http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&feed=atom&action=history
Team:Heidelberg/Notebook/Killing II/7thweek - Revision history
2024-03-29T10:44:28Z
Revision history for this page on the wiki
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http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&diff=82379&oldid=prev
Pascal.kraemer: /* Sunday 09/21/2008 */
2008-10-28T21:35:18Z
<p><span class="autocomment">Sunday 09/21/2008</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 21:35, 28 October 2008</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 72 °C 10 min</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 72 °C 10 min</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 4 °C constant</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 4 °C constant</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://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/7thweek back]]</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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Team:Heidelberg/Notebook/Killing_II/8thweek | go to 8<sup>th</sup> week]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[Team:Heidelberg/Notebook/Killing_II/8thweek | go to 8<sup>th</sup> week]]</div></td></tr>
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Pascal.kraemer
http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&diff=82376&oldid=prev
Pascal.kraemer: /* Saturday 09/20/2008 */
2008-10-28T21:34:57Z
<p><span class="autocomment">Saturday 09/20/2008</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Gel of digestion: 1% Agarose, 135V, 30 min: [[Image:080920-controldigestion_pQE30-His_small.jpg |500 px | center]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Gel of digestion: 1% Agarose, 135V, 30 min: [[Image:080920-controldigestion_pQE30-His_small.jpg |500 px | center]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Inoculation of ONC of 25 colonies per colicin-pQE30</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Inoculation of ONC of 25 colonies per colicin-pQE30</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://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/7thweek back]]</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>==Sunday 09/21/2008==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Sunday 09/21/2008==</div></td></tr>
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Pascal.kraemer
http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&diff=82371&oldid=prev
Pascal.kraemer: /* Friday 09/19/2008 */
2008-10-28T21:34:34Z
<p><span class="autocomment">Friday 09/19/2008</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Of ONCs, eluted in 30µl H<sub>2</sub>; Qiagen Miniprepkit</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Of ONCs, eluted in 30µl H<sub>2</sub>; Qiagen Miniprepkit</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">[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/7thweek back]]</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>==Saturday 09/20/2008==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Saturday 09/20/2008==</div></td></tr>
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Pascal.kraemer
http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&diff=82367&oldid=prev
Pascal.kraemer: /* Thursday 09/18/2008 */
2008-10-28T21:34:18Z
<p><span class="autocomment">Thursday 09/18/2008</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**BBa_I0500 -> pBAD/araC</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**BBa_I0500 -> pBAD/araC</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**BBa_F1610 -> AHL sender part</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>**BBa_F1610 -> AHL sender part</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://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/7thweek back]]</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>==Friday 09/19/2008==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Friday 09/19/2008==</div></td></tr>
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Pascal.kraemer
http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&diff=82365&oldid=prev
Pascal.kraemer: /* Wednesday 09/17/2008 */
2008-10-28T21:34:00Z
<p><span class="autocomment">Wednesday 09/17/2008</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Probes of ColE9 His cloning were send to GATC for sequencing.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Probes of ColE9 His cloning were send to GATC for sequencing.</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">[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/7thweek back]]</ins></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>==Thursday 09/18/2008==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Thursday 09/18/2008==</div></td></tr>
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Pascal.kraemer
http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&diff=82362&oldid=prev
Pascal.kraemer: /* Tuesday 09/16/2008 */
2008-10-28T21:33:25Z
<p><span class="autocomment">Tuesday 09/16/2008</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 21:33, 28 October 2008</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Gelresults of PCR: There were no psitive bands for each PCR. But we used the wrong reverse primers. Because of that we started a new PCR with the right reverse primers (see protocol at pSB1A3-Receiver cloning)</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Gelresults of PCR: There were no psitive bands for each PCR. But we used the wrong reverse primers. Because of that we started a new PCR with the right reverse primers (see protocol at pSB1A3-Receiver cloning)</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;">[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/7thweek back]]</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>==Wednesday 09/17/2008==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Wednesday 09/17/2008==</div></td></tr>
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Pascal.kraemer
http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&diff=82353&oldid=prev
Pascal.kraemer: /* Monday 09/15/2008 */
2008-10-28T21:31:38Z
<p><span class="autocomment">Monday 09/15/2008</span></p>
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<td colspan='2' style="background-color: white; color:black;">Revision as of 21:31, 28 October 2008</td>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 4 °C constant</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 4 °C constant</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">[[https://2008.igem.org/Team:Heidelberg/Notebook/Killing_II/7thweek back]]</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>==Tuesday 09/16/2008==</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>==Tuesday 09/16/2008==</div></td></tr>
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Pascal.kraemer
http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&diff=72284&oldid=prev
Andreaskuehne at 15:46, 27 October 2008
2008-10-27T15:46:05Z
<p></p>
<a href="http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&diff=72284&oldid=70863">Show changes</a>
Andreaskuehne
http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&diff=70863&oldid=prev
Andreaskuehne: /* HisTag cloning of Colicins for purification */
2008-10-27T08:43:15Z
<p><span class="autocomment">HisTag cloning of Colicins for purification</span></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 2.0 µl PstI</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 2.0 µl PstI</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 5.0 µl BSA 10x</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 5.0 µl BSA 10x</div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div> 16.0 µl H<sub>2</<del class="diffchange diffchange-inline">2sub</del>>O</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> 16.0 µl H<sub>2</<ins class="diffchange diffchange-inline">sub</ins>>O</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> -------</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> -------</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 50.0 µl</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div> 50.0 µl</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Gel of Digestion: 1% Agarose,135 V, 40 min [[Image:080919-controldigestion_pQE-30_ColE9_small.jpg |500 px | center]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Gel of Digestion: 1% Agarose,135 V, 40 min [[Image:080919-controldigestion_pQE-30_ColE9_small.jpg |500 px | center]]</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Gelresults: Maybe there is no insert or the enzymes didn't cut.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*Gelresults: Maybe there is no insert or the enzymes didn't cut.</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 class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Sender cloning: pBAD promoter===</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>===Sender cloning: pBAD promoter===</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*BBa_I0500 ONC of 2008 stock did not grow</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>*BBa_I0500 ONC of 2008 stock did not grow</div></td></tr>
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Andreaskuehne
http://2008.igem.org/wiki/index.php?title=Team:Heidelberg/Notebook/Killing_II/7thweek&diff=70862&oldid=prev
Andreaskuehne: New page: <html> <link rel='stylesheet' href='http://igem-heidelberg.de/fileadmin/Wiki/Heidelberg2.css' type='text/css' /> <link rel="stylesheet" href="http://igem-heidelberg.de/fileadmin/Wiki/Menu....
2008-10-27T08:41:48Z
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'''7<sup>th</sup> week'''<br />
<br />
[[Team:Heidelberg/Notebook/Killing_II/6thweek | go back to 6<sup>th</sup> week]]<br />
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[[Team:Heidelberg/Notebook/Killing_II/Notebook |go back to the overview]]<br />
<br />
==Monday 09/15/2008==<br />
===pSB1A3-Receiver-Colicin cloning===<br />
*Colony-PCR-Screen of transformation for selection of positive clones: 108 colonies<br />
25.0 µl Phusion Master Mix<br />
2.5 µl T9002_lux_pR_rv_SpeI_BamHI_RBS<br />
2.5 µl T9002_fw_XbaI<br />
20.0 µl H<sub>2</sub>O<br />
5 colonies<br />
-------<br />
50.0 µl<br />
<br />
program:<br><br />
98 °C 5 min<br />
98 °C 10 sec |<br />
58 °C 30 sec | 25 cycles<br />
72 °C 45 sec |<br />
72 °C 5 min<br />
4 °C constant<br />
*Gel of Colony-PCR-Screen: 1% Agarose, 135 V, 30 min [[Image:080915_PCR_controll_screen_pSB1A3-Receiver_small.jpg |500 px | center]]<br />
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<br />
<br />
<br />
<br />
===HisTag cloning of Colicins for purification===<br />
*Results of Transformation: Some colonies were grown on the LB-Ampicilin plates. Possibly some positive clones. Verification by Colony-PCR-Screen.<br />
*Colony-PCR-Screen of transformation for selection of positive clones: 12 x colE1, 12 x colE9<br />
25.0 µl Phusion Master Mix<br />
2.5 µl ColE1_prot_fw_BamHI/ColE9_prot_fw_BamHI<br />
2.5 µl ColE1_prot_rv_XmaI/ColE9_prot_rv_HindIII<br />
20.0 µl H<sub>2</sub>O<br />
5 colonies<br />
-------<br />
50.0 µl<br />
<br />
program:<br><br />
98 °C 5 min<br />
98 °C 10 sec |<br />
59 °C 30 sec | 25 cycles<br />
72 °C 45 sec |<br />
72 °C 5 min<br />
4 °C constant<br />
*Gel of Colony-PCR-Screen: 1% Agarose, 135 V, 30 min [[Image:080915_HisTag_colony_PCR_screen_small.jpg |500 px | center]]<br />
*Colony-PCR-Screen of colony 1-10 colE9His to identify the positive clone<br />
25.0 µl Phusion Master Mix<br />
2.5 µl ColE1_prot_fw_BamHI/ColE9_prot_fw_BamHI<br />
2.5 µl ColE1_prot_rv_XmaI/ColE9_prot_rv_HindIII<br />
20.0 µl H<sub>2</sub>O<br />
1 colony<br />
-------<br />
50.0 µl<br />
<br />
program:<br><br />
98 °C 5 min<br />
98 °C 10 sec |<br />
68 °C 30 sec | 25 cycles<br />
72 °C 45 sec |<br />
72 °C 5 min<br />
4 °C constant<br />
<br />
*Colony-PCR-Screen of colony 1-10 colE9His to identify the positive clone<br />
25.0 µl Phusion Master Mix<br />
2.5 µl ColE1_prot_fw_BamHI/ColE9_prot_fw_BamHI<br />
2.5 µl ColE1_prot_rv_XmaI/ColE9_prot_rv_HindIII<br />
20.0 µl H<sub>2</sub>O<br />
1 colony<br />
-------<br />
50.0 µl<br />
<br />
program:<br><br />
98 °C 5 min<br />
98 °C 10 sec |<br />
68 °C 30 sec | 25 cycles<br />
72 °C 45 sec |<br />
72 °C 5 min<br />
4 °C constant<br />
<br />
<br />
<br />
==Tuesday 09/16/2008==<br />
===pSB1A3-Receiver-Colicin cloning===<br />
*Miniprep of ONC: eluted in 30 µl H<sub>2</sub>O (Qiagen, Miniprepkit)<br />
*Controldigestion of Minipreps: 2 h -> 37 °C, 15 min -> 65 °C<br />
10.0 µl DNA<br />
3.0 µl H<sub>2</sub>O<br />
2.0 µl NEBuffer I (NEB)<br />
1.5 µl XbaI (20 000 U/ml, NEB)<br />
1.5 µl SpeI (10 000 U/ml, NEB)<br />
2.0 µl BSA 10x (NEB)<br />
-------<br />
20.0 µl<br />
*Gel of digestion: 1% Agarose, 37 min, 135V [[Image:080916-control_digestion_pSB1A2-receiver_cloning_small.jpg |500 px | center]]<br />
*Gelresults:<br />
**Expected Fragments:<br />
***pSB1A3 ~2000 bp<br />
***T9002_without_GFP ~1088 bp<br />
**The expected fragments were not visible. maybe the cloning was not succesfull.<br />
*Digestion of Miniprpes with SpeI only for further cloninf, because double digestion with BamHI is not recommed. 2 h -> 37 °C, 10 min -> 65 °C<br />
*Gel of Digestion: 1% Agarose, 30 min, 135 V [[Image:080916-control_digestion_pSB1A2-receiver_cloning_SpeI_small.jpg |500 px | center]]<br />
*PCR of Minipreps of "positive" clones: To check if we have positive clones we make PCR of the Minipreps we made.<br />
25.0 µl Phusion MasterMix (Finnzymes, NEB)<br />
2.5 µl Primer fw (T9002_Lux_pR_SpeI_BamHI_RBS)<br />
2.5 µl Primer rv (T9002_fw_XbaI)<br />
18.0 µl H<sub>2</sub>O<br />
2.0 µl DNA-Template<br />
-------<br />
50.0 µl<br />
<br />
program:<br />
98 °C 30 sec<br />
98 °C 10 sec |<br />
58 °C 30 sec | 25 cycles<br />
72 °C 45 sec |<br />
72 °C 5 min<br />
4 °C constant<br />
*Gel of PCR products: 1% Agarose, 135 V, 30 min [[Image:080916-PCR_of_mini_pSB1A3-rec-cloning_small.jpg |500 px | center]]<br />
*Gelresults:<br />
**Expected Fragments:<br />
***T9002_without_GFP ~1088 bp<br />
**In each probe there is a fragment of ~1500 bp. We expect that this is a form of undigested plasmid. That means that if the PCR worked properly we have no positive clones. To recheck the Minipreps we try an additional PCR with our old protocoll (no Phusion MasterMix).<br />
*PCR of Minipreps of "positive" clones: To check if we have positive clones we make PCR of the Minipreps we made.<br />
10.0 µl 5x Phusion HF Buffer (Finnzymes, NEB)<br />
1.0 µl 10 mM dNTPs (Invitrogen)<br />
2.5 µl Primer fw (T9002_Lux_pR_SpeI_BamHI_RBS)<br />
2.5 µl Primer rv (T9002_fw_XbaI)<br />
31.5 µl H<sub>2</sub>O<br />
2.0 µl DNA-Template<br />
0.5 µl Phusion DNA-Polymerase (Finnzymes, NEB)<br />
-------<br />
50.0 µl<br />
<br />
program:<br />
98 °C 30 sec<br />
98 °C 10 sec |<br />
59 °C 30 sec | 25 cycles<br />
72 °C 45 sec |<br />
72 °C 5 min<br />
4 °C constant<br />
<br />
===HisTag cloning of Colicins for purification===<br />
*Digestion of Minipreps with BamHI/HindIII for Colicin E1 and BamHI/XmaI for ColicinE9His: We want to select the positivt clones.<br />
*Gel of Digestion: 1% Agarose, 135 V, 30 min [[Image:080916-control_digestion_Hiscloning_small.jpg |500 px | center]]<br />
*Gelresults:<br />
**Expected Fragments ColE1:<br />
***~1580 bp (Insert)<br />
***~3400 bp (Backbone)<br />
**Expected Fragments ColE9:<br />
***~1580 bp (Insert)<br />
***~3400 bp (Backbone)<br />
**None of the inserts is there. Maybe we have only religated backbone. Or maybe only one enzyme cutted the DNA.<br />
*PCR of Minipreps to check if there are any positive clones. Programm see pSB1A2-Receiver-Cloning. Primers:<br />
**Colicin E1:<br />
***ColE1_prot_fw_BamHI<br />
***ColE1_kil_prot_rv_SpeI<br />
**Colicin E9:<br />
***ColE9_prot_fw_BamHI<br />
***ColE9_prot_rv_HindIII<br />
*Gel of ColicinE1 PCR: 1% Agarose, 135V, 30 min [[Image:080916-PCR_of_mini_colE1His_small.jpg |500 px | center]]<br />
*Gel of ColicinE9 PCR: 1% Agarose, 135V, 30 min [[Image:080916-PCR_of_mini_colE9His_small.jpg |500 px | center]]<br />
*Gelresults of PCR: There were no psitive bands for each PCR. But we used the wrong reverse primers. Because of that we started a new PCR with the right reverse primers (see protocol at pSB1A3-Receiver cloning)<br />
<br />
<br />
==Wednesday 09/17/2008==<br />
===pSB1A3-Receiver-Colicin cloning===<br />
*Kontrolgel of PCR (09/16/2008) to select positive clones. [[Image:080917-T9002_without_GFP_PCR_controlgel_small.jpg |500 px | center]]<br />
*Gelresults: positive clones should contain fragments at 1088 bp. Only the colonies 76-80 & 81 - 85 have a dimm band with this size. This could be a positive clone.<br />
*Minipreps of these ten cultures: eluted in 30 µl H<sub>2</sub>O, Qiagen Miniprepkit<br />
*Send plasmid DNA of colonies 78, 86, 87 & 88 to GATC for sequencing:<br />
*Inoculation of ONC with Reference_promoter cells (BBa_I20260), Colicin E1 cells, Colicin E9 cells and MG1655 in 10 ml TB media.<br />
<br />
<br />
<br />
<br />
<br />
<br />
===HisTag cloning of Colicins for purification===<br />
*Gel of ColE9 (top) ColE1 (bottom left) PCR screen (09/15/2008):1% Agarose, 30 min, 135 V [[Image:080917-pQE30His_PCR_controlgel_small.jpg |500 px | center]]<br />
*Gel of ColE1 and ColE9 His PCR screen (09/15/2008): 1% Agarose, 30 min, 135 V [[Image:080917-pQE30His_PCR_controlgel_3_small.jpg |500 px | center]]<br />
*Gel of ColE1 His PCR screen (09/15/2008): 1% Agarose, 30 min, 135 V [[Image:080917-pQE30His_PCR_controlgel_2_small.jpg |500 px | center]]<br />
*Gelresults: Only dimm bands with the right fragment size. This could be the insert or a supercoiled form of the plasmid.<br />
*ColE9 His PCR-Screen for selection of positive clones: 1% Agarose , 135 V, 30 min, expected fragment size ~630 bp [[Image:080917-pQE30His_PCR_controlgel_4_small.jpg |500 px | center]]<br />
*Gelrsults: probes 1, 2, 3, 4, 5, 7 & 8 have bands with the right fragment size.<br />
*Miniprepes of liquid ONC ColE9 His cultures 1-10 ColE1 His cultures 6-10: 1% Agarose, 135 V, 30 min<br />
*Controldigestion of ColE1His 1 & 2 with EcoRI & PstI: 1% Agarose, 135 V, 30 min [[Image:080917-pQE30E1His_digestion_controlgel_small.jpg |500 px | center]]<br />
*Results: fragments are to small. Fragments have the size of the pQE30-vector. -> Religated vector.<br />
*Probes of ColE9 His cloning were send to GATC for sequencing.<br />
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<br />
==Thursday 09/18/2008==<br />
===pSB1A2-Receiver-Colicin cloning===<br />
*Sequencing results:<br />
**only results from forward primer<br />
**sequence is from backbone -> cloning was not succesfull<br />
**maybe the backbone we used was already religated<br />
**->new start of cloning<br />
*PCR of T9002 without GFP:<br />
2.5 µl Primer reverse (T9002_Lux_pR_SpeI_BamHI_RBS)<br />
2.5 µl Primer forward (T9002_fw_XbaI)<br />
25.0 µl Phusion MM<br />
18.0 µl H<sub>2</sub>O<br />
2.0 µl Template DNA<br />
-------<br />
50.0 µl<br />
<br />
program:<br />
98 °C 30 sec<br />
98 °C 10 sec |<br />
58 °C 30 sec | 30 cycles<br />
72 °C 45 sec |<br />
72 °C 5 min<br />
4 °C constant<br />
*Gelextraction of PCR: 0.7% Agarose, 45 min, 135 V [[Image:080918-gelextraction_T9002_wo_GFP_PCR_small.jpg |500 px | center]]<br />
*Digestion of Insert & Backbone: 1 h -> 37 °C, 10 min -> 65 °C<br />
Insert<br />
37.0 µl DNA of Gelex<br />
2.0 µl SpeI<br />
1.0 µl XbaI<br />
5.0 µl NEBuffer 2 10x<br />
5.0 µl BSA 10x<br />
-------<br />
50.0 µl<br />
<br />
Backbone<br />
37.0 µl T9002 Maxi (1 µg/µl)<br />
2.0 µl SpeI<br />
1.0 µl XbaI<br />
5.0 µl NEBuffer 2 10x<br />
5.0 µl BSA 10x<br />
-------<br />
50.0 µl<br />
*Gelextraction of pSB1A3: 0.7% Agarose, 65 min, 100 V, eluated in 50 µl H<sub>2</sub>O [[Image:080918-gelextraction_pSB1A2_small.jpg |500 px | center]]<br />
*Sapping of pSB1A3 backbone:<br />
34 µl DNA<br />
1 µl SAP-Enzymes (Fermentas) <br />
4 µl SAP-Buffer (Fermentas)<br />
-----<br />
39 µl <br />
*ON-Ligation: 14 h -> 16 °C, 20 min -> 65 °C, forever -> 4 °C<br />
5:1 Insert:Vector<br />
15.0 µl Insert<br />
1.0 µl Vector<br />
2.0 µl T4 DNA Ligase<br />
2.0 µl T4 DNA Ligase Buffer<br />
-------<br />
20.0 µl<br />
<br />
3:1 Insert:Vector<br />
14.5 µl Insert<br />
1.5 µl Vector<br />
2.0 µl T4 DNA Ligase<br />
2.0 µl T4 DNA Ligase Buffer<br />
-------<br />
20.0 µl<br />
====Colicin-Test with eucaryotic cells====<br />
*HeLa and MCF7 cells were treated with supernatants of prior tests. There was no effect in the first hour. Maybe the supernatant was uneffective because it was freezed.<br />
<br />
<br />
===HisTag cloning of Colicins for purification===<br />
*Sequencing results:<br />
**religated pQE-30 -> no positive clones<br />
**->restart the cloning<br />
*PCR of colicin E1/E9 proteins:<br />
2.0 µl pColE1/pColE9-J plasmid<br />
2.5 µl Primer fw(ColE1_prot_fw_BamHI/ColE9_prot_fw_BamHI)<br />
2.5 µl Primer rv(ColE1_prot_rv_HindIII/ColE9_prot_rv_XmaI)<br />
18.0 µl H<sub>2</sub>O<br />
25.0 µl Phusion MM<br />
-------<br />
50.0 µl<br />
*Gelextraction of PCR: 0,7% Agarose, 50 min, 100 V; eluted in 30 µl H<sub>2</sub>O, Qiagen Gelextraction Kit [[Image:080918-PCR_of_colicin_proteins_small.jpg |500 px | center]]<br />
*1st Digestion of Vectors and inserts with HindIII/XmaI for ColE1/E9 HisTag cloning: 1h -> 37 °C, 10 min -> 65 °C<br />
vector:<br />
39.8 µl pQE30 (30 ng/µl)<br />
0.2 µl HindIII/XmaI<br />
5.0 µl NEBuffer 2/4 10x<br />
5.0 µl BSA 10x<br />
-------<br />
50.0 µl<br />
<br />
insert:<br />
10.0 µl colicinE1/E9<br />
0.2 µl HindIII/XmaI<br />
5.0 µl NEBuffer 2/4 10x<br />
5.0 µl BSA 10x<br />
29.8 µl H<sub>2</sub>O<br />
-------<br />
50.0 µl<br />
*Purification of probes with Qiagen PCR Purification Kit<br />
*2nd Digestion of Vectors and inserts with BamHIfor ColE1/E9 HisTag cloning: 1h -> 37 °C, 10 min -> 65 °C<br />
39.0 µl pQE30 (30 ng/µl)<br />
1.0 µl BamHI<br />
5.0 µl NEBuffer 3 10x<br />
5.0 µl BSA 10x<br />
-------<br />
50.0 µl<br />
<br />
insert:<br />
39.0 µl colicinE1/E9<br />
1.0 µl BamHI<br />
5.0 µl NEBuffer 2/4 10x<br />
5.0 µl BSA 10x<br />
29.8 µl H<sub>2</sub>O<br />
-------<br />
50.0 µl<br />
*Gelextraction of digested vector and insert: 0.7% Agarose, 45 min, 100 V [[Image:080918-histag_digestion_gelex_small.jpg |500 px | center]]<br />
*ON Ligation of pQE-30 vector and colicin inserts: 14 h -> 16 °C, 20 min -> 65 °C, forever -> 4 °C<br />
12.0 µl Insert<br />
4.0 µl Vector<br />
2.0 µl T4 DNA Ligase<br />
2.0 µl T4 DNA Ligase Buffer<br />
-------<br />
20.0 µl<br />
===Other===<br />
*Inoculation of ONC with<br />
**BBa_I23107 -> medium constitutive promoter<br />
**BBa_I13600 -> CFP cassette<br />
**BBa_I13602 -> CFP cassette<br />
**BBa_B0015 -> Terminator<br />
**BBa_I0500 -> pBAD/araC<br />
**BBa_F1610 -> AHL sender part<br />
<br />
==Friday 09/19/2008==<br />
===pSB1A3-Receiver-Colicin cloning===<br />
*Transformation of pSB1A3-T9002-without-GFP ON-Ligations (Transformation protocol Chris)<br />
2x 5 µl DNA 5:1 on 50 µl TOP 10 cells + 200 ml LB -> LB-Amp plates<br />
2x 5 µl DNA 3:1 on 50 µl TOP 10 cells + 200 ml LB -> LB-Amp plates<br />
===HisTag cloning of Colicins for purification===<br />
*Digestion of old ColE9His-pQE-30 cloning with BamHI/PstI to verify that there are no positive clones: colonies 1 + 4; 1h 10min -> 37 °C<br />
20.0 µl DNA<br />
5.0 µl NEBuffer 3<br />
2.0 µl BamHI<br />
2.0 µl PstI<br />
5.0 µl BSA 10x<br />
16.0 µl H<sub>2</2sub>O<br />
-------<br />
50.0 µl<br />
*Transformation of ColE9/E1 His tag cloning ON-Ligations (Transformation protocol Chris)<br />
2x 5 µl DNA 5:1 on 50 µl TOP 10 cells + 200 ml LB -> LB-Amp plates<br />
*Gel of Digestion: 1% Agarose,135 V, 40 min [[Image:080919-controldigestion_pQE-30_ColE9_small.jpg |500 px | center]]<br />
*Gelresults: Maybe there is no insert or the enzymes didn't cut.<br />
===Sender cloning: pBAD promoter===<br />
*BBa_I0500 ONC of 2008 stock did not grow<br />
*Inoculation of BBa_I0500 of Glycerolstock 2008 & 2004<br />
===Other===<br />
====Team-Meeting====<br />
*Presentationpreparation for Team-Meeting: Which parts can be submitted to the registry?<br />
*Presentation:<br />
**ColicinE1 and E9 as part whole cassette<br />
**Mutagenesis of ColicinE1 Receiver (EcoRI)<br />
====Minipreps====<br />
*Of ONCs, eluted in 30µl H<sub>2</sub>; Qiagen Miniprepkit<br />
<br />
<br />
<br />
==Saturday 09/20/2008==<br />
<br />
===pSB1A3-Receiver-Colicin cloning===<br />
*Colony PCR-Screen of pSB1A2-T9002-GFP:<br />
25.0 µl Phusion MasterMix (Finnzymes, NEB)<br />
2.5 µl Primer_fw (pSB_ins_fw)<br />
2.5 µl Primer_rv (pSB_ins_rv)<br />
20.0 µl H<sub>2</sub>O<br />
5 colonies<br />
-------<br />
50.0 µl<br />
<br />
program:<br />
98 °C 5 min<br />
98 °C 10 sec |<br />
58 °C 30 sec | 25 cycles<br />
72 °C 45 sec |<br />
72 °C 5 min<br />
4 °C constant<br />
*Gel of PCR screen: No bands were visible. Retry PCR-Screen with Taq-Polymerase and other primers. [[Image:080920-PCR-screen-T9002-GFP_small.jpg |500 px | center]]<br />
<br />
<br />
===HisTag cloning of Colicins for purification===<br />
*Digestion of former Cloning with ColE9His-pQE30 EcoRI/PstI and BamHI/PstI: 1h 37 °C -> Gel<br />
20.0 µl DNA<br />
5.0 µl NEBuffer 3<br />
2.0 µl BamHI<br />
2.0 µl PstI<br />
5.0 µl BSA 10x<br />
16.0 µl H<sub>2</sub>O<br />
-------<br />
50.0 µl<br />
<br />
20.0 µl DNA<br />
5.0 µl NEBuffer EcoRI<br />
2.0 µl EcoRI<br />
2.0 µl PstI<br />
5.0 µl BSA 10x<br />
16.0 µl H<sub>2</sub>O<br />
-------<br />
50.0 µl<br />
<br />
20.0 µl DNA<br />
5.0 µl NEBuffer 3<br />
2.0 µl BamHI<br />
5.0 µl BSA 10x<br />
18.0 µl H<sub>2</sub>O<br />
-------<br />
50.0 µl<br />
<br />
20.0 µl DNA<br />
5.0 µl NEBuffer EcoRI<br />
2.0 µl EcoRI<br />
5.0 µl BSA 10x<br />
18.0 µl H<sub>2</sub>O<br />
-------<br />
50.0 µl<br />
<br />
*Gel of digestion: 1% Agarose, 135V, 30 min: [[Image:080920-controldigestion_pQE30-His_small.jpg |500 px | center]]<br />
*Inoculation of ONC of 25 colonies per colicin-pQE30<br />
<br />
==Sunday 09/21/2008==<br />
===pSB1A3-Receiver-Colicin cloning===<br />
*2nd PCR Screening of pSB1A2-T9002-GFP cloning<br />
25.0 µl Taq-Polymerase MasterMix (Fermentas)<br />
2.5 µl Primer_rv (T9002_Lux_pR_SpeI_BamHI_RBS)<br />
2.5 µl Primer_fw (T9002_fw_XbaI)<br />
20.0 µl H<sub>2</sub>O<br />
5 colonies<br />
-------<br />
50.0 µl<br />
<br />
program:<br />
95 °C 3 min<br />
95 °C 1 min |<br />
54 °C 1 min | 30 cycles<br />
72 °C 1 min |<br />
72 °C 10 min<br />
4 °C constant<br />
<br />
*Gel of colony PCR screen: 1% Agarose, 135 V, 30 min [[Image:080921_colony_PCR_screen_pSB1A2-T9002-GFP_small.jpg |500 px | center]]<br />
*Gelresults: Many bands with the right fragment size. New colony PCR screen with single colonies of colonies 31-45 and 66-75<br />
*Gel of single colony PCR screen: 1% Agarose, 135 V, 30 min [[Image:080921_single_colony_PCR_screen_pSB1A2-T9002-GFP_small.jpg |500 px | center]]<br />
*Gelresults: Colonies 31, 35, 36, 39, 40, 42, 45, 66, 69, 71, 72 & 73 have the right fragment size. This could be the positive clones<br />
*Inoculation of LB-Amp ONC from these colonies<br />
<br />
<br />
===HisTag cloning of Colicins for purification===<br />
*Colony PCR screen to select positive clones:<br />
25.0 µl Taq-Polymerase MasterMix (Fermentas)<br />
2.5 µl Primer_rv<br />
2.5 µl Primer_fw<br />
20.0 µl H<sub>2</sub>O<br />
5 colonies<br />
-------<br />
50.0 µl<br />
<br />
program:<br />
95 °C 3 min<br />
95 °C 1 min |<br />
54 °C 1 min | 30 cycles<br />
72 °C 1 min |<br />
72 °C 10 min<br />
4 °C constant<br />
[[Team:Heidelberg/Notebook/Killing_II/8thweek | go to 8<sup>th</sup> week]]</div>
Andreaskuehne