller;"><div>===13/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===13/10/2010===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>measurement construct for single binding sites: <br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>measurement construct for single binding sites: <br/></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* repetition of cloning from <del class="diffchange diffchange-inline">[[Igem2010/Main/synthetic miR Kit/October#11/10/2010 | </del>11/10/2010<del class="diffchange diffchange-inline">]]</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* repetition of cloning from 11/10/2010</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* <u>note:</u> vector concentration of pSMB_miMeasure is okay but after digestion and nucleotide removal it is always pretty low </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* <u>note:</u> vector concentration of pSMB_miMeasure is okay but after digestion and nucleotide removal it is always pretty low </div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** (loss of 75% of DNA on average whereas other DNA yields to 90% of purified DNA as compared to digested amount)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** (loss of 75% of DNA on average whereas other DNA yields to 90% of purified DNA as compared to digested amount)</div></td></tr>
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<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: #eee; color:black; font-size: smaller;"><div>===18/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===18/10/2010===</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* [<del class="diffchange diffchange-inline">[Igem2010</del>/<del class="diffchange diffchange-inline">Main</del>/<del class="diffchange diffchange-inline">Protocols</del>/Colony_PCR <del class="diffchange diffchange-inline">| </del>colony-PCR<del class="diffchange diffchange-inline">]</del>] of 4 colonies for each plate of [[Igem2010/Main/synthetic miR Kit/October#17/10/2010 | previous day transformations]] using reverse binding site oligo and forward hAAT primer expecting a fragment round about 1300 bp</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* [<ins class="diffchange diffchange-inline">https:</ins>//<ins class="diffchange diffchange-inline">2010.igem.org</ins>/<ins class="diffchange diffchange-inline">Team:Heidelberg/Notebook/Methods#</ins>Colony_PCR colony-PCR] of 4 colonies for each plate of [[Igem2010/Main/synthetic miR Kit/October#17/10/2010 | previous day transformations]] using reverse binding site oligo and forward hAAT primer expecting a fragment round about 1300 bp</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** gel 101018-1 reveals almost positive samples except two (data not yet shown)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** gel 101018-1 reveals almost positive samples except two (data not yet shown)</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>
</table>Olahttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/miRNA_Kit/October&diff=175025&oldid=prevOla: /* 04/10/2010 */2010-10-27T06:17:35Z<p><span class="autocomment">04/10/2010</span></p>
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<td colspan='2' style="background-color: white; color:black;">← Older revision</td>
<td colspan='2' style="background-color: white; color:black;">Revision as of 06:17, 27 October 2010</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===04/10/2010===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===04/10/2010===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>repressor construct:<br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>repressor construct:<br/></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* [[Igem2010/Main/Protocols/Colony_PCR | colony-PCR]] of 6 colonies for each plate of <del class="diffchange diffchange-inline">[[Igem2010/Main/synthetic miR Kit/October#03/10/2010 | </del>previous day transformations<del class="diffchange diffchange-inline">]]</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* [[Igem2010/Main/Protocols/Colony_PCR | colony-PCR]] of 6 colonies for each plate of previous day transformations</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** gel 101004-1 reveals almost positive samples</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** gel 101004-1 reveals almost positive samples</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* submission of each a promising mini-prep for sequencing of binding site and correct insert</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* submission of each a promising mini-prep for sequencing of binding site and correct insert</div></td></tr>
</table>Olahttp://2010.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/miRNA_Kit/October&diff=174984&oldid=prevOla: /* 02/10/2010 */2010-10-27T06:16:04Z<p><span class="autocomment">02/10/2010</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 06:16, 27 October 2010</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>repressor construct:<br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>repressor construct:<br/></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>* [[Igem2010/Main/Protocols/Colony_PCR | colony-PCR]] of 6 colonies for each plate of <del class="diffchange diffchange-inline">[https://2010.igem.org/Team:Heidelberg/Notebook/miRNA_Kit/October#01.2F10.2F2010 </del>previous day transformations<del class="diffchange diffchange-inline">]</del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>* [[Igem2010/Main/Protocols/Colony_PCR | colony-PCR]] of 6 colonies for each plate of previous day transformations</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** analysis, see gel 101002-1</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>** analysis, see gel 101002-1</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* twice test digestion of promising mini-preps, following [https://2010.igem.org/3A_Assembly standard protocol recommendations] <br/></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>* twice test digestion of promising mini-preps, following [https://2010.igem.org/3A_Assembly standard protocol recommendations] <br/></div></td></tr>
</table>Ola