http://2012.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=500&target=Jakobmatthes&year=&month=2012.igem.org - User contributions [en]2024-03-29T12:55:14ZFrom 2012.igem.orgMediaWiki 1.16.0http://2012.igem.org/Regions/Europe/Jamboree/PracticeSessionsRegions/Europe/Jamboree/PracticeSessions2012-09-30T12:18:12Z<p>Jakobmatthes: </p>
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<h2>Practice Sessions</h2><br />
<br />
<p>Use this sign-up sheet to sign up for a slot on Friday to practice your talk. Note that there will NOT be any audio/visual support on staff. All classrooms will be unlocked and you should use them and leave them as you found them.<br />
</p><br />
<p> There are a limited number of time slots available on a first-come first-serve basis so please only choose one slot. We cannot match the room that you will ultimately give your presentation in with the practice room. This should, however, give you a chance to practice your talk in a new environment. Please keep in mind that there will be teams waiting to use the room after you, so make sure that your practice finishes on time. <br />
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<table border=1 class="calendar" style="width:800px;" align="center"><br />
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<th>Time</th><br />
<th>HG-0G08</th><br />
<th>HG-0G10</th><br />
<th>HG-0G11</th><br />
<th>HG-0G13</th><br />
<th>HG-0G23</th><br />
<th>HG-OG25</th><br />
<th>HG-0G28</th><br />
<th>HG-0G30</th><br />
<th>HG-1G08</th><br />
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</thead><br />
<tbody align="center"><br />
<tr class="odd"><br />
<th>15:30-16:00 h</th><br />
<td>A1</td><br />
<td>A2</td><br />
<td>A3</td><br />
<td>A4</td><br />
<td>A5</td><br />
<td>A6</td><br />
<td>A7</td><br />
<td>A8</td><br />
<td>A9</td><br />
</tr><br />
<tr class="even"><br />
<th>16:00-16:30 h</th><br />
<td>Chalmers, Gothenburg</td><br />
<td>B2</td><br />
<td>Trieste</td><br />
<td>B4</td><br />
<td>B5</td><br />
<td>B6</td><br />
<td>B7</td><br />
<td>B8</td><br />
<td>B9</td><br />
</tr><br />
<tr class="even"><br />
<th>16:30-17:00 h</th><br />
<td>Groningen</td><br />
<td>Freiburg</td><br />
<td>C3</td><br />
<td>C4</td><br />
<td>C5</td><br />
<td>C6</td><br />
<td>C7</td><br />
<td>C8</td><br />
<td></td><br />
</tr><br />
<tr class="odd"><br />
<th>17:00-17:30 h</th><br />
<td>University College London</td><br />
<td>D2</td><br />
<td>D3</td><br />
<td>D4</td><br />
<td>D5</td><br />
<td>D6</td><br />
<td>D7</td><br />
<td>Fatih-Medical</td><br />
<td>D9</td><br />
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<tr class="even"><br />
<th>17:30-18:00 h</th><br />
<td>University of Westminster</td><br />
<td>E2</td><br />
<td>Uppsala University</td><br />
<td>Cambridge</td><br />
<td>E5</td><br />
<td>E6</td><br />
<td>E7</td><br />
<td>E8</td><br />
<td>E9</td><br />
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<tr class="odd"><br />
<th>18:00-18:30 h</th><br />
<td>SDU-Denmark</td><br />
<td>Goettingen</td><br />
<td>Lyon INSA</td><br />
<td>Tuebingen</td><br />
<td>F5</td><br />
<td>TU Darmstadt</td><br />
<td>F7</td><br />
<td>F8</td><br />
<td>F9</td><br />
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<tr class="even"><br />
<th>18:30-19:00 h</th><br />
<td>Grenoble</td><br />
<td>Evry</td><br />
<td>UniTN-Trento</td><br />
<td>Eindhoven University of Technology</td><br />
<td>LMU-Munich</td><br />
<td>Technion</td><br />
<td>Marburg_<br />
SYNMIKRO</td><br />
<td>TU Munich</td><br />
<td>Bielefeld-Germany</td><br />
</tr><br />
<tr class="odd"><br />
<th>19:00-19:30 h</th><br />
<td>Valencia Biocampus</td><br />
<td>Paris Bettencourt</td><br />
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<tr class="even"><br />
<th>19:30-20:00 h</th><br />
<td bgcolor=#DF0101>Dundee</td><br />
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<th>20:00-20:30 h</th><br />
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<th>20:30-21:00 h</th><br />
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<th>21:00-21:30 h</th><br />
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<th>21:30-22:00 h</th><br />
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</html></div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultsTeam:Tuebingen/Results2012-09-26T12:37:00Z<p>Jakobmatthes: /* Identified Problems */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Results =<br />
__TOC__<br />
<br />
== Parts Statuses ==<br />
<br />
* '''Pfet3, Panb1, lacZ''': PCR was not successful after many tries. For Pfet3, new primers were designed but there was no time left to complete this part.<br />
* '''Tadh1''': Tadh1 has been successfully amplified and ligation in pGEM vectors is under way.<br />
* '''pRS313, pRS315, pRS316''': The shuttle plasmids are available and digested.<br />
* '''Padh1, Psuc2, rox1, mig1, luciferase, mPR ''Danio rerio'', mPR ''Xenopus laevis''''': These parts are all available and digested. The ligation in both pSB1C3 and our pRS shuttle plasmids was after many tries not yet successful.<br />
<br />
== Identified Problems ==<br />
<br />
* '''PCR problems:''' Some PCRs yielded only products in very low concentrations. Some primers also amplified multiple fragments. New primers partially solved these troubles.<br />
* '''Transformation efficiency:''' The first batch of chemocompetent cells had a very low transformation efficiency. Newly prepared cells showed much better efficiency.<br />
* '''Ligation problems:''' Due to our assembly strategy (only XbaI/SpeI digestion) we had troubles with very low efficiency of insert ligation. Further complication is that the insert can be ligated in the wrong direction. This assembly was necessary because the shuttle plasmids feature only the XbaI/SpeI pair in its multiple cloning site.<br />These problems are the main point why assembly and part submission failed.<br />
* '''Chloramphenicol resistancy and pSB1C3:''' Determining the tolerance of Chloramphenicol of our cells was difficult. Common literature concentrations did not work. In addition to ligation problems, this resulted in no positive pSB1C3 constructs.<br />
<br />
== Progress Illustraion ==<br />
The following illustraion gives a quick overview of the final status of our lab work.<br />
[[File:Tue-labmap-results.png|600px|thumb|center|final status of our parts]]</div>Jakobmattheshttp://2012.igem.org/File:Tue-labmap-results.pngFile:Tue-labmap-results.png2012-09-26T12:34:07Z<p>Jakobmatthes: uploaded a new version of &quot;File:Tue-labmap-results.png&quot;</p>
<hr />
<div></div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultsTeam:Tuebingen/Results2012-09-26T12:28:28Z<p>Jakobmatthes: /* Results */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Results =<br />
__TOC__<br />
<br />
== Parts Statuses ==<br />
<br />
* '''Pfet3, Panb1, lacZ''': PCR was not successful after many tries. For Pfet3, new primers were designed but there was no time left to complete this part.<br />
* '''Tadh1''': Tadh1 has been successfully amplified and ligation in pGEM vectors is under way.<br />
* '''pRS313, pRS315, pRS316''': The shuttle plasmids are available and digested.<br />
* '''Padh1, Psuc2, rox1, mig1, luciferase, mPR ''Danio rerio'', mPR ''Xenopus laevis''''': These parts are all available and digested. The ligation in both pSB1C3 and our pRS shuttle plasmids was after many tries not yet successful.<br />
<br />
== Identified Problems ==<br />
<br />
* '''PCR problems:''' Some PCRs yielded only products in very low concentrations. Some primers also amplified multiple fragments. New primers partially solved these troubles.<br />
* '''Transformation efficiency:''' The first batch of chemocompetent had a very low transformation efficiency. Newly prepared cells showed much better efficiency.<br />
* '''Ligation problems:''' Due to our assembly strategy (only XbaI/SpeI digestion) we had troubles with very low efficiency of insert ligation. Further complication is that the insert can be ligated in the wrong direction. This assembly was necessary because the shuttle plasmids feature only the XbaI/SpeI pair in its multiple cloning site.<br />These problems are the main point why assembly and part submission failed.<br />
<br />
== Progress Illustraion ==<br />
The following illustraion gives a quick overview of the final status of our lab work.<br />
[[File:Tue-labmap-results.png|600px|thumb|center|final status of our parts]]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultPersonalTeam:Tuebingen/ResultPersonal2012-09-26T12:14:26Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Impressions =<br />
{{:Team:Tuebingen/Templates/Quote|Jan Rudolph|When me and Jakob first dreamt about participating in iGEM we put it away as mission impossible. But with the help of our advisors and the motivated team we found the iGEM experience turned to be real. I am glad that I was able to gain experience in the wet lab which is usually not part of bioinformatics B.sc. I am looking forward to finish our project next year.}}<br />
<br />
{{:Team:Tuebingen/Templates/Quote|Simon Heumos|When Jan and Jakob introduced me to iGEM, my first thought was: „Wow, now I have the opportunity to do something “out of the usual” compared to my “normal” bioinformatics B.sc. stuff!“<br />
Fortunately iGEM turned out to be a lot of fun in and out of the lab, like learning how to do a PCR or getting to know people of other faculties.<br /><br />
I would enjoy the participation in the future progress of our project.}}<br />
<br />
{{:Team:Tuebingen/Templates/Quote|Lukas Zimmermann|Almost without any lab experience, iGEM gave me the opportunity to learn the basics and to get a feeling for scientific work. Furthermore, it was possible for me to spend the summer with meaningful employment. In a nutshell, iGEM was a very valuable experience. Everything speaks for the next year to participate.}}<br />
<br />
{{:Team:Tuebingen/Templates/Quote|Simeon Roßmann|I spent many days of this summer in the lab. One of the most exciting vacations and I didn’t even have to travel! Thank you iGEM and Team Tübingen for a great experience, see you next year.}}<br />
<br />
{{:Team:Tuebingen/Templates/Quote|Jakob Matthes|Establishing an iGEM team for the first time at University of Tuebingen was a major success. It was a pleasure to work with fellow motivated students and advisors. The long hours in the lab have taught me a solid foundation in lab procedures, project documentation, team management and independent work. iGEM 2012 was a great experience!}}</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultsTeam:Tuebingen/Results2012-09-26T11:54:09Z<p>Jakobmatthes: /* Identified Problems */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Results =<br />
__TOC__<br />
<br />
== Parts Statuses ==<br />
<br />
* '''Pfet3, Panb1, lacZ''': PCR was not successful for many tries. For Pfet3, new primers were designed but there was no time left to complete this part.<br />
* '''Tadh1''': Tadh1 has been successfully amplified and ligation in pGEM vectors is under way.<br />
* '''pRS313, pRS315, pRS316''': The shuttle plasmids are available and digested.<br />
* '''Padh1, Psuc2, rox1, mig1, luciferase, mPR ''Danio rerio'', mPR ''Xenopus laevis''''': These parts are all available and digested. The ligation in both pSB1C3 and our pRS shuttle plasmids was so far not successful.<br />
<br />
== Identified Problems ==<br />
<br />
* '''PCR problems:''' Some PCRs yielded only products in very low concentrations. Some primers also amplified multiple fragments. New primers partially solved these troubles.<br />
* '''Transformation efficiency:''' The first batch of chemocompetent had a very low transformation efficiency. Newly prepared cells showed much better efficiency.<br />
* '''Ligation problems:''' Due to our assembly strategy (only XbaI/SpeI digestion) we had troubles with very low efficiency of insert ligation. Further complication is that the insert can be ligated in the wrong direction. This assembly was necessary because the shuttle plasmids feature only the XbaI/SpeI pair in its multiple cloning site.<br />These problems are the main point why assembly and part submission failed.<br />
<br />
== Progress Illustraion ==<br />
The following illustraion gives a quick overview of the final status of our lab work.<br />
[[File:Tue-labmap-results.png|600px|thumb|center|final status of our parts]]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultsTeam:Tuebingen/Results2012-09-26T11:53:47Z<p>Jakobmatthes: /* Identified Problems */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Results =<br />
__TOC__<br />
<br />
== Parts Statuses ==<br />
<br />
* '''Pfet3, Panb1, lacZ''': PCR was not successful for many tries. For Pfet3, new primers were designed but there was no time left to complete this part.<br />
* '''Tadh1''': Tadh1 has been successfully amplified and ligation in pGEM vectors is under way.<br />
* '''pRS313, pRS315, pRS316''': The shuttle plasmids are available and digested.<br />
* '''Padh1, Psuc2, rox1, mig1, luciferase, mPR ''Danio rerio'', mPR ''Xenopus laevis''''': These parts are all available and digested. The ligation in both pSB1C3 and our pRS shuttle plasmids was so far not successful.<br />
<br />
== Identified Problems ==<br />
<br />
* '''PCR problems''': Some PCRs yielded only products in very low concentrations. Some primers also amplified multiple fragments. New primers partially solved these troubles.<br />
* '''Transformation efficiency''': The first batch of chemocompetent had a very low transformation efficiency. Newly prepared cells showed much better efficiency.<br />
* '''Ligation problems''': Due to our assembly strategy (only XbaI/SpeI digestion) we had troubles with very low efficiency of insert ligation. Further complication is that the insert can be ligated in the wrong direction. This assembly was necessary because the shuttle plasmids feature only the XbaI/SpeI pair in its multiple cloning site.<br />These problems are the main point why assembly and part submission failed.<br />
<br />
== Progress Illustraion ==<br />
The following illustraion gives a quick overview of the final status of our lab work.<br />
[[File:Tue-labmap-results.png|600px|thumb|center|final status of our parts]]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultsTeam:Tuebingen/Results2012-09-26T10:27:44Z<p>Jakobmatthes: /* Identified Problems */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Results =<br />
__TOC__<br />
<br />
== Parts Statuses ==<br />
<br />
* '''Pfet3, Panb1, lacZ''': PCR was not successful for many tries. For Pfet3, new primers were designed but there was no time left to complete this part.<br />
* '''Tadh1''': Tadh1 has been successfully amplified and ligation in pGEM vectors is under way.<br />
* '''pRS313, pRS315, pRS316''': The shuttle plasmids are available and digested.<br />
* '''Padh1, Psuc2, rox1, mig1, luciferase, mPR ''Danio rerio'', mPR ''Xenopus laevis''''': These parts are all available and digested. The ligation in both pSB1C3 and our pRS shuttle plasmids was so far not successful.<br />
<br />
== Identified Problems ==<br />
<br />
* '''PCR problems''': Some PCRs yielded only products in very low concentrations. Some primers also amplified multiple fragments. New primers partially solved these troubles.<br />
*<br />
<br />
== Progress Illustraion ==<br />
The following illustraion gives a quick overview of the final status of our lab work.<br />
[[File:Tue-labmap-results.png|600px|thumb|center|final status of our parts]]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultsTeam:Tuebingen/Results2012-09-26T10:25:09Z<p>Jakobmatthes: /* Results */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Results =<br />
__TOC__<br />
<br />
== Parts Statuses ==<br />
<br />
* '''Pfet3, Panb1, lacZ''': PCR was not successful for many tries. For Pfet3, new primers were designed but there was no time left to complete this part.<br />
* '''Tadh1''': Tadh1 has been successfully amplified and ligation in pGEM vectors is under way.<br />
* '''pRS313, pRS315, pRS316''': The shuttle plasmids are available and digested.<br />
* '''Padh1, Psuc2, rox1, mig1, luciferase, mPR ''Danio rerio'', mPR ''Xenopus laevis''''': These parts are all available and digested. The ligation in both pSB1C3 and our pRS shuttle plasmids was so far not successful.<br />
<br />
== Identified Problems ==<br />
<br />
== Progress Illustraion ==<br />
The following illustraion gives a quick overview of the final status of our lab work.<br />
[[File:Tue-labmap-results.png|600px|thumb|center|final status of our parts]]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultsTeam:Tuebingen/Results2012-09-26T10:23:27Z<p>Jakobmatthes: /* Results */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Results =<br />
__TOC__<br />
<br />
* '''Pfet3, Panb1, lacZ''': PCR was not successful for many tries. For Pfet3, new primers were designed but there was no time left to complete this part.<br />
* '''Tadh1''': Tadh1 has been successfully amplified and ligation in pGEM vectors is under way.<br />
* '''pRS313, pRS315, pRS316''': The shuttle plasmids are available and digested.<br />
* '''Padh1, Psuc2, rox1, mig1, luciferase, mPR ''Danio rerio'', mPR ''Xenopus laevis''''': These parts are all available and digested. The ligation in both pSB1C3 and our pRS shuttle plasmids was so far not successful.<br />
<br />
[[File:Tue-labmap-results.png|600px|thumb|center|final status of our parts]]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultsTeam:Tuebingen/Results2012-09-26T10:23:05Z<p>Jakobmatthes: /* Results */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Results =<br />
__TOC__<br />
<br />
[[File:Tue-labmap-results.png|200px|thumb|right|final status of our parts]]<br />
* '''Pfet3, Panb1, lacZ''': PCR was not successful for many tries. For Pfet3, new primers were designed but there was no time left to complete this part.<br />
* '''Tadh1''': Tadh1 has been successfully amplified and ligation in pGEM vectors is under way.<br />
* '''pRS313, pRS315, pRS316''': The shuttle plasmids are available and digested.<br />
* '''Padh1, Psuc2, rox1, mig1, luciferase, mPR ''Danio rerio'', mPR ''Xenopus laevis''''': These parts are all available and digested. The ligation in both pSB1C3 and our pRS shuttle plasmids was so far not successful.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultsTeam:Tuebingen/Results2012-09-26T10:22:40Z<p>Jakobmatthes: /* Results */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Results =<br />
__TOC__<br />
<br />
* '''Pfet3, Panb1, lacZ''': PCR was not successful for many tries. For Pfet3, new primers were designed but there was no time left to complete this part.<br />
* '''Tadh1''': Tadh1 has been successfully amplified and ligation in pGEM vectors is under way.<br />
* '''pRS313, pRS315, pRS316''': The shuttle plasmids are available and digested.<br />
* '''Padh1, Psuc2, rox1, mig1, luciferase, mPR ''Danio rerio'', mPR ''Xenopus laevis''''': These parts are all available and digested. The ligation in both pSB1C3 and our pRS shuttle plasmids was so far not successful.<br />
<br />
[[File:Tue-labmap-results.png|600px|thumb|center|final status of our parts]]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-26T10:17:39Z<p>Jakobmatthes: /* Parts, Plasmids and Constructs */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<br />
Both parts and vectors had to be worked on separately until they are available as digested fragments with XbaI/SpeI overhangs. Then parts could be ligated into the shipping plasmid pSB1C3 to submit them to the Parts Registry. To assemble our parts into the desired constructs ligation in correct order in the pRS plasmids were necessary. The final step is the transformation of all three plasmids into yeast strains.<br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! length [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen <br /> (University of Tuebingen) || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || 317 || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed. <br/><br />
For each part we made a 7 times preparation with a gradient of 45°C - 58,9°C with the following intermediate stages:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient<br />
|-<br />
| A || 60,0°C<br />
|-<br />
| B || 58,9°C<br />
|-<br />
| C || 57,0°C<br />
|-<br />
| D || 54,1°C<br />
|-<br />
| E || 50,5°C<br />
|-<br />
| F || 47,9°C<br />
|-<br />
| G || 46,0°C<br />
|-<br />
| H || 45,0°C<br />
|}<br />
<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably damaged. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
The purification of the parts mig1 and mPR ''Danio rerio'' and the pRS vectors was performed with a [[Team:Tuebingen/NotebookProtocols#Genaxxon_PCR_DNA_Purification_Mini_Prep_Kit|PCR Purification Kit]] in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
4. A [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|scaled up restriction digest]] and preparative [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] of the parts 3 and 8 was executed to prepare them for later ligation into pRS.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 7 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient !! Part name<br />
|-<br />
| A || 60,0°C ||<br />
|-<br />
| B || 58,9°C || Tadh1<br />
|-<br />
| C || 57,0°C || Tadh1<br />
|-<br />
| D || 54,1°C ||<br />
|-<br />
| E || 50,5°C || Tadh1<br />
|-<br />
| F || 47,9°C || Pfet3<br />
|-<br />
| G || 46,0°C || Pfet3<br />
|-<br />
| H || 45,0°C || Pfet3<br />
|}<br />
<br />
The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of part 7 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|scaled-up restriction digest]] of the parts 3, 4 and 10 was executed, because the digested stocks of these parts were empty.<br />
<br />
4. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
5. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==<br />
<br />
1. Some more [[Team:Tuebingen/NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|mini-preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]].<br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. We finished our Wiki, designed our poster and made our presentation for the Jamboree in Amsterdam.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-26T10:11:18Z<p>Jakobmatthes: /* Procedure */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<br />
Both parts and vectors had to be worked on separately until they are available as digested fragments with XbaI/SpeI overhangs. Then parts could be ligated into the shipping plasmid pSB1C3 to submit them to the Parts Registry. To assemble our parts into the desired constructs ligation in correct order in the pRS plasmids were necessary. The final step is the transformation of all three plasmids into yeast strains.<br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! length [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen <br /> (University of Tuebingen) || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed. <br/><br />
For each part we made a 7 times preparation with a gradient of 45°C - 58,9°C with the following intermediate stages:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient<br />
|-<br />
| A || 60,0°C<br />
|-<br />
| B || 58,9°C<br />
|-<br />
| C || 57,0°C<br />
|-<br />
| D || 54,1°C<br />
|-<br />
| E || 50,5°C<br />
|-<br />
| F || 47,9°C<br />
|-<br />
| G || 46,0°C<br />
|-<br />
| H || 45,0°C<br />
|}<br />
<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
The purification of the parts mig1 and mPR ''Danio rerio'' and the pRS vectors was performed with a [[Team:Tuebingen/NotebookProtocols#Genaxxon_PCR_DNA_Purification_Mini_Prep_Kit|PCR Purification Kit]] in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
4. A [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|scaled up restriction digest]] and preparative [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] of the parts 3 and 8 was executed to prepare them for later ligation into pRS.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 7 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient !! Part name<br />
|-<br />
| A || 60,0°C ||<br />
|-<br />
| B || 58,9°C || Tadh1<br />
|-<br />
| C || 57,0°C || Tadh1<br />
|-<br />
| D || 54,1°C ||<br />
|-<br />
| E || 50,5°C || Tadh1<br />
|-<br />
| F || 47,9°C || Pfet3<br />
|-<br />
| G || 46,0°C || Pfet3<br />
|-<br />
| H || 45,0°C || Pfet3<br />
|}<br />
<br />
The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of part 7 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|scaled-up restriction digest]] of the parts 3, 4 and 10 was executed, because the digested stocks of these parts were empty.<br />
<br />
4. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
5. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==<br />
<br />
1. Some more [[Team:Tuebingen/NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|mini-preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]].<br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. We finished our Wiki, designed our poster and made our presentation for the Jamboree in Amsterdam.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-26T10:07:45Z<p>Jakobmatthes: /* Week 5 (8/06 - 8/12) */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! length [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen <br /> (University of Tuebingen) || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed. <br/><br />
For each part we made a 7 times preparation with a gradient of 45°C - 58,9°C with the following intermediate stages:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient<br />
|-<br />
| A || 60,0°C<br />
|-<br />
| B || 58,9°C<br />
|-<br />
| C || 57,0°C<br />
|-<br />
| D || 54,1°C<br />
|-<br />
| E || 50,5°C<br />
|-<br />
| F || 47,9°C<br />
|-<br />
| G || 46,0°C<br />
|-<br />
| H || 45,0°C<br />
|}<br />
<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
The purification of the parts mig1 and mPR ''Danio rerio'' and the pRS vectors was performed with a [[Team:Tuebingen/NotebookProtocols#Genaxxon_PCR_DNA_Purification_Mini_Prep_Kit|PCR Purification Kit]] in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
4. A [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|scaled up restriction digest]] and preparative [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] of the parts 3 and 8 was executed to prepare them for later ligation into pRS.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 7 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient !! Part name<br />
|-<br />
| A || 60,0°C ||<br />
|-<br />
| B || 58,9°C || Tadh1<br />
|-<br />
| C || 57,0°C || Tadh1<br />
|-<br />
| D || 54,1°C ||<br />
|-<br />
| E || 50,5°C || Tadh1<br />
|-<br />
| F || 47,9°C || Pfet3<br />
|-<br />
| G || 46,0°C || Pfet3<br />
|-<br />
| H || 45,0°C || Pfet3<br />
|}<br />
<br />
The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of part 7 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|scaled-up restriction digest]] of the parts 3, 4 and 10 was executed, because the digested stocks of these parts were empty.<br />
<br />
4. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
5. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==<br />
<br />
1. Some more [[Team:Tuebingen/NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|mini-preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]].<br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. We finished our Wiki, designed our poster and made our presentation for the Jamboree in Amsterdam.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-26T10:01:17Z<p>Jakobmatthes: /* Week 7 (8/20 - 8/26) */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! length [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen <br /> (University of Tuebingen) || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed. <br/><br />
For each part we made a 7 times preparation with a gradient of 45°C - 58,9°C with the following intermediate stages:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient<br />
|-<br />
| A || 60,0°C<br />
|-<br />
| B || 58,9°C<br />
|-<br />
| C || 57,0°C<br />
|-<br />
| D || 54,1°C<br />
|-<br />
| E || 50,5°C<br />
|-<br />
| F || 47,9°C<br />
|-<br />
| G || 46,0°C<br />
|-<br />
| H || 45,0°C<br />
|}<br />
<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
The purification of the parts Mig 1 and mPR D.r. and the pRS vectors was performed with a PCR purification Kit in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
4. A [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|scaled up restriction digest]] and preparative [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] of the parts 3 and 8 was executed to prepare them for later ligation into pRS.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 7 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient !! Part name<br />
|-<br />
| A || 60,0°C ||<br />
|-<br />
| B || 58,9°C || Tadh1<br />
|-<br />
| C || 57,0°C || Tadh1<br />
|-<br />
| D || 54,1°C ||<br />
|-<br />
| E || 50,5°C || Tadh1<br />
|-<br />
| F || 47,9°C || Pfet3<br />
|-<br />
| G || 46,0°C || Pfet3<br />
|-<br />
| H || 45,0°C || Pfet3<br />
|}<br />
<br />
The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of part 7 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|scaled-up restriction digest]] of the parts 3, 4 and 10 was executed, because the digested stocks of these parts were empty.<br />
<br />
4. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
5. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==<br />
<br />
1. Some more [[Team:Tuebingen/NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|mini-preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]].<br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. We finished our Wiki, designed our poster and made our presentation for the Jamboree in Amsterdam.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-26T09:57:29Z<p>Jakobmatthes: /* Week 11 (9/17 - 9/23) */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! length [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen <br /> (University of Tuebingen) || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed. <br/><br />
For each part we made a 7 times preparation with a gradient of 45°C - 58,9°C with the following intermediate stages:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient<br />
|-<br />
| A || 60,0°C<br />
|-<br />
| B || 58,9°C<br />
|-<br />
| C || 57,0°C<br />
|-<br />
| D || 54,1°C<br />
|-<br />
| E || 50,5°C<br />
|-<br />
| F || 47,9°C<br />
|-<br />
| G || 46,0°C<br />
|-<br />
| H || 45,0°C<br />
|}<br />
<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
The purification of the parts Mig 1 and mPR D.r. and the pRS vectors was performed with a PCR purification Kit in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
4. A great restriction digest and preparative gel-electrophoresis of the parts 3 and 8 was executed to prepare them for later ligation into pRS.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 7 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient !! Part name<br />
|-<br />
| A || 60,0°C ||<br />
|-<br />
| B || 58,9°C || Tadh1<br />
|-<br />
| C || 57,0°C || Tadh1<br />
|-<br />
| D || 54,1°C ||<br />
|-<br />
| E || 50,5°C || Tadh1<br />
|-<br />
| F || 47,9°C || Pfet3<br />
|-<br />
| G || 46,0°C || Pfet3<br />
|-<br />
| H || 45,0°C || Pfet3<br />
|}<br />
<br />
The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of part 7 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|scaled-up restriction digest]] of the parts 3, 4 and 10 was executed, because the digested stocks of these parts were empty.<br />
<br />
4. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
5. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==<br />
<br />
1. Some more [[Team:Tuebingen/NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|mini-preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]].<br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. We finished our Wiki, designed our poster and made our presentation for the Jamboree in Amsterdam.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-26T09:55:28Z<p>Jakobmatthes: /* Parts, Plasmids and Constructs */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! length [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen <br /> (University of Tuebingen) || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed. <br/><br />
For each part we made a 7 times preparation with a gradient of 45°C - 58,9°C with the following intermediate stages:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient<br />
|-<br />
| A || 60,0°C<br />
|-<br />
| B || 58,9°C<br />
|-<br />
| C || 57,0°C<br />
|-<br />
| D || 54,1°C<br />
|-<br />
| E || 50,5°C<br />
|-<br />
| F || 47,9°C<br />
|-<br />
| G || 46,0°C<br />
|-<br />
| H || 45,0°C<br />
|}<br />
<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
The purification of the parts Mig 1 and mPR D.r. and the pRS vectors was performed with a PCR purification Kit in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
4. A great restriction digest and preparative gel-electrophoresis of the parts 3 and 8 was executed to prepare them for later ligation into pRS.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 7 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient !! Part name<br />
|-<br />
| A || 60,0°C<br />
|-<br />
| B || 58,9°C || Tadh1<br />
|-<br />
| C || 57,0°C || Tadh1<br />
|-<br />
| D || 54,1°C<br />
|-<br />
| E || 50,5°C || Tadh1<br />
|-<br />
| F || 47,9°C || Pfet3<br />
|-<br />
| G || 46,0°C || Pfet3<br />
|-<br />
| H || 45,0°C || Pfet3<br />
|}<br />
<br />
The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of part 7 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|scaled-up restriction digest]] of the parts 3, 4 and 10 was executed, because the digested stocks of these parts were empty.<br />
<br />
4. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
5. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==<br />
<br />
1. Some more [[Team:Tuebingen/NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|mini-preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]].<br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. We finished our Wiki, designed our poster and made our presentation for the Jamboree in Amsterdam.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-26T09:47:31Z<p>Jakobmatthes: /* Weekly Reports */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! lenght [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen <br /> (University of Tuebingen) || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed. <br/><br />
For each part we made a 7 times preparation with a gradient of 45°C - 58,9°C with the following intermediate stages:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient<br />
|-<br />
| A || 60,0°C<br />
|-<br />
| B || 58,9°C<br />
|-<br />
| C || 57,0°C<br />
|-<br />
| D || 54,1°C<br />
|-<br />
| E || 50,5°C<br />
|-<br />
| F || 47,9°C<br />
|-<br />
| G || 46,0°C<br />
|-<br />
| H || 45,0°C<br />
|}<br />
<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
The purification of the parts Mig 1 and mPR D.r. and the pRS vectors was performed with a PCR purification Kit in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
4. A great restriction digest and preparative gel-electrophoresis of the parts 3 and 8 was executed to prepare them for later ligation into pRS.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 7 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient !! Part name<br />
|-<br />
| A || 60,0°C<br />
|-<br />
| B || 58,9°C || Tadh1<br />
|-<br />
| C || 57,0°C || Tadh1<br />
|-<br />
| D || 54,1°C<br />
|-<br />
| E || 50,5°C || Tadh1<br />
|-<br />
| F || 47,9°C || Pfet3<br />
|-<br />
| G || 46,0°C || Pfet3<br />
|-<br />
| H || 45,0°C || Pfet3<br />
|}<br />
<br />
The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of part 7 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|scaled-up restriction digest]] of the parts 3, 4 and 10 was executed, because the digested stocks of these parts were empty.<br />
<br />
4. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
5. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==<br />
<br />
1. Some more [[Team:Tuebingen/NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|mini-preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]].<br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. We finished our Wiki, designed our poster and made our presentation for the Jamboree in Amsterdam.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-26T09:45:09Z<p>Jakobmatthes: /* Weekly Reports */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! lenght [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen <br /> (University of Tuebingen) || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed. <br/><br />
For each part we made a 7 times preparation with a gradient of 45°C - 58,9°C with the following intermediate stages:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient<br />
|-<br />
| A || 60,0°C<br />
|-<br />
| B || 58,9°C<br />
|-<br />
| C || 57,0°C<br />
|-<br />
| D || 54,1°C<br />
|-<br />
| E || 50,5°C<br />
|-<br />
| F || 47,9°C<br />
|-<br />
| G || 46,0°C<br />
|-<br />
| H || 45,0°C<br />
|}<br />
<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
The purification of the parts Mig 1 and mPR D.r. and the pRS vectors was performed with a PCR purification Kit in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
4. A great restriction digest and preparative gel-electrophoresis of the parts 3 and 8 was executed to prepare them for later ligation into pRS.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 7 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Position in the thermocycler !! gradient !! Part name<br />
|-<br />
| A || 60,0°C<br />
|-<br />
| B || 58,9°C || Tadh1<br />
|-<br />
| C || 57,0°C || Tadh1<br />
|-<br />
| D || 54,1°C<br />
|-<br />
| E || 50,5°C || Tadh1<br />
|-<br />
| F || 47,9°C || Pfet3<br />
|-<br />
| G || 46,0°C || Pfet3<br />
|-<br />
| H || 45,0°C || Pfet3<br />
|}<br />
<br />
The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of part 7 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. A new great restriction digest of the parts 3,4 and 10 was executed, because the cut stocks of these parts were empty.<br />
<br />
4. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
5. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==<br />
<br />
1. Some more mini-preps of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following restriction digest.<br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. We finished our Wiki, designed our poster and made our presentation for the Jamboree in Amsterdam.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultsTeam:Tuebingen/Results2012-09-26T09:43:31Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Results =<br />
__TOC__<br />
<br />
[[File:Tue-labmap-results.png|600px|thumb|center|final status of our parts]]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookPreparationsTeam:Tuebingen/NotebookPreparations2012-09-26T09:40:35Z<p>Jakobmatthes: /* Identification of genes */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Preparations =<br />
__TOC__<br />
After determining our principal project idea we had to design our system prior to any work in the wet lab. Several steps were involved:<br />
<br />
== Identification of plasmids ==<br />
<br />
We decided to use a shuttle vector which works in ''E. coli'' and ''S. cerevisiae''. The advantage is that we can assemble and build our target constructs in the rapid growing ''E. coli''. The shuttle vectors must meet the following demands:<br />
* Multiple cloning site with XbaI and SpeI restriction sites<br />
* Multiple cloning site with beta-galactosidase, so blue-white screening is possible<br />
* Ampicillin resistance<br />
* amino-acid genes for selection<br />
* Integration site for integration in the yeast genome<br />
<br />
After consultation with Prof. Jansen, our yeast expert, we decided to use the shuttle vectors pRS313, pRS315 and pRS316.<br />
<br />
Due to several EcoRI and PstI restriction sites in the pRS plasmids (not located around the multiple cloning site), we can only use XbaI and SpeI for assembly.<br />
<br />
== Identification of genes ==<br />
<br />
'''Receptors:''' Aiming at aquatic environment we chose to use the membrane progesterone receptor (mPR) from the model organism ''Danio rerio'' (zebrafish). The second organism was ''Salmo salar'' (salmon) but we could not locate a homologous gene due to missing full genome sequence data. Following a database-wide BLAST search we selected the membrane progesterone receptor from ''Xenopus laevis'' (African clawed frog), another well-studied model organism, as our second receptor.<br />
<br />
'''Signalling:''' The fet3 promoter targeted by the membrane receptors was already determined and proven working by J Smith et al. (2008). We could not obtain the sequence used by Smith and decided to take the upstream sequence (approx. 600bp) of the fet3 gene.<br />
<br />
Since Pfet3 is regulated negatively by the mPR we decided on inverting our signal with an additional signaling step to have more sensitive measurement results. So the second part of the signalling system needs a repressor and its target. Additionally, knock-out strains not containing a working repressor have to be viable. We chose the mig1 (repressor) / Psuc2 (repressor target) pair and the rox1 (repressor) / Panb1 (repressor target) pair.<br />
<br />
'''Reporter:''' The targets of our signalling system regulate our reporter. We have access to the plasmid storage of our lab. Common reporters used in this yeast-based environment are firefly luciferase and beta-galactosidase. Both are available to us and have no legal issues concerning the publishing in the PartsRegistry.<br />
<br />
== Sequence analysis and primer design ==<br />
<br />
Next was the check of all sequences for unwanted restriction sites.<br />
<br />
As noted above, due to several unwanted restriction sites (EcoRI and PstI) in the pRS vectors, we can only use XbaI and SpeI for assembly.<br />
<br />
The firefly luciferase has an unwanted XbaI site so we had to use NheI/SpeI restriction instead. The XbaI site could be removed by gene synthesis.<br />
<br />
Three weeks before the official lab time started, we designed our primers with [http://de-de.invitrogen.com/site/de/de/home/Products-and-Services/Applications/Cloning/vector-nti-software.html Vector NTI]. The programm was basically used to compute good primers. We then manually optimized the primer sequences by setting G and C nucleotides at the 3'-tail so that the development of hydrogen bridge bonds is increased, which leads to stronger bindings.<br />
In addition the primer sequences were modified so that the related primer pairs have preferably the same or close annealing temperatures.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! forward primer !! reverse primer<br />
|-<br />
| 1 || lacZ || 5’-GCTAGCATGGTGCTGCGTTGG || 5’-ACTAGTTTATTTTTGACACCAG<br />
|-<br />
| 2 || luciferase || 5’-GCTAGCATGGAAGACGCCAAA || 5’-ACTAGTTTAAAGCTTCTTTCCGCC<br />
|-<br />
| 3 || Padh1 || 5’-TCTAGAAAGAAATGATGGTAAA || 5’-ACTAGTAGTTGATTGTATGCTT<br />
|-<br />
| 4 || Psuc2 || 5’-TCTAGACATACTAAGACATTTACCG || 5’-ACTAGTCATATACGTTAGTGAAAA <br />
|-<br />
| 5 || Pfet3 || 5’-TCTAGACATTACTGCTGTAAAAAGG || 5’-GCAAAAAATTAGAACTAGACTAGT <br />
|-<br />
| 6 || Panb1 || 5’-TCTAGATTTTTTCCTGTGTTCACC || 5’-ACTAGTGTTTTAGTGTGTGAATGA <br />
|-<br />
| 7 || Tadh1 || 5’-TCTAGAGCGAATTTCTTATGATTT || 5’-ACTAGTAGGTGTTGTCCTCTGAGG<br />
|-<br />
| 8 || rox1 || 5’-TCTAGAATGAATCCTAAATCCTCTACAC || 5-ACTAGTAATTGTTCTTTTGAGGCG <br />
|}</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultsTeam:Tuebingen/Results2012-09-26T09:31:53Z<p>Jakobmatthes: Created page with "{{:Team:Tuebingen/Template/Tuebingen}} = Results = final status of our parts"</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Results =<br />
[[File:Tue-labmap-results.png|600px|thumb|center|final status of our parts]]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-26T09:31:34Z<p>Jakobmatthes: /* Procedure */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! lenght [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen <br /> (University of Tuebingen) || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed. <br/><br />
For each part we made a 7 times preparation with a gradient of 45°C - 58,9°C with the following intermediate stages:<br />
<br />
<br />
<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
The purification of the parts Mig 1 and mPR D.r. and the pRS vectors was performed with a PCR purification Kit in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
4. A great restriction digest and preparative gel-electrophoresis of the parts 3 and 8 was executed to prepare them for later ligation into pRS.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 7 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of part 7 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. A new great restriction digest of the parts 3,4 and 10 was executed, because the cut stocks of these parts were empty.<br />
<br />
4. Some more unsuccessful ligation and transformation of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
5. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==<br />
<br />
1. Some more mini-preps of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following restriction digest.<br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. We finished our Wiki, designed our poster and made our presentation for the Jamboree in Amsterdam.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/Templates/NavigationTeam:Tuebingen/Templates/Navigation2012-09-26T09:29:23Z<p>Jakobmatthes: </p>
<hr />
<div><!-- the old navigation bar<br />
<div id="nav"><br />
* [[Team:Tuebingen|Home]]<br />
* [[Team:Tuebingen/Team|Team]]<br />
* [[Team:Tuebingen/Project|Project]]<br />
* [[Team:Tuebingen/Activities|Activities]]<br />
* [[Team:Tuebingen/Notebook|Notebook]]<br />
* [[Team:Tuebingen/Attributions|Attributions]]<br />
* [[Team:Tuebingen/Safety|Safety]]<br />
* [[Team:Tuebingen/Parts|Submitted Parts]]<br />
* [[Team:Tuebingen/Sponsorship|Sponsorship]]<br />
* [[Team:Tuebingen/Contact|Contact us]]<br />
</div><br />
--><br />
<div id="nav"><br />
* [[Team:Tuebingen|Home]]<br />
* <span>Team</span><br />
** [[Team:Tuebingen/TeamWho|Who we are]]<br />
** [[Team:Tuebingen/TeamWhat|What we did]]<br />
** [[Team:Tuebingen/TeamWhere|Where we're from]]<br />
** [[Team:Tuebingen/Attributions|Attributions]]<br />
** [[Team:Tuebingen/ResultExp|Experience]]<br />
** [[Team:Tuebingen/ResultPersonal|Impressions]]<br />
** [[Team:Tuebingen/ResultAcks|Acknowledgment]]<br />
* <span>Project</span><br />
** [[Team:Tuebingen/ProjectOverview|Overview]]<br />
** [[Team:Tuebingen/ProjectQuestions|Questions]]<br />
** [[Team:Tuebingen/ProjectMechanism|Mechanism]]<br />
** [[Team:Tuebingen/ProjectImplementation|Implementation]]<br />
** [[Team:Tuebingen/Parts|Submitted Parts]]<br />
** [[Team:Tuebingen/Application|Application]]<br />
** [[Team:Tuebingen/References|References]]<br />
* <span>Notebook</span><br />
** [[Team:Tuebingen/NotebookPreparations|Preparations]]<br />
** [[Team:Tuebingen/NotebookReports|Reports]]<br />
** [[Team:Tuebingen/Results|Results]]<br />
** [[Team:Tuebingen/NotebookProtocols|Protocols]]<br />
** [[Team:Tuebingen/NotebookAppendix|Appendix]]<br />
** [[Team:Tuebingen/NotebookCalendar|Calendar]]<br />
* [[Team:Tuebingen/Activities|Activities & Press]]<br />
* [[Team:Tuebingen/Safety|Safety]]<br />
* [[Team:Tuebingen/Sponsorship|Sponsorship]]<br />
* [[Team:Tuebingen/Contact|Contact us]]<br />
</div></div>Jakobmattheshttp://2012.igem.org/File:Tue-labmap.pngFile:Tue-labmap.png2012-09-26T09:26:01Z<p>Jakobmatthes: uploaded a new version of &quot;File:Tue-labmap.png&quot;</p>
<hr />
<div></div>Jakobmattheshttp://2012.igem.org/File:Tue-labmap-results.pngFile:Tue-labmap-results.png2012-09-26T09:22:59Z<p>Jakobmatthes: </p>
<hr />
<div></div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultExpTeam:Tuebingen/ResultExp2012-09-26T09:15:06Z<p>Jakobmatthes: /* The Experience */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Experience =<br />
<br />
We have come a long way since we first met to form a team and discuss possible projects. We learned how to make an idea into something big, that we can really accomplish things with motivation and hard work, how to use a lab without constant instructions and most of all that as a team even disappointments can be fun. <br />
<br />
We had a great time with iGEM, made new friends, met people from all over Germany and we are looking forward to meet even more in Amsterdam and in the coming years.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/Templates/NavigationTeam:Tuebingen/Templates/Navigation2012-09-26T08:09:15Z<p>Jakobmatthes: </p>
<hr />
<div><!-- the old navigation bar<br />
<div id="nav"><br />
* [[Team:Tuebingen|Home]]<br />
* [[Team:Tuebingen/Team|Team]]<br />
* [[Team:Tuebingen/Project|Project]]<br />
* [[Team:Tuebingen/Activities|Activities]]<br />
* [[Team:Tuebingen/Notebook|Notebook]]<br />
* [[Team:Tuebingen/Attributions|Attributions]]<br />
* [[Team:Tuebingen/Safety|Safety]]<br />
* [[Team:Tuebingen/Parts|Submitted Parts]]<br />
* [[Team:Tuebingen/Sponsorship|Sponsorship]]<br />
* [[Team:Tuebingen/Contact|Contact us]]<br />
</div><br />
--><br />
<div id="nav"><br />
* [[Team:Tuebingen|Home]]<br />
* <span>Team</span><br />
** [[Team:Tuebingen/TeamWho|Who we are]]<br />
** [[Team:Tuebingen/TeamWhat|What we did]]<br />
** [[Team:Tuebingen/TeamWhere|Where we're from]]<br />
** [[Team:Tuebingen/Attributions|Attributions]]<br />
** [[Team:Tuebingen/ResultExp|Experience]]<br />
** [[Team:Tuebingen/ResultPersonal|Impressions]]<br />
** [[Team:Tuebingen/ResultAcks|Acknowledgment]]<br />
* <span>Project</span><br />
** [[Team:Tuebingen/ProjectOverview|Overview]]<br />
** [[Team:Tuebingen/ProjectQuestions|Questions]]<br />
** [[Team:Tuebingen/ProjectMechanism|Mechanism]]<br />
** [[Team:Tuebingen/ProjectImplementation|Implementation]]<br />
** [[Team:Tuebingen/Parts|Submitted Parts]]<br />
** [[Team:Tuebingen/Application|Application]]<br />
** [[Team:Tuebingen/References|References]]<br />
* <span>Notebook</span><br />
** [[Team:Tuebingen/NotebookPreparations|Preparations]]<br />
** [[Team:Tuebingen/NotebookReports|Reports]]<br />
** [[Team:Tuebingen/NotebookProtocols|Protocols]]<br />
** [[Team:Tuebingen/NotebookAppendix|Appendix]]<br />
** [[Team:Tuebingen/NotebookCalendar|Calendar]]<br />
* [[Team:Tuebingen/Activities|Activities & Press]]<br />
* [[Team:Tuebingen/Safety|Safety]]<br />
* [[Team:Tuebingen/Sponsorship|Sponsorship]]<br />
* [[Team:Tuebingen/Contact|Contact us]]<br />
</div></div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookProtocolsTeam:Tuebingen/NotebookProtocols2012-09-26T08:06:31Z<p>Jakobmatthes: /* Protocols */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Protocols =<br />
__TOC__<br />
== Chemo-competent cells ==<br />
'''Inoue buffer'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| MnCl<sub>2</sub> * 2H<sub>2</sub>0 || 9.67 g<br />
|-<br />
| CaCl<sub>2</sub> * 2H<sub>2</sub>0 || 2.2 g<br />
|-<br />
| KCl || 18.65 g<br />
|-<br />
| PIPES (0.5 M, pH 6.7) || 20 ml<br />
|-<br />
| H<sub>2</sub>0 || ad 1 l<br />
|}<br />
Sterilize through filtration (0.45 µm filter) and store at -20 °C.<br />
<br />
'''Cells'''<br />
# Pick an ''E. coli'' colony and inoculate 25 ml SOB.<br />
# Let bacteria grow for 8 hours at 37 °C and 250 rpm.<br />
# Inoculate three 100 ml SOB volumes with 1 ml, 2 ml and 4 ml of the prepared pre-culture.<br />
# Incubate over night at 18 - 22 °C and 200 rpm.<br />
# At OD<sub>600</sub> = 0.55, put culture for 10 min on ice.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Resuspend cell pellet in 30 ml 0 °C Inoue buffer.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Repeat the previous two steps.<br />
# Resuspend cells in 8 ml 0 °C Inoue buffer. Add 1.5 ml DMSO and incubate on ice for 10 min.<br />
# Aliquot cells à 100 µl and freeze in liquid nitrogen. Store at -80 °C.<br />
<br />
<br />
== pGEM Ligation ==<br />
Ligation for TA-cloning of PCR products<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 2X Rapid Ligation Buffer || 5 µl<br />
|-<br />
| pGEM vector || 0.5 µl (25ng)<br />
|-<br />
| PCR product || 3.5 µl<br />
|-<br />
| T4 DNA ligase || 1 µl (3 Weiss units)<br />
|}<br />
<br />
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.<br />
<br />
<br />
<br />
== Ligation ==<br />
Ligation for digested parts and vectors<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 10X T4 DNA Ligase Buffer || 1 µl<br />
|-<br />
| vector DNA || 1 µl (20-100 ng)<br />
|-<br />
| insert DNA || 5 µl (up to 5:1 molar ratio insert to vector)<br />
|-<br />
| T4 DNA ligase || 1 µl (1 unit)<br />
|-<br />
| water || 2.5 µl<br />
|}<br />
Mix all reagents and incubate at 22°C for 1 hour.<br />
<br />
<br />
<br />
== Chemotransformation ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| chemo-competent ''E. coli'' || 100 µl<br />
|-<br />
| plasmid DNA || up to 10 µl (max. 1/10 of volume)<br />
|}<br />
# Add plasmid DNA to cell culture.<br />
# Incubate for 30 min on ice.<br />
# Heat shock for 90 sec at 42°C.<br />
# Add 900 µl LB.<br />
# Let the bacteria grow at 37°C for at least 1 hour.<br />
<br />
<br />
<br />
== Restriction digest ==<br />
<br />
<br />
<br />
=== control digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 1 µl<br />
|-<br />
| XbaI (RE) || 0.5 µl (5 units)<br />
|-<br />
| SpeI (RE) || 0.5 µl (5 units)<br />
|-<br />
| DNA || 1 µl (up to 1 µg)<br />
|- <br />
| water || 7 µl<br />
|}<br />
# Incubate at least for 1 hour at 37°C.<br />
<br />
<br />
=== preparative double digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 5 µl (50 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for 8 hours at 37°C.<br />
# After 3 hours add 2 µl SpeI.<br />
# Add 7 µl XbaI and incubate for another 8 hours.<br />
<br />
<br />
=== plasmid linearization ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 7 µl (70 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for at least 8 hours at 37°C.<br />
<br />
<br />
== PCR ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Taq/Pfu buffer || 5 µl<br />
|-<br />
| Taq/Pfu polymerase || 1 µl<br />
|-<br />
| primer forward || 0.5 µl (100 pmol/µl)<br />
|-<br />
| primer reverse || 0.5 µl (100 pmol/µl)<br />
|-<br />
| dNTPs || 2.5 µl (200 µM)<br />
|-<br />
| template DNA || 1 µl<br />
|-<br />
| water || 36 µl<br />
|}<br />
<br />
<br />
<br />
'''PCR conditions'''<br />
{| class="wikitable"<br />
|-<br />
! Step !! Duration !! Settings<br />
|-<br />
| 1 || 2 min || 94°C<br />
|-<br />
| 2 || 45 sec || 94°C<br />
|-<br />
| 3 || 30 sec || gradient or annealing temperature<br />
|-<br />
| 4 || 90 sec || 72°C<br />
|-<br />
| || || steps 2-4: 30 cycles<br />
|-<br />
| 5 || 7 min || 72°C<br />
|-<br />
| 6 || (hold) || 4°C<br />
|}<br />
<br />
<br />
<br />
== Gel electrophoresis ==<br />
<br />
'''TAE buffer 50x'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 0.05 M EDTA || 18.61 g<br />
|-<br />
| 1 M acetic acid || 60.05 g<br />
|-<br />
| 2 M Tris || 242.28 g<br />
|-<br />
| water || 1 l<br />
|}<br />
Adjust to pH 8.5.<br />
<br />
<br />
<br />
'''Gel'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| TAE 1x buffer || 120 ml<br />
|-<br />
| Agarose || 1.2 g<br />
|}<br />
Solve agarose in TAE 1x buffer and boil until solution is clear.<br />
<br />
<br />
<br />
'''Well loading'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| PCR product or DNA || 5 µl<br />
|-<br />
| Loading dye 6x || 1 µl<br />
|}<br />
Can be scaled up linearly.<br />
<br />
<br />
<br />
== LB medium ==<br />
<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 10 g<br />
|-<br />
| yeast extract || 5 g<br />
|-<br />
| NaCl || 5 g<br />
|-<br />
| water || 1 l<br />
|}<br />
Adjust to pH 7.0.<br />
<br />
<br />
<br />
'''Agar-plates'''<br />
<br />
# Solve 16 g Agar-Agar in 1 l LB buffer and boil until solution is clear. <br />
# If it is nearly cold pour it into petri dishes (approx. 25 ml per dish).<br />
<br />
<br />
<br />
== SOB medium ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 20 g<br />
|-<br />
| yeast-extract || 5 g<br />
|-<br />
| NaCl || 0.5 g<br />
|-<br />
| 250mM KCl || 10 ml<br />
|-<br />
| water MiliQ || 1 l<br />
|}<br />
<br />
# Solve the components in 1 l water.<br />
# Autoclave.<br />
# After autoclaving add 5 ml MgCl<sub>2</sub>.<br />
<br />
<br />
<br />
<br />
== Genaxxon Plasmid DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon Gel Extraction Mini Prep Kit ==<br />
[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon PCR DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== QIAGEN Plasmid Midi Kit ==<br />
[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookProtocolsTeam:Tuebingen/NotebookProtocols2012-09-26T08:04:17Z<p>Jakobmatthes: /* Protocols */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Protocols =<br />
__TOC__<br />
== Chemo-competent cells ==<br />
'''Inoue buffer'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| MnCl<sub>2</sub> * 2H<sub>2</sub>0 || 9.67 g<br />
|-<br />
| CaCl<sub>2</sub> * 2H<sub>2</sub>0 || 2.2 g<br />
|-<br />
| KCl || 18.65 g<br />
|-<br />
| PIPES (0.5 M, pH 6.7) || 20 ml<br />
|-<br />
| H<sub>2</sub>0 || ad 1 l<br />
|}<br />
Sterilize through filtration (0.45 µm filter) and store at -20 °C.<br />
<br />
'''Cells'''<br />
# Pick an ''E. coli'' colony and inoculate 25 ml SOB.<br />
# Let bacteria grow for 8 hours at 37 °C and 250 rpm.<br />
# Inoculate three 100 ml SOB volumes with 1 ml, 2 ml and 4 ml of the prepared pre-culture.<br />
# Incubate over night at 18 - 22 °C and 200 rpm.<br />
# At OD<sub>600</sub> = 0.55, put culture for 10 min on ice.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Resuspend cell pellet in 30 ml 0 °C Inoue buffer.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Repeat the previous two steps.<br />
# Resuspend cells in 8 ml 0 °C Inoue buffer. Add 1.5 ml DMSO and incubate on ice for 10 min.<br />
# Aliquot cells à 100 µl and freeze in liquid nitrogen. Store at -80 °C.<br />
<br />
<br />
== pGEM Ligation ==<br />
Ligation for TA-cloning of PCR products<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 2X Rapid Ligation Buffer || 5 µl<br />
|-<br />
| pGEM vector || 0.5 µl (25ng)<br />
|-<br />
| PCR product || 3.5 µl<br />
|-<br />
| T4 DNA ligase || 1 µl (3 Weiss units)<br />
|}<br />
<br />
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.<br />
<br />
<br />
<br />
<br />
== Ligation ==<br />
Ligation for digested parts and vectors<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 10X T4 DNA Ligase Buffer || 1 µl<br />
|-<br />
| vector DNA || 1 µl (20-100 ng)<br />
|-<br />
| insert DNA || 5 µl (up to 5:1 molar ratio insert to vector)<br />
|-<br />
| T4 DNA ligase || 1 µl (1 unit)<br />
|-<br />
| water || 2.5 µl<br />
|}<br />
Mix all reagents and incubate at 22°C for 1 hour.<br />
<br />
<br />
<br />
<br />
== Chemotransformation ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| chemo-competent ''E. coli'' || 100 µl<br />
|-<br />
| plasmid DNA || up to 10 µl (max. 1/10 of volume)<br />
|}<br />
# Add plasmid DNA to cell culture.<br />
# Incubate for 30 min on ice.<br />
# Heat shock for 90 sec at 42°C.<br />
# Add 900 µl LB.<br />
# Let the bacteria grow at 37°C for at least 1 hour.<br />
<br />
<br />
<br />
<br />
== Restriction digest ==<br />
<br />
<br />
<br />
=== control digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 1 µl<br />
|-<br />
| XbaI (RE) || 0.5 µl (5 units)<br />
|-<br />
| SpeI (RE) || 0.5 µl (5 units)<br />
|-<br />
| DNA || 1 µl (up to 1 µg)<br />
|- <br />
| water || 7 µl<br />
|}<br />
# Incubate at least for 1 hour at 37°C.<br />
<br />
<br />
=== preparative double digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 5 µl (50 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for 8 hours at 37°C.<br />
# After 3 hours add 2 µl SpeI.<br />
# Add 7 µl XbaI and incubate for another 8 hours.<br />
<br />
<br />
=== plasmid linearization ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 7 µl (70 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for at least 8 hours at 37°C.<br />
<br />
<br />
== PCR ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Taq/Pfu buffer || 5 µl<br />
|-<br />
| Taq/Pfu polymerase || 1 µl<br />
|-<br />
| primer forward || 0.5 µl (100 pmol/µl)<br />
|-<br />
| primer reverse || 0.5 µl (100 pmol/µl)<br />
|-<br />
| dNTPs || 2.5 µl (200 µM)<br />
|-<br />
| template DNA || 1 µl<br />
|-<br />
| water || 36 µl<br />
|}<br />
<br />
<br />
<br />
'''PCR conditions'''<br />
{| class="wikitable"<br />
|-<br />
! Step !! Duration !! Settings<br />
|-<br />
| 1 || 2 min || 94°C<br />
|-<br />
| 2 || 45 sec || 94°C<br />
|-<br />
| 3 || 30 sec || gradient or annealing temperature<br />
|-<br />
| 4 || 90 sec || 72°C<br />
|-<br />
| || || steps 2-4: 30 cycles<br />
|-<br />
| 5 || 7 min || 72°C<br />
|-<br />
| 6 || (hold) || 4°C<br />
|}<br />
<br />
<br />
<br />
<br />
== Gel electrophoresis ==<br />
<br />
'''TAE buffer 50x'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 0.05 M EDTA || 18.61 g<br />
|-<br />
| 1 M acetic acid || 60.05 g<br />
|-<br />
| 2 M Tris || 242.28 g<br />
|-<br />
| water || 1 l<br />
|}<br />
Adjust to pH 8.5.<br />
<br />
<br />
<br />
'''Gel'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| TAE 1x buffer || 120 ml<br />
|-<br />
| Agarose || 1.2 g<br />
|}<br />
Solve agarose in TAE 1x buffer and boil until solution is clear.<br />
<br />
<br />
<br />
'''Well loading'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| PCR product or DNA || 5 µl<br />
|-<br />
| Loading dye 6x || 1 µl<br />
|}<br />
Can be scaled up linearly.<br />
<br />
<br />
<br />
== LB medium ==<br />
<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 10,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 5,0 g<br />
|-<br />
| water || 1,0 l<br />
|}<br />
Adjust to pH 7.0.<br />
<br />
<br />
<br />
'''Agar-plates'''<br />
<br />
# Solve 16g Agar-Agar in 1l LB buffer and boil until solution is clear. <br />
# If it is nearly cold pour it into some petri dish.<br />
<br />
<br />
<br />
<br />
== SOB medium ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 20,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 0,5 g<br />
|-<br />
| 250mM KCl || 10ml<br />
|-<br />
| water MiliQ || 1l<br />
|}<br />
<br />
# Solve the components in 1 l water.<br />
# Autoclave.<br />
# After autoclaving add 5 ml MgCl<sub>2</sub>.<br />
<br />
<br />
<br />
<br />
== Genaxxon Plasmid DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon Gel Extraction Mini Prep Kit ==<br />
[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon PCR DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== QIAGEN Plasmid Midi Kit ==<br />
[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookProtocolsTeam:Tuebingen/NotebookProtocols2012-09-26T08:03:15Z<p>Jakobmatthes: /* Protocols */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Protocols =<br />
__TOC__<br />
== Chemo-competent cells ==<br />
'''Inoue buffer'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| MnCl<sub>2</sub> * 2H<sub>2</sub>0 || 9.67 g<br />
|-<br />
| CaCl<sub>2</sub> * 2H<sub>2</sub>0 || 2.2 g<br />
|-<br />
| KCl || 18.65 g<br />
|-<br />
| PIPES (0.5 M, pH 6.7) || 20 ml<br />
|-<br />
| H<sub>2</sub>0 || ad 1 l<br />
|}<br />
Sterilize through filtration (0.45 µm filter) and store at -20 °C.<br />
<br />
'''Cells'''<br />
# Pick an ''E. coli'' colony and inoculate 25 ml SOB.<br />
# Let bacteria grow for 8 hours at 37 °C and 250 rpm.<br />
# Inoculate three 100 ml SOB volumes with 1 ml, 2 ml and 4 ml of the prepared pre-culture.<br />
# Incubate over night at 18 - 22 °C and 200 rpm.<br />
# At OD<sub>600</sub> = 0.55, put culture for 10 min on ice.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Resuspend cell pellet in 30 ml 0 °C Inoue buffer.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Repeat the previous two steps.<br />
# Resuspend cells in 8 ml 0 °C Inoue buffer. Add 1.5 ml DMSO and incubate on ice for 10 min.<br />
# Aliquot cells à 100 µl and freeze in liquid nitrogen. Store at -80 °C.<br />
<br />
<br />
== pGEM Ligation ==<br />
Ligation for TA-cloning of PCR products<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 2X Rapid Ligation Buffer || 5 µl<br />
|-<br />
| pGEM vector || 0.5 µl (25ng)<br />
|-<br />
| PCR product || 3.5 µl<br />
|-<br />
| T4 DNA ligase || 1 µl (3 Weiss units)<br />
|}<br />
<br />
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.<br />
<br />
<br />
<br />
<br />
== Ligation ==<br />
Ligation for digested parts and vectors<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 10X T4 DNA Ligase Buffer || 1 µl<br />
|-<br />
| vector DNA || 1 µl (20-100 ng)<br />
|-<br />
| insert DNA || 5 µl (up to 5:1 molar ratio insert to vector)<br />
|-<br />
| T4 DNA ligase || 1 µl (1 unit)<br />
|-<br />
| water || 2.5 µl<br />
|}<br />
Mix all reagents and incubate at 22°C for 1 hour.<br />
<br />
<br />
<br />
<br />
== Chemotransformation ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| chemo-competent ''E. coli'' || 100 µl<br />
|-<br />
| plasmid DNA || up to 10 µl (max. 1/10 of volume)<br />
|}<br />
# Add plasmid DNA to cell culture.<br />
# Incubate for 30 min on ice.<br />
# Heat shock for 90 sec at 42°C.<br />
# Add 900 µl LB.<br />
# Let the bacteria grow at 37°C for at least 1 hour.<br />
<br />
<br />
<br />
<br />
== Restriction digest ==<br />
<br />
<br />
<br />
=== control digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 1 µl<br />
|-<br />
| XbaI (RE) || 0.5 µl (5 units)<br />
|-<br />
| SpeI (RE) || 0.5 µl (5 units)<br />
|-<br />
| DNA || 1 µl (up to 1 µg)<br />
|- <br />
| water || 7 µl<br />
|}<br />
# Incubate at least for 1 hour at 37°C.<br />
<br />
<br />
=== preparative double digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 5 µl (50 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for 8 hours at 37°C.<br />
# After 3 hours add 2 µl SpeI.<br />
# Add 7 µl XbaI and incubate for another 8 hours.<br />
<br />
<br />
=== plasmid linearization ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 7 µl (70 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for at least 8 hours at 37°C.<br />
<br />
<br />
== PCR ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Taq/Pfu buffer || 5 µl<br />
|-<br />
| Taq/Pfu polymerase || 1 µl<br />
|-<br />
| primer forward || 0.5 µl (100 pmol/µl)<br />
|-<br />
| primer reverse || 0.5 µl (100 pmol/µl)<br />
|-<br />
| dNTPs || 2.5 µl (200 µM)<br />
|-<br />
| template DNA || 1 µl<br />
|-<br />
| water || 36 µl<br />
|}<br />
<br />
<br />
<br />
'''PCR conditions'''<br />
{| class="wikitable"<br />
|-<br />
! Step !! Duration !! Settings<br />
|-<br />
| 1 || 2 min || 94°C<br />
|-<br />
| 2 || 45 sec || 94°C<br />
|-<br />
| 3 || 30 sec || gradient or annealing temperature<br />
|-<br />
| 4 || 90 sec || 72°C<br />
|-<br />
| || || steps 2-4: 30 cycles<br />
|-<br />
| 5 || 7 min || 72°C<br />
|-<br />
| 6 || (hold) || 4°C<br />
|}<br />
<br />
<br />
<br />
<br />
== Gel electrophoresis ==<br />
<br />
'''TAE buffer 50x'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 0.05 M EDTA || 18.61 g<br />
|-<br />
| 1 M acetic acid || 60.05 g<br />
|-<br />
| 2 M Tris || 242.28 g<br />
|-<br />
| water || 1 l<br />
|}<br />
Adjust to pH 8.5.<br />
<br />
<br />
<br />
'''Gel'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| TAE 1x buffer || 120 ml<br />
|-<br />
| Agarose || 1.2 g<br />
|}<br />
Solve agarose in TAE 1x buffer and boil until solution is clear.<br />
<br />
<br />
<br />
'''Well loading'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| PCR product or DNA || 5 µl<br />
|-<br />
| Loading dye 6x || 1 µl<br />
|}<br />
Can be scaled up linearly.<br />
<br />
<br />
<br />
== LB medium ==<br />
<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 10,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 5,0 g<br />
|-<br />
| water || 1,0 l<br />
|}<br />
Adjust to pH 7.0.<br />
<br />
<br />
<br />
'''Agar-plates'''<br />
<br />
# Solve 16g Agar-Agar in 1l LB buffer and boil until solution is clear. <br />
# If it is nearly cold pour it into some petri dish.<br />
<br />
<br />
<br />
<br />
== SOB medium ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 20,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 0,5 g<br />
|-<br />
| 250mM KCl || 10ml<br />
|-<br />
| water MiliQ || 1l<br />
|}<br />
<br />
# Solve the components in 1l water.<br />
# autoclave<br />
# After autoclaving add 5ml MgCl2<br />
<br />
<br />
<br />
<br />
== Genaxxon Plasmid DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon Gel Extraction Mini Prep Kit ==<br />
[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon PCR DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== QIAGEN Plasmid Midi Kit ==<br />
[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookProtocolsTeam:Tuebingen/NotebookProtocols2012-09-26T08:02:43Z<p>Jakobmatthes: /* Restriction digest */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Protocols =<br />
__TOC__<br />
== Chemo-competent cells ==<br />
'''Inoue buffer'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| MnCl<sub>2</sub> * 2H<sub>2</sub>0 || 9.67 g<br />
|-<br />
| CaCl<sub>2</sub> * 2H<sub>2</sub>0 || 2.2 g<br />
|-<br />
| KCl || 18.65 g<br />
|-<br />
| PIPES (0.5 M, pH 6.7) || 20 ml<br />
|-<br />
| H<sub>2</sub>0 || ad 1 l<br />
|}<br />
Sterilize through filtration (0.45 µm filter) and store at -20 °C.<br />
<br />
'''Cells'''<br />
# Pick an ''E. coli'' colony and inoculate 25 ml SOB.<br />
# Let bacteria grow for 8 hours at 37 °C and 250 rpm.<br />
# Inoculate three 100 ml SOB volumes with 1 ml, 2 ml and 4 ml of the prepared pre-culture.<br />
# Incubate over night at 18 - 22 °C and 200 rpm.<br />
# At OD<sub>600</sub> = 0.55, put culture for 10 min on ice.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Resuspend cell pellet in 30 ml 0 °C Inoue buffer.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Repeat the previous two steps.<br />
# Resuspend cells in 8 ml 0 °C Inoue buffer. Add 1.5 ml DMSO and incubate on ice for 10 min.<br />
# Aliquot cells à 100 µl and freeze in liquid nitrogen. Store at -80 °C.<br />
<br />
<br />
== pGEM Ligation ==<br />
Ligation for TA-cloning of PCR products<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 2X Rapid Ligation Buffer || 5 µl<br />
|-<br />
| pGEM vector || 0.5 µl (25ng)<br />
|-<br />
| PCR product || 3.5 µl<br />
|-<br />
| T4 DNA ligase || 1 µl (3 Weiss units)<br />
|}<br />
<br />
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.<br />
<br />
<br />
<br />
<br />
== Ligation ==<br />
Ligation for digested parts and vectors<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 10X T4 DNA Ligase Buffer || 1 µl<br />
|-<br />
| vector DNA || 1 µl (20-100 ng)<br />
|-<br />
| insert DNA || 5 µl (up to 5:1 molar ratio insert to vector)<br />
|-<br />
| T4 DNA ligase || 1 µl (1 unit)<br />
|-<br />
| water || 2.5 µl<br />
|}<br />
Mix all reagents and incubate at 22°C for 1 hour.<br />
<br />
<br />
<br />
<br />
== Chemotransformation ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| chemo-competent ''E. coli'' || 100 µl<br />
|-<br />
| plasmid DNA || up to 10 µl (max. 1/10 of volume)<br />
|}<br />
# Add plasmid DNA to cell culture.<br />
# Incubate for 30 min on ice.<br />
# Heat shock for 90 sec at 42°C.<br />
# Add 900 µl LB.<br />
# Let the bacteria grow at 37°C for at least 1 hour.<br />
<br />
<br />
<br />
<br />
== Restriction digest ==<br />
<br />
<br />
<br />
=== control digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 1 µl<br />
|-<br />
| XbaI (RE) || 0.5 µl (5 units)<br />
|-<br />
| SpeI (RE) || 0.5 µl (5 units)<br />
|-<br />
| DNA || 1 µl (up to 1 µg)<br />
|- <br />
| water || 7 µl<br />
|}<br />
# Incubate at least for 1 hour at 37°C.<br />
<br />
<br />
=== preparative double digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 5 µl (50 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for 8 hours at 37°C.<br />
# After 3 hours add 2 µl SpeI.<br />
# Add 7 µl XbaI and incubate for another 8 hours.<br />
<br />
<br />
=== plasmid linearization ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 7 µl (70 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for at least 8 hours at 37°C.<br />
<br />
== PCR ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Taq/Pfu buffer || 5 µl<br />
|-<br />
| Taq/Pfu polymerase || 1 µl<br />
|-<br />
| primer forward || 0.5 µl (100 pmol/µl)<br />
|-<br />
| primer reverse || 0.5 µl (100 pmol/µl)<br />
|-<br />
| dNTPs || 2.5 µl (200 µM)<br />
|-<br />
| template DNA || 1 µl<br />
|-<br />
| water || 36 µl<br />
|}<br />
<br />
<br />
<br />
'''PCR conditions'''<br />
{| class="wikitable"<br />
|-<br />
! Step !! Duration !! Settings<br />
|-<br />
| 1 || 2 min || 94°C<br />
|-<br />
| 2 || 45 sec || 94°C<br />
|-<br />
| 3 || 30 sec || gradient or annealing temperature<br />
|-<br />
| 4 || 90 sec || 72°C<br />
|-<br />
| || || steps 2-4: 30 cycles<br />
|-<br />
| 5 || 7 min || 72°C<br />
|-<br />
| 6 || (hold) || 4°C<br />
|}<br />
<br />
<br />
<br />
<br />
== Gel electrophoresis ==<br />
<br />
'''TAE buffer 50x'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 0.05 M EDTA || 18.61 g<br />
|-<br />
| 1 M acetic acid || 60.05 g<br />
|-<br />
| 2 M Tris || 242.28 g<br />
|-<br />
| water || 1 l<br />
|}<br />
Adjust to pH 8.5.<br />
<br />
<br />
<br />
'''Gel'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| TAE 1x buffer || 120 ml<br />
|-<br />
| Agarose || 1.2 g<br />
|}<br />
Solve agarose in TAE 1x buffer and boil until solution is clear.<br />
<br />
<br />
<br />
'''Well loading'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| PCR product or DNA || 5 µl<br />
|-<br />
| Loading dye 6x || 1 µl<br />
|}<br />
Can be scaled up linearly.<br />
<br />
<br />
<br />
== LB medium ==<br />
<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 10,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 5,0 g<br />
|-<br />
| water || 1,0 l<br />
|}<br />
Adjust to pH 7.0.<br />
<br />
<br />
<br />
'''Agar-plates'''<br />
<br />
# Solve 16g Agar-Agar in 1l LB buffer and boil until solution is clear. <br />
# If it is nearly cold pour it into some petri dish.<br />
<br />
<br />
<br />
<br />
== SOB medium ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 20,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 0,5 g<br />
|-<br />
| 250mM KCl || 10ml<br />
|-<br />
| water MiliQ || 1l<br />
|}<br />
<br />
# Solve the components in 1l water.<br />
# autoclave<br />
# After autoclaving add 5ml MgCl2<br />
<br />
<br />
<br />
<br />
== Genaxxon Plasmid DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon Gel Extraction Mini Prep Kit ==<br />
[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon PCR DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== QIAGEN Plasmid Midi Kit ==<br />
[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultPersonalTeam:Tuebingen/ResultPersonal2012-09-26T07:59:10Z<p>Jakobmatthes: </p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Impressions =<br />
{{:Team:Tuebingen/Templates/Quote|Jan Rudolph|When me and Jakob first dreamt about participating in iGEM we put it away as mission impossible. But with the help of our advisors and the motivated team we found the iGEM experience turned to be real. I am glad that I was able to gain experience in the wet lab which is usually not part of bioinformatics B.sc. I am looking forward to finish our project next year.}}<br />
<br />
{{:Team:Tuebingen/Templates/Quote|Simon Heumos|When Jan and Jakob introduced me to iGEM, my first thought was: „Wow, now I have the opportunity to do something “out of the usual” compared to my “normal” bioinformatics B.sc. stuff!“<br />
Fortunately iGEM turned out to be a lot of fun in and out of the lab, like learning how to do a PCR or getting to know people of other faculties.<br /><br />
I would enjoy the participation in the future progress of our project.}}<br />
<br />
{{:Team:Tuebingen/Templates/Quote|Lukas Zimmermann|Almost without any lab experience, iGEM gave me the opportunity to learn the basics and to get a feeling for scientific work. Furthermore, it was possible for me to spend the summer with meaningful employment. In a nutshell, iGEM was a very valuable experience. Everything speaks for the next year to participate.}}<br />
<br />
{{:Team:Tuebingen/Templates/Quote|Simeon Roßmann|I spent many days of this summer in the lab. One of the most exciting vacations and I didn’t even have to travel! Thank you iGEM and Team Tübingen for a great experience, see you next year.}}</div>Jakobmattheshttp://2012.igem.org/File:Tue-labmap.pngFile:Tue-labmap.png2012-09-25T17:21:49Z<p>Jakobmatthes: uploaded a new version of &quot;File:Tue-labmap.png&quot;</p>
<hr />
<div></div>Jakobmattheshttp://2012.igem.org/File:Tue-labmap.pngFile:Tue-labmap.png2012-09-25T17:16:41Z<p>Jakobmatthes: uploaded a new version of &quot;File:Tue-labmap.png&quot;</p>
<hr />
<div></div>Jakobmattheshttp://2012.igem.org/File:Tue-labmap.pngFile:Tue-labmap.png2012-09-25T17:13:49Z<p>Jakobmatthes: uploaded a new version of &quot;File:Tue-labmap.png&quot;</p>
<hr />
<div></div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-25T13:48:52Z<p>Jakobmatthes: /* Parts, Plasmids and Constructs */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<!--<br />
{|<br />
| [[File:Tue-map-parts.png|200px|thumb|preparation of parts]]<br />
| [[File:Tue-map-vector.png|200px|thumb|preparation of vectors]]<br />
|-<br />
| [[File:Tue-map-assembly.png|200px|thumb|assembly]]<br />
| [[File:Tue-map-shipping.png|200px|thumb|parts for shipping]]<br />
|}<br />
--><br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! lenght [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen <br /> (University of Tuebingen) || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed.<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
A mini plasmid preparation was performed afterwards to purify DNA in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 6 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of part 6 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookPreparationsTeam:Tuebingen/NotebookPreparations2012-09-25T12:27:03Z<p>Jakobmatthes: /* Sequence analysis and primer design */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Preparations =<br />
__TOC__<br />
After determining our principal project idea we had to design our system prior to any work in the wet lab. Several steps were involved:<br />
<br />
== Identification of plasmids ==<br />
<br />
We decided to use a shuttle vector which works in ''E. coli'' and ''S. cerevisiae''. The advantage is that we can assemble and build our target constructs in the rapid growing ''E. coli''. The shuttle vectors must meet the following demands:<br />
* Multiple cloning site with XbaI and SpeI restriction sites<br />
* Multiple cloning site with beta-galactosidase, so blue-white screening is possible<br />
* Ampicillin resistance<br />
* amino-acid genes for selection<br />
* Integration site for integration in the yeast genome<br />
<br />
After consultation with Prof. Jansen, our yeast expert, we decided to use the shuttle vectors pRS313, pRS315 and pRS316.<br />
<br />
Due to several EcoRI and PstI restriction sites in the pRS plasmids (not located around the multiple cloning site), we can only use XbaI and SpeI for assembly.<br />
<br />
== Identification of genes ==<br />
<br />
'''Receptors:''' Aiming at aquatic environment we chose to use the membrane progesterone receptor (mPR) from the model organism ''Danio rerio'' (zebra fish). The second organism was ''Salmo salar'' (salmon) but we could not locate a homologous gene due to missing full genome sequence data. Following a database-wide BLAST search we selected the membrane progesterone receptor from ''Xenopus laevis'' (african clawed frog), another well-studied model organism, as our second receptor.<br />
<br />
'''Signalling:''' The fet3 promoter targeted by the membrane receptors was already determined and proven working by J Smith et al. (2008). We could not obtain the sequence used by Smith and decided to take the upstream sequence (approx. 600bp) of the fet3 gene.<br />
<br />
Since Pfet3 is regulated negatively by the mPR we decided on inverting our signal with an additional signaling step to have more sensitive measurement results. So the second part of the signalling system needs a repressor its target. Additionally, knock-out strains not containing a working repressor have to be viable. We chose the mig1 (repressor) / Psuc2 (repressor target) pair and the rox1 (repressor) / Panb1 (repressor target) pair.<br />
<br />
'''Reporter:''' The targets of our signalling system regulate our reporter. We have access to the plasmid storage of our lab. Common reporters used in this yeast-based environment are firefly luciferase and beta-galactosidase. Both are available to us and have no legal issues concerning the publishing in the PartsRegistry.<br />
<br />
== Sequence analysis and primer design ==<br />
<br />
Next was the check of all sequences for unwanted restriction sites.<br />
<br />
As noted above, due to several unwanted restriction sites (EcoRI and PstI) in the pRS vectors, we can only use XbaI and SpeI for assembly.<br />
<br />
The firefly luciferase has an unwanted XbaI site so we had to use NheI/SpeI restriction instead. The XbaI site could be removed by gene synthesis.<br />
<br />
Three weeks before the official lab time started, we designed our primers with [http://de-de.invitrogen.com/site/de/de/home/Products-and-Services/Applications/Cloning/vector-nti-software.html Vector NTI]. The programm was basically used to compute good primers. We then manually optimized the primer sequences by setting G and C nucleotides at the 3'-tail so that the development of hydrogen bridge bonds is increased, which leads to stronger bindings.<br />
In addition the primer sequences were modified so that the related primer pairs have preferably the same or close annealing temperatures.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! forward primer !! reverse primer<br />
|-<br />
| 1 || lacZ || 5’-GCTAGCATGGTGCTGCGTTGG || 5’-ACTAGTTTATTTTTGACACCAG<br />
|-<br />
| 2 || luciferase || 5’-GCTAGCATGGAAGACGCCAAA || 5’-ACTAGTTTAAAGCTTCTTTCCGCC<br />
|-<br />
| 3 || Padh1 || 5’-TCTAGAAAGAAATGATGGTAAA || 5’-ACTAGTAGTTGATTGTATGCTT<br />
|-<br />
| 4 || Psuc2 || 5’-TCTAGACATACTAAGACATTTACCG || 5’-ACTAGTCATATACGTTAGTGAAAA <br />
|-<br />
| 5 || Pfet3 || 5’-TCTAGACATTACTGCTGTAAAAAGG || 5’-GCAAAAAATTAGAACTAGACTAGT <br />
|-<br />
| 6 || Panb1 || 5’-TCTAGATTTTTTCCTGTGTTCACC || 5’-ACTAGTGTTTTAGTGTGTGAATGA <br />
|-<br />
| 7 || Tadh1 || 5’-TCTAGAGCGAATTTCTTATGATTT || 5’-ACTAGTAGGTGTTGTCCTCTGAGG<br />
|-<br />
| 8 || rox1 || 5’-TCTAGAATGAATCCTAAATCCTCTACAC || 5-ACTAGTAATTGTTCTTTTGAGGCG <br />
|}</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookPreparationsTeam:Tuebingen/NotebookPreparations2012-09-25T12:26:04Z<p>Jakobmatthes: /* Sequence analysis and primer design */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Preparations =<br />
__TOC__<br />
After determining our principal project idea we had to design our system prior to any work in the wet lab. Several steps were involved:<br />
<br />
== Identification of plasmids ==<br />
<br />
We decided to use a shuttle vector which works in ''E. coli'' and ''S. cerevisiae''. The advantage is that we can assemble and build our target constructs in the rapid growing ''E. coli''. The shuttle vectors must meet the following demands:<br />
* Multiple cloning site with XbaI and SpeI restriction sites<br />
* Multiple cloning site with beta-galactosidase, so blue-white screening is possible<br />
* Ampicillin resistance<br />
* amino-acid genes for selection<br />
* Integration site for integration in the yeast genome<br />
<br />
After consultation with Prof. Jansen, our yeast expert, we decided to use the shuttle vectors pRS313, pRS315 and pRS316.<br />
<br />
Due to several EcoRI and PstI restriction sites in the pRS plasmids (not located around the multiple cloning site), we can only use XbaI and SpeI for assembly.<br />
<br />
== Identification of genes ==<br />
<br />
'''Receptors:''' Aiming at aquatic environment we chose to use the membrane progesterone receptor (mPR) from the model organism ''Danio rerio'' (zebra fish). The second organism was ''Salmo salar'' (salmon) but we could not locate a homologous gene due to missing full genome sequence data. Following a database-wide BLAST search we selected the membrane progesterone receptor from ''Xenopus laevis'' (african clawed frog), another well-studied model organism, as our second receptor.<br />
<br />
'''Signalling:''' The fet3 promoter targeted by the membrane receptors was already determined and proven working by J Smith et al. (2008). We could not obtain the sequence used by Smith and decided to take the upstream sequence (approx. 600bp) of the fet3 gene.<br />
<br />
Since Pfet3 is regulated negatively by the mPR we decided on inverting our signal with an additional signaling step to have more sensitive measurement results. So the second part of the signalling system needs a repressor its target. Additionally, knock-out strains not containing a working repressor have to be viable. We chose the mig1 (repressor) / Psuc2 (repressor target) pair and the rox1 (repressor) / Panb1 (repressor target) pair.<br />
<br />
'''Reporter:''' The targets of our signalling system regulate our reporter. We have access to the plasmid storage of our lab. Common reporters used in this yeast-based environment are firefly luciferase and beta-galactosidase. Both are available to us and have no legal issues concerning the publishing in the PartsRegistry.<br />
<br />
== Sequence analysis and primer design ==<br />
<br />
Next was the check of all sequences for unwanted restriction sites.<br />
<br />
As noted above, due to several unwanted restriction sites (EcoRI and PstI) in the pRS vectors, we can only use XbaI and SpeI for assembly.<br />
<br />
The firefly luciferase has an unwanted XbaI site so we had to use NheI/SpeI restriction instead. The XbaI site could be removed by gene synthesis.<br />
<br />
Three weeks before the official lab time started, we designed our primers with [http://de-de.invitrogen.com/site/de/de/home/Products-and-Services/Applications/Cloning/vector-nti-software.html Vector NTI]. The programm was basically used to compute good primers. We then manually optimized the primer sequences by setting G and C nucleotides at the 3'-tail so that the development of hydrogen bridge bonds is increased, which leads to stronger bindings.<br />
In addition the primer sequences were modified so much that the related primer pairs have preferably the same annealing temperature.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! Forward Primer !! Reverse Primer<br />
|-<br />
| 1 || lacZ || 5’-GCTAGCATGGTGCTGCGTTGG || 5’-ACTAGTTTATTTTTGACACCAG<br />
|-<br />
| 2 || luciferase || 5’-GCTAGCATGGAAGACGCCAAA || 5’-ACTAGTTTAAAGCTTCTTTCCGCC<br />
|-<br />
| 3 || Padh1 || 5’-TCTAGAAAGAAATGATGGTAAA || 5’-ACTAGTAGTTGATTGTATGCTT<br />
|-<br />
| 4 || Psuc2 || 5’-TCTAGACATACTAAGACATTTACCG || 5’-ACTAGTCATATACGTTAGTGAAAA <br />
|-<br />
| 5 || Pfet3 || 5’-TCTAGACATTACTGCTGTAAAAAGG || 5’-GCAAAAAATTAGAACTAGACTAGT <br />
|-<br />
| 6 || Panb1 || 5’-TCTAGATTTTTTCCTGTGTTCACC || 5’-ACTAGTGTTTTAGTGTGTGAATGA <br />
|-<br />
| 7 || Tadh1 || 5’-TCTAGAGCGAATTTCTTATGATTT || 5’-ACTAGTAGGTGTTGTCCTCTGAGG<br />
|-<br />
| 8 || rox1 || 5’-TCTAGAATGAATCCTAAATCCTCTACAC || 5-ACTAGTAATTGTTCTTTTGAGGCG <br />
|}</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/AttributionsTeam:Tuebingen/Attributions2012-09-25T12:24:47Z<p>Jakobmatthes: /* Attributions */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Attributions =<br />
<br />
''This page gives credit to all work and the people involved.''<br />
<br />
'''Project idea''' <br />
<br />
The project idea - to detect and/or degrade steroids - was proposed by the student team after lenghty meetings.<br />
<br />
'''System design''' <br />
<br />
The overall design (including selection of vectors, promoters and genes) was conducted in close cooperation with Prof. Jansen. Primer design was performed with students and Dr. Apel. At all times students were present. Each step was explained to all students so everyone has knowledge of the entire process.<br />
<br />
'''Lab supplies'''<br />
<br />
LB medium was initially prepared by the student team to learn the protocol. Later on, LB was provided by the host lab. SOB and TAE buffer were always prepared by the students. Agar plates were produced by the students with exception of the first batch which was provided by the host lab.<br />
<br />
'''Lab hardware'''<br />
<br />
All lab hardware was operated by the students. Thermocycler programs were initially set up with advise.<br />
<br />
'''Sequencing'''<br />
<br />
Sequencing was performed by an external provider. All sequencing results were evaluated by the students. In some cases, secquencing primers were created in collaboration with advisors.<br />
<br />
'''Lab work'''<br />
<br />
Lab work was conducted by the students. Advisors or host lab personnel did not perform any experiments. Their involvement was advise and consultion only.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/ResultAcksTeam:Tuebingen/ResultAcks2012-09-25T12:24:18Z<p>Jakobmatthes: /* Acknowledgment */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Acknowledgment =<br />
<br />
Our project would not exist in this form without the support of the University of Tuebingen as an institute that not only taught us the basics we needed and showed us how much fun science is but also gave us the financial support for our own scientific adventure. <br />
<br />
Especially Dr. Apel and the team of Prof. Jansen helped us out throughout the whole process with ideas, a lab, material, expertise, supervising and believe in us. Prof. Nordheim and Prof. Kohlbacher were important contact partners for a wide range of obstacles in organization and administration that we would not have been able to overcome without them.<br />
<br />
We also want to thank the Reinhold-and-Maria-Teufel-Stiftung and Prof. Oecking for helping to finance our trip to the Jamboree in Amsterdam.</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/Templates/NavigationTeam:Tuebingen/Templates/Navigation2012-09-25T12:23:44Z<p>Jakobmatthes: </p>
<hr />
<div><!-- the old navigation bar<br />
<div id="nav"><br />
* [[Team:Tuebingen|Home]]<br />
* [[Team:Tuebingen/Team|Team]]<br />
* [[Team:Tuebingen/Project|Project]]<br />
* [[Team:Tuebingen/Activities|Activities]]<br />
* [[Team:Tuebingen/Notebook|Notebook]]<br />
* [[Team:Tuebingen/Attributions|Attributions]]<br />
* [[Team:Tuebingen/Safety|Safety]]<br />
* [[Team:Tuebingen/Parts|Submitted Parts]]<br />
* [[Team:Tuebingen/Sponsorship|Sponsorship]]<br />
* [[Team:Tuebingen/Contact|Contact us]]<br />
</div><br />
<br />
<hr /><br />
--><br />
<div id="nav"><br />
* [[Team:Tuebingen|Home]]<br />
* <span>Team</span><br />
** [[Team:Tuebingen/TeamWho|Who we are]]<br />
** [[Team:Tuebingen/TeamWhat|What we did]]<br />
** [[Team:Tuebingen/TeamWhere|Where we're from]]<br />
** [[Team:Tuebingen/Attributions|Attributions]]<br />
** [[Team:Tuebingen/ResultPersonal|Impressions]]<br />
* <span>Project</span><br />
** [[Team:Tuebingen/ProjectOverview|Overview]]<br />
** [[Team:Tuebingen/ProjectQuestions|Questions]]<br />
** [[Team:Tuebingen/ProjectMechanism|Mechanism]]<br />
** [[Team:Tuebingen/ProjectImplementation|Implementation]]<br />
** [[Team:Tuebingen/Parts|Submitted Parts]]<br />
** [[Team:Tuebingen/Application|Application]]<br />
** [[Team:Tuebingen/References|References]]<br />
* <span>Results</span><br />
** [[Team:Tuebingen/ResultAcks|Acknowledgment]]<br />
** [[Team:Tuebingen/ResultExp|Experience]]<br />
* <span>Notebook</span><br />
** [[Team:Tuebingen/NotebookPreparations|Preparations]]<br />
** [[Team:Tuebingen/NotebookReports|Reports]]<br />
** [[Team:Tuebingen/NotebookProtocols|Protocols]]<br />
** [[Team:Tuebingen/NotebookAppendix|Appendix]]<br />
** [[Team:Tuebingen/NotebookCalendar|Calendar]]<br />
* [[Team:Tuebingen/Activities|Activities & Press]]<br />
* [[Team:Tuebingen/Safety|Safety]]<br />
* [[Team:Tuebingen/Sponsorship|Sponsorship]]<br />
* [[Team:Tuebingen/Contact|Contact us]]<br />
</div></div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-25T12:22:03Z<p>Jakobmatthes: /* Week 11 (9/17 - 9/23) */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<!--<br />
{|<br />
| [[File:Tue-map-parts.png|200px|thumb|preparation of parts]]<br />
| [[File:Tue-map-vector.png|200px|thumb|preparation of vectors]]<br />
|-<br />
| [[File:Tue-map-assembly.png|200px|thumb|assembly]]<br />
| [[File:Tue-map-shipping.png|200px|thumb|parts for shipping]]<br />
|}<br />
--><br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! lenght [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen University Tuebingen || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed.<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
A mini plasmid preparation was performed afterwards to purify DNA in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 6 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of part 6 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-25T12:21:05Z<p>Jakobmatthes: /* Week 11 (9/17 - 9/23) */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<!--<br />
{|<br />
| [[File:Tue-map-parts.png|200px|thumb|preparation of parts]]<br />
| [[File:Tue-map-vector.png|200px|thumb|preparation of vectors]]<br />
|-<br />
| [[File:Tue-map-assembly.png|200px|thumb|assembly]]<br />
| [[File:Tue-map-shipping.png|200px|thumb|parts for shipping]]<br />
|}<br />
--><br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! lenght [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen University Tuebingen || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed.<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
A mini plasmid preparation was performed afterwards to purify DNA in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
1. Some [[NotebookProtocols#Genaxxon_Gel_Extraction_Mini_Prep_Kit|Mini-Preps]] of the parts 3, 4, 8, 9, 10 and 11 in pSB1C3 were executed with a following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]]. <br />
But we had still problems with the Chloramphenicol concentration, because the bacterial densitity was very low in the LB medium. Therefore no bands were visible on the gel after the mini-prep.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] of the parts 5 and 6 was performed with the new primers in order to determine the optimal annealing temperature.<br />
<br />
The PCR of part 6 was successful. After the purification with the [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|PCR purification kit]] a [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in ''E. coli TOP10'' was executed. <br />
Due to the fact that the time was running out we decided not to continue to work with this parts.<br />
<br />
3. The T-shirts for the Jamboree in Amsterdam were designed. Aside, a lot of work on the wiki took place.<br />
<br />
== Week 12 (9/24 - 9/30) ==</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-25T12:12:45Z<p>Jakobmatthes: /* Week 10 (9/10 - 9/16) */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<!--<br />
{|<br />
| [[File:Tue-map-parts.png|200px|thumb|preparation of parts]]<br />
| [[File:Tue-map-vector.png|200px|thumb|preparation of vectors]]<br />
|-<br />
| [[File:Tue-map-assembly.png|200px|thumb|assembly]]<br />
| [[File:Tue-map-shipping.png|200px|thumb|parts for shipping]]<br />
|}<br />
--><br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! lenght [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen University Tuebingen || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed.<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
A mini plasmid preparation was performed afterwards to purify DNA in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primers for parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
<br />
== Week 12 (9/24 - 9/30) ==</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookReportsTeam:Tuebingen/NotebookReports2012-09-25T12:12:27Z<p>Jakobmatthes: /* Week 10 (9/10 - 9/16) */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Weekly Reports =<br />
__TOC__<br />
== Procedure ==<br />
The following illustration gives a general overview to our approach of lab work.<br />
[[File:Tue-labmap.png|600px|thumb|center|lab procedures]]<br />
<!--<br />
{|<br />
| [[File:Tue-map-parts.png|200px|thumb|preparation of parts]]<br />
| [[File:Tue-map-vector.png|200px|thumb|preparation of vectors]]<br />
|-<br />
| [[File:Tue-map-assembly.png|200px|thumb|assembly]]<br />
| [[File:Tue-map-shipping.png|200px|thumb|parts for shipping]]<br />
|}<br />
--><br />
<br />
== Parts, Plasmids and Constructs ==<br />
<br />
To understand all referenced parts and their enumeration, here is a full listing:<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! source !! lenght [bp] !! annealing temperature !! Registry Part<br />
|-<br />
| 1 || lacZ || plasmid of AG Jansen University Tuebingen || 2514 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950005 BBa_K950005]<br />
|-<br />
| 2 || luciferase || plasmid of AG Jansen || 1650 || 46.4 °C || [http://partsregistry.org/wiki/index.php?title=BBa_K950004 BBa_K950004]<br />
|-<br />
| 3 || Padh1 || plasmid of the iGEM Kit || 1457 || 48.0 °C || [http://partsregistry.org/wiki/index.php/Part:BBa_K165015 BBa_K165015]<br />
|-<br />
| 4 || Psuc2 || genomic yeast DNA || 711 || 45.6 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950003 BBa_K950003]<br />
|-<br />
| 5 || Pfet3 || genomic yeast DNA || 587 || 47.1 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950000 BBa_K950000]<br />
|-<br />
| 6 || Panb1 || genomic yeast DNA || 412 || 48.0 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950002 BBa_K950002]<br />
|-<br />
| 7 || Tadh1 || genomic yeast DNA || || 51.8 °C ||<br />
|-<br />
| 8 || rox1 || genomic yeast DNA || 1237 || 49.9 °C || [http://partsregistry.org/wiki/index.php?title=Part:BBa_K950001 BBa_K950001]<br />
|-<br />
| 9 || mPR ''Danio rerio'' || ''Danio rerio'', synthesized by IDT || 1077 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950006 BBa_K950006]<br />
|-<br />
| 10 || mig1 || ''Saccharomyces cerevisiae'', synthesized by IDT || 1527 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950009 BBa_K950009]<br />
|-<br />
| 11 || mPR ''Xenopus laevis'' || ''Xenopus laevis'', synthesized by IDT || 1074 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950007 BBa_K950007]<br />
|-<br />
|<br />
|-<br />
| || pGEM-T Easy vector || pGEM-T Easy Vector Kit || 3015 || ||<br />
|-<br />
|<br />
|-<br />
| || pRS313 vector || vector of AG Jansen || 4967 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950008 BBa_K950008]<br />
|-<br />
| || pRS315 vector || vector of AG Jansen || 6018 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950010 BBa_K950010]<br />
|-<br />
| || pRS316 vector || vector of AG Jansen || 4887 || ||[http://partsregistry.org/wiki/index.php?title=Part:BBa_K950011 BBa_K950011]<br />
|}<br />
<br />
For the full information of these parts in the Parts Registry, refer to [[Team:Tuebingen/Parts|Submitted Parts]].<br />
<br />
== Week 1 (7/9 - 7/15) ==<br />
<br />
[[File:Tue-medium.jpg|thumb|right|LB, SOB, TAE]]<br />
[[File:Chemo-competent-cells.jpg|thumb|right|TOP10 cells on plate]]<br />
<br />
Thursday the 12th of July was the first day in our laboratory.<br />
<br />
1. At first different substances, for example LB, SOB and TAE buffer 50x, which would be necessary for the further practice, were prepared.<br />
<br />
2. To determine the optimal annealing temperature, a [[Team:Tuebingen/NotebookProtocols#PCR|gradient PCR]] for the parts 1-8 was performed.<br />
Doing a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]] the PCR results were tested.<br />
<br />
3. Preparing chemocompetent ''E. coli TOP 10'' cells after [[Team:Tuebingen/NotebookProtocols#Chemo-competent_cells|Inoue protocol]].<br />
<br />
== Week 2 (7/16 - 7/22) ==<br />
<br />
1. A successful [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] for the Parts 1-7 with the optimal annealing temperature was performed. It was controlled by a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel-electrophoresis]].<br />
<br />
The Parts 3-7 were cleaned with a PCR-DNA-Purification-Kit. After that the concentration of the purified parts was measured with NanoDrop.<br />
<br />
2. To test the competence of the chemocompetent ''E. coli TOP 10'' cells a [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] with pRS313 and a negative control was done. Due to the fact that the competent cells didn't work, new chemocompetent ''E. coli TOP 10'' cells were prepared. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of pRS313, pRS315 and pRS316 in these competent cells was successful.<br />
<br />
== Week 3 (7/23 - 7/29) ==<br />
<br />
[[File:Tue-geldoku.jpg|200px|thumb|right|gel electrophoresis documentation system]]<br />
<br />
1. The first [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] of the parts 1-8 in pGEM with following [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] in the competent ''E. coli TOP10'' was done. But unfortunately only a few colonies grew on the inoculated agar-plates, which were incubated over night at 37°C. <br />
<br />
[[File:Tue-insertgel.png|200px|thumb|right|gel with pGEM bands and a few inserts]]<br />
The [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] was performed on the extracted plasmids of the grown colonies to control the ligation of the insert. The following gel electrophoresis showed that the ligation was not successful, because only bands of 3000bp for the pGEM vector was visible, but no bands for the insert.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 8 was executed. Using a PCR-DNA-Purification-Kit the PCR-product of part 8 was purified. The PCR-product of part 1 was purified with a preparative gel. The concentration of the final products was measured with NanoDrop.<br />
<br />
== Week 4 (7/30 - 8/05) ==<br />
<br />
1. The shipment with the synthesized parts (mPR of ''Danio rerio'' and mig1) arrived.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of the parts mPR ''Danio rerio'' and mig1 was performed using the competent ''E. coli TOP10'' cells. Another transformation of the backbone plasmids pRS313, pRS315 and pRS316 was executed. Both were successful.<br />
<br />
The first attempt to isolate the plasmids was through usage of a plasmid preparation kit, but this try failed. Therefore the plasmid isolation was successfully repeated using alkaline lysis.<br />
<br />
3. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 with Taq/Pfu polymerase was performed applicating new yeast DNA. As an effect of the frequent freezing and defrosting the old yeast DNA was probably destroyed. Therefore some earlier PCRs did not work.<br />
<br />
== Week 5 (8/06 - 8/12) ==<br />
<br />
[[File:Tue-freezer.jpg|thumb|right|freezer with most of our reagents]]<br />
<br />
1. A [[Team:Tuebingen/NotebookProtocols#control_digest|small restriction digest]] of the shuttle vectors pRS313, pRS315 and pRS316 was performed with XbaI and SpeI in order to examine the capability to linearize with the right overhangs for a ligation to take place later.<br />
The restriction digest was executed with the parts mig1 and mPR of ''Danio rerio'', too.<br />
Due to unclean plasmids and DNA (perhaps to much salt) this step had to be repeated several times, because the restriction digests were incomplete.<br />
<br />
Therefore the plasmids (pRS313, pRS315, pRS316 and the parts mig1, mPR ''Danio rerio'') were purified again with a Midi Prep DNA purification kit. Now the restriction digest was executed completely. We estimate that max. 30 µg DNA can be digested with our reaction.<br />
<br />
A mini plasmid preparation was performed afterwards to purify DNA in order to prepare the DNA for ligation.<br />
<br />
2. A new [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1-8 was executed using Herculase in order to obtain a higher amount of PCR product. The polymerase Herculase was used due to its precision and productivity. Indeed the result of the PCR was better than with the Pfu/Taq polymerase.<br />
<br />
A preparative gel for PCR products 3, 4, 5, 6, 7, 8 (from PCR with Herculase) delivered new template DNA for another PCR with Taq/Pfu Polymerase.<br />
<br />
== Week 6 (8/13 - 8/19) ==<br />
<br />
[[File:Tue-prepgel.png|200px|thumb|right|preparative gel to isolate digested inserts]]<br />
1. The first successful [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligation]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into pGEM vector of part 4 in ''E. coli TOP10'' was executed. A lot of colonies grew on the agar-plate. After a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI and the control with a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 711bp was visible. The sequencing of the DNA confirmed that part 4 has the expected nucleotide sequence.<br />
A Midi-Prep, restriction digest and preparative gel electrophoresis followed in order to prepare them for later ligation into pRS vectors.<br />
<br />
[[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|Ligation]] of part 3, 6, 7 and 8 in pGEM vector was performed. Reaction took place over night at 4 °C. The [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] of these parts was executed into ''E. coli TOP10''.<br />
After growth over night, a mini plasmid preparation was performed. After a colony-PCR with parts 3, 6, 7, 8 did not work, we had to go back to the restriction digest for insert controllin. Positive samples were prepared for sequencing. The parts 3 and 8 were sequenced successfully and yielded a good sequence. The ligation of parts 6 and 7 failed, so we decided to skip part 6, because we may use Psuc2 as an alternative promotor for luciferase. <br />
<br />
2. We received the synthesized receptor of ''Xenopus laevis''. It was successfully transformed in ''E. coli TOP10'' and purified with a Midi-Prep.<br />
<br />
== Week 7 (8/20 - 8/26) ==<br />
<br />
[[File:Tue-etbr.jpg|thumb|right|preparing one of many gel electrophoreses]]<br />
1. The [[Team:Tuebingen/NotebookProtocols#PCR|PCR]] of the parts 1 and 2 with Herculase polymerase was executed.<br />
The PCR products were checked with an analytical gel afterwards. The PCR of part 1 failed again, so we decided to reject part 1 and continue working only with luciferase (part 2), because we only need one reporter gene.<br />
<br />
2. Since we ran out of luciferase plasmid DNA, we decided to [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] the remaining DNA of luciferase into ''E. coli TOP10''. The transformation was successful. A Midi-Prep yielded new plasmid DNA.<br />
<br />
3. The receptors (mPR ''Danio rerio'', mPR ''Xenopus laevis'') and mig1 were initially [[Team:Tuebingen/NotebookProtocols#Ligation|ligated into pRS vectors]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10''. But no colonies grew on the agar-plates.<br />
<br />
== Week 8 (8/27 - 9/02) ==<br />
<br />
[[File:Evalseq.jpg|thumb|right|evaluating sequences via BLAST]]<br />
1. Part 5 was [[Team:Tuebingen/NotebookProtocols#pGEM_Ligation|ligated into pGEM vector]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformed]] into ''E. coli TOP10'' afterwards. After performing a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] it was obvious that the insert did not have the correct length and therefore has to be discarded.<br />
<br />
2. Second [[Team:Tuebingen/NotebookProtocols#Ligation|ligation]] of mPR ''Danio rerio'', mPR ''Xenopus laevis'' and mig1 into pRS vectors and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformation]] into ''E. coli TOP10'' was executed. Some colonies grew on the agar-plates. Therefore a mini-prep and a [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI with a following [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] was conducted. But there was only ligation of the insert into pGEM, not into pRS. The hypothesis was that the pGEM constructs were contamination.<br />
<br />
3. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligation]] of the parts 3 and 4 in pRS vectors with following transformation into ''E. coli TOP10'' was performed. But it was not successful.<br />
<br />
== Week 9 (9/03 - 9/09) ==<br />
<br />
1. [[Team:Tuebingen/NotebookProtocols#Chemotransformation|Transformations]] of the vector pSB1C3 with the insert RFP into ''E. coli TOP10''. The cells were plated on agar with different Chloramphenicol concentrations in order to find out the right concentration of the antibiotic.<br />
<br />
'''Concentration results:'''<br />
{| class="wikitable"<br />
|-<br />
! Chloramphenicol concentration !! results (after transformation) !! results (already selected colonies)<br />
|-<br />
| 30 µg/ml || no growth || viable<br />
|-<br />
| 15 µg/ml || no growth || viable<br />
|-<br />
| 5 µg/ml || up to 30 colonies || viable <br />
|-<br />
| 1 µg/ml || lawn || viable <br />
|-<br />
| 0.1 µg/ml || lawn || viable <br />
|}<br />
<br />
2. Some more unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of the parts 3, 4, 8, 9, 10, 11 into pRS vectors. There grew no colonies on the agar-plates or the ligation failed.<br />
<br />
== Week 10 (9/10 - 9/16) ==<br />
<br />
1. After many [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 5 into ''E. coli TOP10'' without any result, we have decided to order new primers for Pfet3, to achieve annealing temperatures closer to each other.<br />
<br />
New primers were also ordered for Tadh1, because the old primers did not fit to the yeast-DNA.<br />
<br />
2. A [[Team:Tuebingen/NotebookProtocols#QIAGEN_Plasmid_Midi_Kit|Midi-Prep]] of the pSB1C3 vector with a following [[Team:Tuebingen/NotebookProtocols#preparative_double_digest|restriction digest]] was executed.<br />
<br />
3. After a lot of unsuccessful [[Team:Tuebingen/NotebookProtocols#Ligation|ligations]] and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] of part 2 in pGEM some colonies grew on the plate. Therefore a [[Team:Tuebingen/NotebookProtocols#Genaxxon_Plasmid_DNA_Purification_Mini_Prep_Kit|Mini-Prep]] with following [[Team:Tuebingen/NotebookProtocols#control_digest|restriction digest]] with XbaI and SpeI was performed. After a [[Team:Tuebingen/NotebookProtocols#Gel_electrophoresis|gel electrophoresis]] the right band of 1650bp was visible.<br />
<br />
Part 2 was sequenced, but the primer SP6 and T7 did not fit to the DNA. Perhaps the insert (Part 2) was not in the pGEM vector. To achieve sequencing results we designed custom sequencing primers: We aimed at 150bp overlap in the center of the luciferase gene. The two sequencing results reach from the center of the gene to approx. 100bp outside of the gene.<br />
[[File:Luciferase primer.png|thumb|center|749px|custom sequencing primers]]<br />
<br />
4. [[Team:Tuebingen/NotebookProtocols#Ligation|Ligations]] of the parts 3, 4, 8, 9, 10, 11 in pSB1C3 and [[Team:Tuebingen/NotebookProtocols#Chemotransformation|transformations]] into E. coli. A lot of colonies grew on the plates.<br />
<br />
5. The new primer of the parts 5 and 6 arrived at the end of the week.<br />
<br />
== Week 11 (9/17 - 9/23) ==<br />
<br />
== Week 12 (9/24 - 9/30) ==</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookPreparationsTeam:Tuebingen/NotebookPreparations2012-09-25T10:12:46Z<p>Jakobmatthes: /* Sequence analysis and primer design */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Preparations =<br />
__TOC__<br />
After determining our principal project idea we had to design our system prior to any work in the wet lab. Several steps were involved:<br />
<br />
== Identification of plasmids ==<br />
<br />
We decided to use a shuttle vector which works in ''E. coli'' and ''S. cerevisiae''. The advantage is that we can assemble and build our target constructs in the rapid growing ''E. coli''. The shuttle vectors must meet the following demands:<br />
* Multiple cloning site with XbaI and SpeI restriction sites<br />
* Multiple cloning site with beta-galactosidase, so blue-white screening is possible<br />
* Ampicillin resistance<br />
* amino-acid genes for selection<br />
* Integration site for integration in the yeast genome<br />
<br />
After consultation with Prof. Jansen, our yeast expert, we decided to use the shuttle vectors pRS313, pRS315 and pRS316.<br />
<br />
Due to several EcoRI and PstI restriction sites in the pRS plasmids (not located around the multiple cloning site), we can only use XbaI and SpeI for assembly.<br />
<br />
== Identification of genes ==<br />
<br />
'''Receptors:''' Aiming at aquatic environment we chose to use the membrane progesterone receptor (mPR) from the model organism ''Danio rerio'' (zebra fish). The second organism was ''Salmo salar'' (salmon) but we could not locate a homologous gene due to missing full genome sequence data. Following a database-wide BLAST search we selected the membrane progesterone receptor from ''Xenopus laevis'' (african clawed frog), another well-studied model organism, as our second receptor.<br />
<br />
'''Signalling:''' The fet3 promoter targeted by the membrane receptors was already determined and proven working by J Smith et al. (2008). We could not obtain the sequence used by Smith and decided to take the upstream sequence (approx. 600bp) of the fet3 gene.<br />
<br />
Since Pfet3 is regulated negatively by the mPR we decided on inverting our signal with an additional signaling step to have more sensitive measurement results. So the second part of the signalling system needs a repressor its target. Additionally, knock-out strains not containing a working repressor have to be viable. We chose the mig1 (repressor) / Psuc2 (repressor target) pair and the rox1 (repressor) / Panb1 (repressor target) pair.<br />
<br />
'''Reporter:''' The targets of our signalling system regulate our reporter. We have access to the plasmid storage of our lab. Common reporters used in this yeast-based environment are firefly luciferase and beta-galactosidase. Both are available to us and have no legal issues concerning the publishing in the PartsRegistry.<br />
<br />
== Sequence analysis and primer design ==<br />
<br />
Next was the check of all sequences for unwanted restriction sites.<br />
<br />
As noted above, due to several unwanted restriction sites (EcoRI and PstI) in the pRS vectors, we can only use XbaI and SpeI for assembly.<br />
<br />
The firefly luciferase has an unwanted XbaI site so we had to use NheI/SpeI restriction instead. The XbaI site could be removed by gene synthesis.<br />
<br />
Three weeks before the official lab time started, we designed our primers with [http://de-de.invitrogen.com/site/de/de/home/Products-and-Services/Applications/Cloning/vector-nti-software.html Vector NTI] (commercial). The programm was basically used to compute our "optimal" primers. We then manually optimized the primer sequences by setting a lot of Gs and Cs at the 3'-tail so that the development of hydrogen bridge bonds is increased, which leads to stronger bindings.<br />
In addition the primer sequences were modified so much that the related primer pairs have preferably the same annealing temperature.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Part # !! Part name !! Primer_Sequence_F !! Primer_Sequence_R<br />
|-<br />
| 1 || lacZ || 5’-GCTAGCATGGTGCTGCGTTGG || 5’-ACTAGTTTATTTTTGACACCAG<br />
|-<br />
| 2 || luciferase || 5’-GCTAGCATGGAAGACGCCAAA || 5’-ACTAGTTTAAAGCTTCTTTCCGCC<br />
|-<br />
| 3 || Padh1 || 5’-TCTAGAAAGAAATGATGGTAAA || 5’-ACTAGTAGTTGATTGTATGCTT<br />
|-<br />
| 4 || Psuc2 || 5’-TCTAGACATACTAAGACATTTACCG || 5’-ACTAGTCATATACGTTAGTGAAAA <br />
|-<br />
| 5 || Pfet3 || 5’-TCTAGACATTACTGCTGTAAAAAGG || 5’-ACTAGTTCTAATTTTTTGC <br />
|-<br />
| 6 || Panb1 || 5’-TCTAGATTTTTTCCTGTGTTCACC || 5’-ACTAGTGTTTTAGTGTGTGAATGA <br />
|-<br />
| 7 || Tadh1 || 5’-TCTAGAGCATGTGCTCTGTATG || 5’-ACTAGTCTCCATGGTGGCGGCG<br />
|-<br />
| 8 || rox1 || 5’-TCTAGAATGAATCCTAAATCCTCTACAC || 5-ACTAGTAATTGTTCTTTTGAGGCG <br />
|}</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookProtocolsTeam:Tuebingen/NotebookProtocols2012-09-25T10:11:15Z<p>Jakobmatthes: /* Protocols */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Protocols =<br />
__TOC__<br />
== Chemo-competent cells ==<br />
'''Inoue buffer'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| MnCl<sub>2</sub> * 2H<sub>2</sub>0 || 9.67 g<br />
|-<br />
| CaCl<sub>2</sub> * 2H<sub>2</sub>0 || 2.2 g<br />
|-<br />
| KCl || 18.65 g<br />
|-<br />
| PIPES (0.5 M, pH 6.7) || 20 ml<br />
|-<br />
| H<sub>2</sub>0 || ad 1 l<br />
|}<br />
Sterilize through filtration (0.45 µm filter) and store at -20 °C.<br />
<br />
'''Cells'''<br />
# Pick an ''E. coli'' colony and inoculate 25 ml SOB.<br />
# Let bacteria grow for 8 hours at 37 °C and 250 rpm.<br />
# Inoculate three 100 ml SOB volumes with 1 ml, 2 ml and 4 ml of the prepared pre-culture.<br />
# Incubate over night at 18 - 22 °C and 200 rpm.<br />
# At OD<sub>600</sub> = 0.55, put culture for 10 min on ice.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Resuspend cell pellet in 30 ml 0 °C Inoue buffer.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Repeat the previous two steps.<br />
# Resuspend cells in 8 ml 0 °C Inoue buffer. Add 1.5 ml DMSO and incubate on ice for 10 min.<br />
# Aliquot cells à 100 µl and freeze in liquid nitrogen. Store at -80 °C.<br />
<br />
<br />
== pGEM Ligation ==<br />
Ligation for TA-cloning of PCR products<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 2X Rapid Ligation Buffer || 5 µl<br />
|-<br />
| pGEM vector || 0.5 µl (25ng)<br />
|-<br />
| PCR product || 3.5 µl<br />
|-<br />
| T4 DNA ligase || 1 µl (3 Weiss units)<br />
|}<br />
<br />
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.<br />
<br />
<br />
<br />
<br />
== Ligation ==<br />
Ligation for digested parts and vectors<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 10X T4 DNA Ligase Buffer || 1 µl<br />
|-<br />
| vector DNA || 1 µl (20-100 ng)<br />
|-<br />
| insert DNA || 5 µl (up to 5:1 molar ratio insert to vector)<br />
|-<br />
| T4 DNA ligase || 1 µl (1 unit)<br />
|-<br />
| water || 2.5 µl<br />
|}<br />
Mix all reagents and incubate at 22°C for 1 hour.<br />
<br />
<br />
<br />
<br />
== Chemotransformation ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| chemo-competent ''E. coli'' || 100 µl<br />
|-<br />
| plasmid DNA || up to 10 µl (max. 1/10 of volume)<br />
|}<br />
# Add plasmid DNA to cell culture.<br />
# Incubate for 30 min on ice.<br />
# Heat shock for 90 sec at 42°C.<br />
# Add 900 µl LB.<br />
# Let the bacteria grow at 37°C for at least 1 hour.<br />
<br />
<br />
<br />
<br />
== Restriction digest ==<br />
<br />
<br />
<br />
=== control digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 1 µl<br />
|-<br />
| XbaI (RE) || 0.5 µl (5 units)<br />
|-<br />
| SpeI (RE) || 0.5 µl (5 units)<br />
|-<br />
| DNA || 1 µl (up to 1 µg)<br />
|- <br />
| water || 7 µl<br />
|}<br />
# Incubate at least for 1 hour at 37°C.<br />
<br />
<br />
<br />
=== preparative double digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 5 µl (50 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for 8 hours at 37°C.<br />
# After 3 hours add 2 µl SpeI.<br />
# Add 7 µl XbaI and incubate for another 8 hours.<br />
<br />
=== plasmid linearization ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 7 µl (70 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for at least 8 hours at 37°C.<br />
<br />
<br />
== PCR ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Taq/Pfu buffer || 5 µl<br />
|-<br />
| Taq/Pfu polymerase || 1 µl<br />
|-<br />
| primer forward || 0.5 µl (100 pmol/µl)<br />
|-<br />
| primer reverse || 0.5 µl (100 pmol/µl)<br />
|-<br />
| dNTPs || 2.5 µl (200 µM)<br />
|-<br />
| template DNA || 1 µl<br />
|-<br />
| water || 36 µl<br />
|}<br />
<br />
<br />
<br />
'''PCR conditions'''<br />
{| class="wikitable"<br />
|-<br />
! Step !! Duration !! Settings<br />
|-<br />
| 1 || 2 min || 94°C<br />
|-<br />
| 2 || 45 sec || 94°C<br />
|-<br />
| 3 || 30 sec || gradient or annealing temperature<br />
|-<br />
| 4 || 90 sec || 72°C<br />
|-<br />
| || || steps 2-4: 30 cycles<br />
|-<br />
| 5 || 7 min || 72°C<br />
|-<br />
| 6 || (hold) || 4°C<br />
|}<br />
<br />
<br />
<br />
<br />
== Gel electrophoresis ==<br />
<br />
'''TAE buffer 50x'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 0.05 M EDTA || 18.61 g<br />
|-<br />
| 1 M acetic acid || 60.05 g<br />
|-<br />
| 2 M Tris || 242.28 g<br />
|-<br />
| water || 1 l<br />
|}<br />
Adjust to pH 8.5.<br />
<br />
<br />
<br />
'''Gel'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| TAE 1x buffer || 120 ml<br />
|-<br />
| Agarose || 1.2 g<br />
|}<br />
Solve agarose in TAE 1x buffer and boil until solution is clear.<br />
<br />
<br />
<br />
'''Well loading'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| PCR product or DNA || 5 µl<br />
|-<br />
| Loading dye 6x || 1 µl<br />
|}<br />
Can be scaled up linearly.<br />
<br />
<br />
<br />
== LB medium ==<br />
<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 10,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 5,0 g<br />
|-<br />
| water || 1,0 l<br />
|}<br />
Adjust to pH 7.0.<br />
<br />
<br />
<br />
'''Agar-plates'''<br />
<br />
# Solve 16g Agar-Agar in 1l LB buffer and boil until solution is clear. <br />
# If it is nearly cold pour it into some petri dish.<br />
<br />
<br />
<br />
<br />
== SOB medium ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 20,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 0,5 g<br />
|-<br />
| 250mM KCl || 10ml<br />
|-<br />
| water MiliQ || 1l<br />
|}<br />
<br />
# Solve the components in 1l water.<br />
# autoclave<br />
# After autoclaving add 5ml MgCl2<br />
<br />
<br />
<br />
<br />
== Genaxxon Plasmid DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon Gel Extraction Mini Prep Kit ==<br />
[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon PCR DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== QIAGEN Plasmid Midi Kit ==<br />
[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookProtocolsTeam:Tuebingen/NotebookProtocols2012-09-25T10:07:13Z<p>Jakobmatthes: /* Protocols */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Protocols =<br />
__TOC__<br />
== Chemo-competent cells ==<br />
'''Inoue buffer'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| MnCl<sub>2</sub> * 2H<sub>2</sub>0 || 9.67 g<br />
|-<br />
| CaCl<sub>2</sub> * 2H<sub>2</sub>0 || 2.2 g<br />
|-<br />
| KCl || 18.65 g<br />
|-<br />
| PIPES (0.5 M, pH 6.7) || 20 ml<br />
|-<br />
| H<sub>2</sub>0 || ad 1 l<br />
|}<br />
Sterilize through filtration (0.45 µm filter) and store at -20 °C.<br />
<br />
'''Cells'''<br />
# Pick an ''E. coli'' colony and inoculate 25 ml SOB.<br />
# Let bacteria grow for 8 hours at 37 °C and 250 rpm.<br />
# Inoculate three 100 ml SOB volumes with 1 ml, 2 ml and 4 ml of the prepared pre-culture.<br />
# Incubate over night at 18 - 22 °C and 200 rpm.<br />
# At OD<sub>600</sub> = 0.55, put culture for 10 min on ice.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Resuspend cell pellet in 30 ml 0 °C Inoue buffer.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Repeat the previous two steps.<br />
# Resuspend cells in 8 ml 0 °C Inoue buffer. Add 1.5 ml DMSO and incubate on ice for 10 min.<br />
# Aliquot cells à 100 µl and freeze in liquid nitrogen. Store at -80 °C.<br />
<br />
<br />
== pGEM Ligation ==<br />
Ligation for TA-cloning of PCR products<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 2X Rapid Ligation Buffer || 5 µl<br />
|-<br />
| pGEM vector || 0.5 µl (25ng)<br />
|-<br />
| PCR product || 3.5 µl<br />
|-<br />
| T4 DNA ligase || 1 µl (3 Weiss units)<br />
|}<br />
<br />
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.<br />
<br />
<br />
<br />
<br />
== Ligation ==<br />
Ligation for digested parts and vectors<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 10X T4 DNA Ligase Buffer || 1 µl<br />
|-<br />
| vector DNA || 1 µl (20-100 ng)<br />
|-<br />
| insert DNA || 5 µl (up to 5:1 molar ratio insert to vector)<br />
|-<br />
| T4 DNA ligase || 1 µl (1 unit)<br />
|-<br />
| water || 2.5 µl<br />
|}<br />
Mix all reagents and incubate at 22°C for 1 hour.<br />
<br />
<br />
<br />
<br />
== Chemotransformation ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| chemo-competent ''E. coli'' || 100 µl<br />
|-<br />
| plasmid DNA || up to 10 µl (max. 1/10 of volume)<br />
|}<br />
# Add plasmid DNA to cell culture.<br />
# Incubate for 30 min on ice.<br />
# Heat shock for 90 sec at 42°C.<br />
# Add 900 µl LB.<br />
# Let the bacteria grow at 37°C for at least 1 hour.<br />
<br />
<br />
<br />
<br />
== Restriction digest ==<br />
<br />
<br />
<br />
=== control digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 1 µl<br />
|-<br />
| XbaI (RE) || 0.5 µl (5 units)<br />
|-<br />
| SpeI (RE) || 0.5 µl (5 units)<br />
|-<br />
| DNA || 1 µl (up to 1 µg)<br />
|- <br />
| water || 7 µl<br />
|}<br />
# Incubate at least for 1 hour at 37°C.<br />
<br />
<br />
<br />
=== preparative double digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 5 µl (50 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for 8 hours at 37°C.<br />
# After 3 hours add 2 µl SpeI.<br />
# Add 7 µl XbaI and incubate for another 8 hours.<br />
<br />
=== plasmid linearization ===<br />
<br />
<br />
<br />
== PCR ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Taq/Pfu buffer || 5 µl<br />
|-<br />
| Taq/Pfu polymerase || 1 µl<br />
|-<br />
| primer forward || 0.5 µl (100 pmol/µl)<br />
|-<br />
| primer reverse || 0.5 µl (100 pmol/µl)<br />
|-<br />
| dNTPs || 2.5 µl (200 µM)<br />
|-<br />
| template DNA || 1 µl<br />
|-<br />
| water || 36 µl<br />
|}<br />
<br />
<br />
<br />
'''PCR conditions'''<br />
{| class="wikitable"<br />
|-<br />
! Step !! Duration !! Settings<br />
|-<br />
| 1 || 2 min || 94°C<br />
|-<br />
| 2 || 45 sec || 94°C<br />
|-<br />
| 3 || 30 sec || gradient or annealing temperature<br />
|-<br />
| 4 || 90 sec || 72°C<br />
|-<br />
| || || steps 2-4: 30 cycles<br />
|-<br />
| 5 || 7 min || 72°C<br />
|-<br />
| 6 || (hold) || 4°C<br />
|}<br />
<br />
<br />
<br />
<br />
== Gel electrophoresis ==<br />
<br />
'''TAE buffer 50x'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 0.05 M EDTA || 18.61 g<br />
|-<br />
| 1 M acetic acid || 60.05 g<br />
|-<br />
| 2 M Tris || 242.28 g<br />
|-<br />
| water || 1 l<br />
|}<br />
Adjust to pH 8.5.<br />
<br />
<br />
<br />
'''Gel'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| TAE 1x buffer || 120 ml<br />
|-<br />
| Agarose || 1.2 g<br />
|}<br />
Solve agarose in TAE 1x buffer and boil until solution is clear.<br />
<br />
<br />
<br />
'''Well loading'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| PCR product or DNA || 5 µl<br />
|-<br />
| Loading dye 6x || 1 µl<br />
|}<br />
Can be scaled up linearly.<br />
<br />
<br />
<br />
== LB medium ==<br />
<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 10,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 5,0 g<br />
|-<br />
| water || 1,0 l<br />
|}<br />
Adjust to pH 7.0.<br />
<br />
<br />
<br />
'''Agar-plates'''<br />
<br />
# Solve 16g Agar-Agar in 1l LB buffer and boil until solution is clear. <br />
# If it is nearly cold pour it into some petri dish.<br />
<br />
<br />
<br />
<br />
== SOB medium ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 20,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 0,5 g<br />
|-<br />
| 250mM KCl || 10ml<br />
|-<br />
| water MiliQ || 1l<br />
|}<br />
<br />
# Solve the components in 1l water.<br />
# autoclave<br />
# After autoclaving add 5ml MgCl2<br />
<br />
<br />
<br />
<br />
== Genaxxon Plasmid DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon Gel Extraction Mini Prep Kit ==<br />
[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon PCR DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== QIAGEN Plasmid Midi Kit ==<br />
[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]</div>Jakobmattheshttp://2012.igem.org/Team:Tuebingen/NotebookProtocolsTeam:Tuebingen/NotebookProtocols2012-09-25T10:06:23Z<p>Jakobmatthes: /* Chemo-competent cells */</p>
<hr />
<div>{{:Team:Tuebingen/Template/Tuebingen}}<br />
= Protocols =<br />
__TOC__<br />
== Chemo-competent cells ==<br />
'''Inoue buffer'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| MnCl<sub>2</sub> * 2H<sub>2</sub>0 || 9.67 g<br />
|-<br />
| CaCl<sub>2</sub> * 2H<sub>2</sub>0 || 2.2 g<br />
|-<br />
| KCl || 18.65 g<br />
|-<br />
| PIPES (0.5 M, pH 6.7) || 20 ml<br />
|-<br />
| H<sub>2</sub>0 || ad 1 l<br />
|}<br />
Sterilize through filtration (0.45 µm filter) and store at -20 °C.<br />
<br />
'''Cells'''<br />
# Pick an ''E. coli'' colony and inoculate 25 ml SOB.<br />
# Let bacteria grow for 8 hours at 37 °C and 250 rpm.<br />
# Inoculate three 100 ml SOB volumes with 1 ml, 2 ml and 4 ml of the prepared pre-culture.<br />
# Incubate over night at 18 - 22 °C and 200 rpm.<br />
# At OD<sub>600</sub> = 0.55, put culture for 10 min on ice.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Resuspend cell pellet in 30 ml 0 °C Inoue buffer.<br />
# Centrifuge cells at 2500 g for 10 min at 4 °C. Discard supernatant completely.<br />
# Repeat the previous two steps.<br />
# Resuspend cells in 8 ml 0 °C Inoue buffer. Add 1.5 ml DMSO and incubate on ice for 10 min.<br />
# Aliquot cells à 100 µl and freeze in liquid nitrogen. Store at -80 °C.<br />
<br />
== pGEM Ligation ==<br />
Ligation for TA-cloning of PCR products<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 2X Rapid Ligation Buffer || 5 µl<br />
|-<br />
| pGEM vector || 0.5 µl (25ng)<br />
|-<br />
| PCR product || 3.5 µl<br />
|-<br />
| T4 DNA ligase || 1 µl (3 Weiss units)<br />
|}<br />
<br />
Mix all reagents in a 0.5 ml tube. Incubate reaction at 4°C over night.<br />
<br />
<br />
<br />
<br />
== Ligation ==<br />
Ligation for digested parts and vectors<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 10X T4 DNA Ligase Buffer || 1 µl<br />
|-<br />
| vector DNA || 1 µl (20-100 ng)<br />
|-<br />
| insert DNA || 5 µl (up to 5:1 molar ratio insert to vector)<br />
|-<br />
| T4 DNA ligase || 1 µl (1 unit)<br />
|-<br />
| water || 2.5 µl<br />
|}<br />
Mix all reagents and incubate at 22°C for 1 hour.<br />
<br />
<br />
<br />
<br />
== Chemotransformation ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| chemo-competent ''E. coli'' || 100 µl<br />
|-<br />
| plasmid DNA || up to 10 µl (max. 1/10 of volume)<br />
|}<br />
# Add plasmid DNA to cell culture.<br />
# Incubate for 30 min on ice.<br />
# Heat shock for 90 sec at 42°C.<br />
# Add 900 µl LB.<br />
# Let the bacteria grow at 37°C for at least 1 hour.<br />
<br />
<br />
<br />
<br />
== Restriction digest ==<br />
<br />
<br />
<br />
=== control digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 1 µl<br />
|-<br />
| XbaI (RE) || 0.5 µl (5 units)<br />
|-<br />
| SpeI (RE) || 0.5 µl (5 units)<br />
|-<br />
| DNA || 1 µl (up to 1 µg)<br />
|- <br />
| water || 7 µl<br />
|}<br />
# Incubate at least for 1 hour at 37°C.<br />
<br />
<br />
<br />
=== preparative double digest ===<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Tango buffer 10x || 10 µl<br />
|-<br />
| SpeI (RE) || 5 µl (50 units)<br />
|-<br />
| DNA || up to 30 µg<br />
|- <br />
| water || ad 150 µl<br />
|}<br />
# Incubate for 8 hours at 37°C.<br />
# After 3 hours add 2 µl SpeI.<br />
# Add 7 µl XbaI and incubate for another 8 hours.<br />
<br />
=== plasmid linearization ===<br />
<br />
<br />
<br />
== PCR ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Taq/Pfu buffer || 5 µl<br />
|-<br />
| Taq/Pfu polymerase || 1 µl<br />
|-<br />
| primer forward || 0.5 µl (100 pmol/µl)<br />
|-<br />
| primer reverse || 0.5 µl (100 pmol/µl)<br />
|-<br />
| dNTPs || 2.5 µl (200 µM)<br />
|-<br />
| template DNA || 1 µl<br />
|-<br />
| water || 36 µl<br />
|}<br />
<br />
<br />
<br />
'''PCR conditions'''<br />
{| class="wikitable"<br />
|-<br />
! Step !! Duration !! Settings<br />
|-<br />
| 1 || 2 min || 94°C<br />
|-<br />
| 2 || 45 sec || 94°C<br />
|-<br />
| 3 || 30 sec || gradient or annealing temperature<br />
|-<br />
| 4 || 90 sec || 72°C<br />
|-<br />
| || || steps 2-4: 30 cycles<br />
|-<br />
| 5 || 7 min || 72°C<br />
|-<br />
| 6 || (hold) || 4°C<br />
|}<br />
<br />
<br />
<br />
<br />
== Gel electrophoresis ==<br />
<br />
'''TAE buffer 50x'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| 0.05 M EDTA || 18.61 g<br />
|-<br />
| 1 M acetic acid || 60.05 g<br />
|-<br />
| 2 M Tris || 242.28 g<br />
|-<br />
| water || 1 l<br />
|}<br />
Adjust to pH 8.5.<br />
<br />
<br />
<br />
'''Gel'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| TAE 1x buffer || 120 ml<br />
|-<br />
| Agarose || 1.2 g<br />
|}<br />
Solve agarose in TAE 1x buffer and boil until solution is clear.<br />
<br />
<br />
<br />
'''Well loading'''<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| PCR product or DNA || 5 µl<br />
|-<br />
| Loading dye 6x || 1 µl<br />
|}<br />
Can be scaled up linearly.<br />
<br />
<br />
<br />
== LB medium ==<br />
<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 10,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 5,0 g<br />
|-<br />
| water || 1,0 l<br />
|}<br />
Adjust to pH 7.0.<br />
<br />
<br />
<br />
'''Agar-plates'''<br />
<br />
# Solve 16g Agar-Agar in 1l LB buffer and boil until solution is clear. <br />
# If it is nearly cold pour it into some petri dish.<br />
<br />
<br />
<br />
<br />
== SOB medium ==<br />
{| class="wikitable"<br />
|-<br />
! Component !! Volume<br />
|-<br />
| Trypton || 20,0 g<br />
|-<br />
| yeast-extract || 5,0 g<br />
|-<br />
| NaCl || 0,5 g<br />
|-<br />
| 250mM KCl || 10ml<br />
|-<br />
| water MiliQ || 1l<br />
|}<br />
<br />
# Solve the components in 1l water.<br />
# autoclave<br />
# After autoclaving add 5ml MgCl2<br />
<br />
<br />
<br />
<br />
== Genaxxon Plasmid DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/Plasmid-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon Gel Extraction Mini Prep Kit ==<br />
[http://www.genaxxon.com/catalogue/DNA-Purification-Kits/PCR-and-Gel-extraction-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== Genaxxon PCR DNA Purification Mini Prep Kit ==<br />
[http://www.genaxxon.de/Katalog/DNA-Reinigungskits/PCR-DNA-Purification-Mini-Prep-Kit-50-columns.html Manual provided by Genaxxon]<br />
<br />
<br />
<br />
<br />
== QIAGEN Plasmid Midi Kit ==<br />
[http://www.qiagen.com/products/plasmid/qiagenplasmidpurificationsystem/qiagenplasmidmidikit.aspx#Tabs=t2 Manual provided by QIAGEN]</div>Jakobmatthes