Team:DTU-Denmark-2/Notebook/Lab notes

From 2011.igem.org

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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 1"class="h2">Week 1</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 1"class="h2">Week 1</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 2 "class="h2"> Week 2</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 2 "class="h2"> Week 2</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 3" class="h2"> Week 3</a><br>
+
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 3"class="h2"> Week 3</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 4"class="h2"> Week 4</a><br><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 4"class="h2"> Week 4</a><br><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#August"class="h1">August</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#August"class="h1">August</a><br>
-
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 5" class="h2"> Week 5</a><br>
+
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 5"class="h2"> Week 5</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 6"class="h2"> Week 6</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 6"class="h2"> Week 6</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 7"class="h2"> Week 7</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#Week 7"class="h2"> Week 7</a><br>
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<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#October"class="h1">October</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#October"class="h1">October</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#November"class="h1">November</a><br>
<a href="https://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes#November"class="h1">November</a><br>
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<br>
<br>
<a name="Week 1"></a><h3><b>Week 1: 04.07.2011 - 10.07.2011</b></h3>
<a name="Week 1"></a><h3><b>Week 1: 04.07.2011 - 10.07.2011</b></h3>
 +
 +
First week! Time was spent on designing the Plug 'n' Play assembly system, figuring out what 48 biobricks to construct, and searching for appropriate DNA templates.
 +
<br>
 +
<br>
<a name="Week 2"></a><h3><b>Week 2: 11.07.2011 - 17.07.2011</b></h3>
<a name="Week 2"></a><h3><b>Week 2: 11.07.2011 - 17.07.2011</b></h3>
<a name="Wednesday 13.07.2011"></a><h4>Wednesday</h4>
<a name="Wednesday 13.07.2011"></a><h4>Wednesday</h4>
-
First primers have been ordered, so we are soon ready for lab.
+
The first batch of primers have been ordered, so we are waiting in excitement for them to arrive, so we can start our work in the lab.
<a name="Week 3"></a><h3><b>Week 3: 18.07.2011 - 24.07.2011</b></h3>
<a name="Week 3"></a><h3><b>Week 3: 18.07.2011 - 24.07.2011</b></h3>
<a name="Tuesday 19.07.2011"></a><h4>Tuesday</h4>
<a name="Tuesday 19.07.2011"></a><h4>Tuesday</h4>
-
First day in the lab and 48 biobricks to go, how exciting!  
+
First day in the lab and 48 biobricks to go. This is exciting!  
<br>
<br>
-
Of the 48 biobricks we will have when the project is finished we had only established 17 of them and ordered primers. We started the day with 11 PCR reactions of the first bash of 17 biobricks, we still need some DNA templates to due all 17. 6 PCR were correct and was purified with a purification Kit (GE Healthcare). So only 42 biobricks to go!
+
We plan to construct 48 new BioBricks. We have now established 17 of them and ordered primers for them with USER tails. We started the day with 11 PCR reactions. 6 of them were correct, and they were purified with a purification Kit (GE Healthcare). Only 42 biobricks to go!
<br>
<br>
<br>
<br>
Line 87: Line 100:
<br>
<br>
<ul>
<ul>
-
<li>pALC</li>
+
<li>pAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>)</li>
-
<li>TtrpC</li>
+
<li>TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036 ">BBa_K678036</a>)</li>
-
<li>Terminator 1</li>
+
<li>Terminator 1 (<a href="http://partsregistry.org/Part:BBa_678037">BBa_K678037</a>)</li>
-
<li>Terminator 2</li>
+
<li>Terminator 2 (<a href="http://partsregistry.org/Part:BBa_K678038">BBa_K678038</a>)</li>
-
<li>Terminator 3</li>
+
<li>Terminator 3 (<a href="http://partsregistry.org/Part:BBa_K678039">BBa_K678039</a>)</li>
-
<li>argB</li>
+
<li>argB (<a href="http://partsregistry.org/Part:BBa_K678043">BBa_K678043</a>)</li>
</ul>
</ul>
-
<img src="https://static.igem.org/mediawiki/2011/a/a7/20.07.2011.png" height="270px" alt="""/>  
+
<img src="https://static.igem.org/mediawiki/2011/a/a7/20.07.2011.png" height="270px" alt="""/> </img>
<br>
<br>
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<a name="Thursday 21.07.2011"></a><h4>Thursday</h4>
<a name="Thursday 21.07.2011"></a><h4>Thursday</h4>
-
Some of the PCR reactions that didn't work Tuesday were repeated with a different annealing temperature and amplification of new biobricks.
+
The failed PCR reactions from tuesday were repeated. This time we used a different annealing temperature, and....IT WORKED!
<br>
<br>
<br>
<br>
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<ul>
<ul>
-
<li>PGK</li>
+
<li>PGK (<a href="http://partsregistry.org/Part:BBa_K678004">BBa_K678004</a>)</li>
-
<li>BGHpA</li>
+
<li>BGHpA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>)</li>
-
<li>eGFP+k_GOI</li>
+
<li>eGFP+k_GOI (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
</ul>
</ul>
-
<img src="https://static.igem.org/mediawiki/2011/e/ee/21.07.2011.png" height="270px" alt="""/>  
+
<img src="https://static.igem.org/mediawiki/2011/e/ee/21.07.2011.png" height="270px" alt="""/> </img>
<br>
<br>
<br>
<br>
-
The fragments will be frozen and purified later.
+
The fragments will be frozen and later purified.
<br>
<br>
<br>
<br>
-
We still have not succeed to make biobricks pyrG and pyrG-DR (use as a marker in fungi), despite two PCR attempts with a high annealing (59°) and low (56°) temperature.
+
We still have not succeed to make BioBricks pyrG and pyrG-DR (used as a marker in fungi), despite different PCR attempts with a high annealing (59°) and low (56°) temperature.
<br>
<br>
<br>
<br>
<b>Work for iGEM Copenhagen team</b>
<b>Work for iGEM Copenhagen team</b>
<br>
<br>
-
We have established collaboration with iGEM Copenhagen team, where we will create some biobricks for them and they can use our plug 'n' play system and they will save a lot of time. So today we transformed competent E.coli cells with some of the standard biological parts from iGEM, BBa_R0010, Bba_B0015 and BBa_J52034, so we later take make biobricks to match our system.  
+
We have established collaboration with the iGEM team from Copenhagen University. We will costumize a number of their BioBricks to use with our Plug 'n' Play system. This is a great way of showing how easy it is to costumize the Plug 'n' Play system for any application, and the Copenhagen team will save a lot of time.  
 +
Today we transformed competent E.coli cells with some of the standard biological parts from iGEM,<a href="http://partsregistry.org/Part:BBa_R0010"> BBa_R0010</a>,<a href="http://partsregistry.org/Part:BBa_R0015"> Bba_B0015</a> and <a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>, and later we will make them fit the Plug 'n' Play with DNA system.  
<br>
<br>
<br>
<br>
<a name="Friday 22.07.2011"></a><h4>Friday</h4>
<a name="Friday 22.07.2011"></a><h4>Friday</h4>
-
The biobrick which we still have not succeed in making with PCR is tried again with small modification. We did the PCR reaction with and without MgCl<sub>2</sub> 50mM in the reactions.
+
We made a PCR of a troublesome BioBrick again - but this time with a small modification. We made a PCR mix with and without MgCl<sub>2</sub> 50mM in the reactions. And...IT WORKED with MgCl<sub>2</sub> 50mM!
<br>
<br>
<br>
<br>
<b>Biobrick obtained:</b>  
<b>Biobrick obtained:</b>  
<ul>
<ul>
-
<li>CMV</li>
+
<li>CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>)</li>
-
<li>hygR</li>
+
<li>hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
</ul>
</ul>
-
<img src="https://static.igem.org/mediawiki/2011/c/ce/22.07.2011_1.png" height="270px" alt="""/>  
+
<img src="https://static.igem.org/mediawiki/2011/c/ce/22.07.2011_1.png" height="270px" alt="""/> </img>
-
<img src="https://static.igem.org/mediawiki/2011/2/28/22.07.2011_2.png" height="270px" alt="""/>
+
<img src="https://static.igem.org/mediawiki/2011/2/28/22.07.2011_2.png" height="270px" alt="""/> </img>
<br>
<br>
<br>
<br>
-
The fragments will be frozen and purified later.
+
The fragments will be frozen and later purified .
<br>
<br>
<br>
<br>
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-
We got a new batch of primers today. They need to be diluted and stocked, before they are ready for use.  
+
We got a new delivery of primers from IDT today. They are to be diluted and stocked, before use.  
We have had some difficulty with obtaining 5 of our new biobricks; SV40+ori, SV40 pA, LacZ, pyrG and pyrG-DR. We ran a touch PCR program, where the annealing temperature ran from 55° to 65° for each sample in each cycle.  
We have had some difficulty with obtaining 5 of our new biobricks; SV40+ori, SV40 pA, LacZ, pyrG and pyrG-DR. We ran a touch PCR program, where the annealing temperature ran from 55° to 65° for each sample in each cycle.  
<br>
<br>
<br>
<br>
-
<b>The PCR reactions were successful, so following biobricks are ready for Plug'n'Play:  </b>
+
<b>The PCR reactions were successful, and following biobricks are ready for Plug'n'Play:  </b>
   
   
<ul>
<ul>
-
<li>SV40 +ori</li>
+
<li>SV40 +ori (<a href="http://partsregistry.org/Part:BBa_K678005">BBa_K678005</a>)</li>
-
<li>SV40 pA</li>
+
<li>SV40 pA (<a href="http://partsregistry.org/Part:BBa_K678012">BBa_K678012</a>)</li>
-
<li>LacZ</li>
+
<li>LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>)</li>
</ul>
</ul>
-
<img src="https://static.igem.org/mediawiki/2011/7/76/26.07.2011_1.png" height="270px" alt="""/>
+
<img src="https://static.igem.org/mediawiki/2011/7/76/26.07.2011_1.png" height="270px" alt="""/> </img>
-
 
+
-
The fragments will be frozen and purified later.
+
-
 
+
-
We still have trouble with obtaining pyrG and pyrG-DR.
+
<br>
<br>
<br>
<br>
<a name="Tuesday 26.07.2011"></a><h4>Tuesday</h4>
<a name="Tuesday 26.07.2011"></a><h4>Tuesday</h4>
-
The new primer batch we recieved yesterday, will be used for amplification of many many many new biobricks today.
+
The primers recieved yesterday will be used for amplification of many many many new biobricks today.
<br>
<br>
<br>
<br>
-
<b>Out of 15 PCR reactions only 5 succeeded:  </b>
+
<b>Out of 15 PCR reactions 5 succeeded:  </b>
<br>
<br>
<ul>
<ul>
-
<li>GFP_GOI </li>
+
<li>GFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678028">BBa_K678028</a>) </li>
-
<li>GFP_modul</li>
+
<li>GFP_modul (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>)</li>
-
<li>GFP_TS</li>
+
<li>GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>)</li>
-
<li>eGFP_TS</li>
+
<li>eGFP_TS (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
-
<li>eGFP_module</li>
+
<li>eGFP_module (<a href="http://partsregistry.org/Part:BBa_K678009">BBa_K678009</a>)</li>
</ul>
</ul>
-
<img src="https://static.igem.org/mediawiki/2011/b/bf/26.07.2011_2.png" height="270px" alt="""/>
+
<img src="https://static.igem.org/mediawiki/2011/b/bf/26.07.2011_2.png" height="270px" alt="""/> </img>
-
<img src="https://static.igem.org/mediawiki/2011/a/ab/26.07.2011_3.png" height="270px" alt="""/>
+
<img src="https://static.igem.org/mediawiki/2011/a/ab/26.07.2011_3.png" height="270px" alt="""/> </img>
   
   
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<a name="Wednesday 27.07.2011"></a><h4>Wednesday</h4>
<a name="Wednesday 27.07.2011"></a><h4>Wednesday</h4>
-
Today we have run 8 reaction, 3 standard and 5 touch PCR reactions on some of the biobricks that we still have difficulties obtaining.  
+
Today we have run 8 reactions, 3 standard and 5 touch PCR reactions on some of the biobricks that we still have difficulties obtaining.  
<br>
<br>
<br>
<br>
-
<b>Only one succeeded:</b>
+
<b>One succeeded:</b>
<ul>
<ul>
-
<li>hgH-polyA </li>
+
<li>hgH-polyA (<a href="http://partsregistry.org/Part:BBa_K678018">BBa_K678018</a>) </li>
</ul>
</ul>
-
<img src="https://static.igem.org/mediawiki/2011/e/e5/27.07.2011.png" height="270px" alt="""/>
+
<img src="https://static.igem.org/mediawiki/2011/e/e5/27.07.2011.png" height="270px" alt="""/> </img>
<br>
<br>
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<a name="Week 5"></a><h3><b>Week 5: 01.08.2011 - 07.08.2011</b></h3>
<a name="Week 5"></a><h3><b>Week 5: 01.08.2011 - 07.08.2011</b></h3>
<a name="Monday 01.08.2011"></a><h4>Monday</h4>
<a name="Monday 01.08.2011"></a><h4>Monday</h4>
-
New week, new energy!
+
New week, new energy, and lots of team spirit!
Today 15 PCR reactions were conducted, including parts for iGEM Copenhagen team. 14 succeeded. The annealing temperature was increased to 63°, and it worked like magic!  
Today 15 PCR reactions were conducted, including parts for iGEM Copenhagen team. 14 succeeded. The annealing temperature was increased to 63°, and it worked like magic!  
<br>
<br>
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<b>New biobricks in the bag:</b>
<b>New biobricks in the bag:</b>
<ul>
<ul>
-
<li>RFP_GOI&nbsp;(<b>20</b>) </li>
+
<li>RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>) </li>
-
<li>RFP_modul&nbsp;(<b>21</b>)</li>
+
<li>RFP_modul (<a href="http://partsregistry.org/Part:BBa_K678030">BBa_K678030</a>)</li>
-
<li>RFP_TS&nbsp;(<b>22</b>)</li>
+
<li>RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>)</li>
-
<li>bleR&nbsp;(<b>29</b>)</li>
+
<li>bleR (<a href="http://partsregistry.org/Part:BBa_K678044">BBa_K678044</a>)</li>
-
<li>pgpdA&nbsp;(<b>26</b>)</li>
+
<li>pgpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>)</li>
-
<li>yA&nbsp;(<b>27</b>)</li>
+
<li>yA (<a href="http://partsregistry.org/Part:BBa_K678035">BBa_K678035</a>)</li>
-
<li>Plasmid_fun&nbsp;(<b>28</b>)</li>
+
<li>Plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>)</li>
-
<li>Plasmid_mam&nbsp;(<b>30</b>)</li>
+
<li>Plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>)</li>
-
<li>Amp cas &nbsp;(<b>C1</b>)</li>
+
<li>Amp cas (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
-
<li>Hygromycin&nbsp;(<b>18</b>)</li>
+
<li>Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
<li>CYP79A2 (Copenhagen part)&nbsp;(<b>C4</b>)</li>
+
<li>CYP79A2 (Copenhagen part) (<a href="http://partsregistry.org/Part:BBa_K527001">BBa_K527001</a>)</li>
-
<li>Terminator (Copenhagen part)&nbsp;(<b>C6</b>)</li>
+
<li>Terminator (Copenhagen part) (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>)</li>
-
<li>pIPTG (Copenhagen part)&nbsp;(<b>C2</b>)</li>
+
<li>pIPTG (Copenhagen part) (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>)</li>
-
<li>CYP79B2(Copenhagen part)&nbsp;(<b>C5</b></li>
+
<li>CYP79B2(Copenhagen part) (<a href="http://partsregistry.org/Part:BBa_K527002">BBa_K527002</a>)</li>
</ul>
</ul>
<br>
<br>
-
<img src="https://static.igem.org/mediawiki/2011/e/e2/1.08.2011_1.png" height="270px" alt="""/>
+
<img src="https://static.igem.org/mediawiki/2011/e/e2/1.08.2011_1.png" height="270px" alt="""/> </img>
-
<img src="https://static.igem.org/mediawiki/2011/4/4d/01.08.2011_2.png" height="270px" alt="""/>
+
<img src="https://static.igem.org/mediawiki/2011/4/4d/01.08.2011_2.png" height="270px" alt="""/> </img>
-
<img src="https://static.igem.org/mediawiki/2011/9/97/01.08.2011_3.png" height="270px" alt="""/>
+
<img src="https://static.igem.org/mediawiki/2011/9/97/01.08.2011_3.png" height="270px" alt="""/> </img>
<a name=" Wednesday 03.08.2011"></a><h4> Wednesday </h4>
<a name=" Wednesday 03.08.2011"></a><h4> Wednesday </h4>
-
We still have 2 biobricks we haven’t been able to obtain with PCR: pyrG and pyrG-DR.  
+
There are still 2 BioBricks that we haven't been able to obtain with PCR: pyrG and pyrG-DR.  
<br>
<br>
-
Today we tried again with gradient PCR to find the optimal melting temperature, but it did not help.   
+
Today we tried again with gradient PCR to find the optimal melting temperature, but no magic happened.   
<br>
<br>
<br>
<br>
<a name="Thursday 04.08.2011"></a><h4>Thursday</h4>
<a name="Thursday 04.08.2011"></a><h4>Thursday</h4>
-
Today we purified 22 biobricks, which have been obtained with PCR during the last week of laboratory work. The purification of DNA was done by using the illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare) according to the manufactures manual for purification of DNA from TAE and TBE agarose gels protocol, see protocol section for more information.<br><br>
+
22 BioBricks, which have been obtained with PCR during the last week of laboratory work, was purified today. The purification of DNA was done by using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). The procedure was carried out according to the manufactures protocol for purification of DNA from TAE and TBE agarose gels, see protocol section for more information.<br><br>
-
Despite several attempts, we have not been able to obtain biobrick pyrG and pyrG-DR. Therefore we made a test with well-known primers without USER-tails (sometimes these USER-tails can be tricky). This was done to verify if something was wrong with the DNA template. The gel electrophoresis did not show any bands, meaning that the DNA template was the source of error.  
+
Despite several attempts we have not been able to obtain BioBrick pyrG and pyrG-DR. Therefore we made a test with well-known primers without USER-tails (sometimes these USER-tails can be tricky). This was done to verify if something was wrong with the DNA template. The gel electrophoresis did not show any bands, and thereby it revealed that the DNA template was the source of error. Problem solved!
<br>
<br>
<br>   
<br>   
-
We also started cultivation of the mammalian cell line, U2OS, which is derived from a 15-year old patient with osteosarcoma (bone cancedr). They were defrosted and cultivated in in DMEM medium containing serum, penicillin and streptomycin. The cell line was then transferred to a 25 cm2 culture flask, see protocol section.
+
We also started cultivation of the mammalian cell line, U2OS, which is derived from a patient with osteosarcoma (bone cancer). They were defrosted and cultivated in DMEM medium containing serum, penicillin and streptomycin. The cell line was then transferred to a 25 cm2 culture flask, see protocol section.
<br>
<br>
<br>
<br>
Line 263: Line 274:
<a name="Monday 08.08.2011"></a><h4>Monday</h4>
<a name="Monday 08.08.2011"></a><h4>Monday</h4>
-
Today we made the first USER cloning, it is very exciting to see if our system works! <br>
+
Today we made the first USER cloning, we are very excited to see if the system works the way, we want it to! <br>
All our biobricks are divided into devices, see protocol for USER MIX. <br>
All our biobricks are divided into devices, see protocol for USER MIX. <br>
 +
Device 1,2,3,5 and 6 was not registrated later.
<br>
<br>
<b>The following devices were cloned today:</b>
<b>The following devices were cloned today:</b>
Line 271: Line 283:
<ul>
<ul>
-
<li>Device 1: plasmid_mam<b>&nbsp;(30)</b>&nbsp;+&nbsp;CMV<b>&nbsp;(3)</b>&nbsp;+&nbsp;eGFP<b>&nbsp;(32)</b>&nbsp;+&nbsp;Hgh pA<b>&nbsp;(33)</b>&nbsp;+&nbsp;Hygromycin<b>&nbsp;(18)</b></li>
+
<li><b>Device 1</b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + eGFP (<a href="http://partsregistry.org/Part:BBa_K678006">BBa_K678006</a>) + hGH pA (<a href="http://partsregistry.org/Part:BBa_K678018">BBa_K678018</a>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
<li>Device 2: plasmid_mam<b>&nbsp;(30)</b>&nbsp;+&nbsp;SV40<b>&nbsp;(2)</b>&nbsp;+&nbsp;eGFP<b>&nbsp;(32)</b>&nbsp;+&nbsp;SV40 pA<b>&nbsp;(4)</b>&nbsp;+&nbsp;Hygromycin<b>&nbsp;(18)</b></li>
+
 
-
<li>Device 3: plasmid_mam<b>&nbsp;(30)</b>&nbsp;+&nbsp;PGK<b>&nbsp;(1)</b>&nbsp;+&nbsp;eGFP<b>&nbsp;(32)</b>&nbsp;+&nbsp;BGH pA<b>&nbsp;(5)</b>&nbsp;+&nbsp;Neomycin<b>&nbsp;(19)</b></li>
+
<li><b>Device 2</b>: <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + SV40+ori (<a href="http://partsregistry.org/Part:BBa_K678005">BBa_K678005</a>) + eGFP (<a href="http://partsregistry.org/Part:BBa_K678006">BBa_K678006</a>) + SV40 pA (<a href="http://partsregistry.org/Part:BBa_K678012">BBa_K678012</a>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
<li>Device 4: plasmid_fun<b>&nbsp;(28)</b>&nbsp;+&nbsp;pAlc<b>&nbsp;(7)</b>&nbsp;+&nbsp;LacZ<b>&nbsp;(8)</b>&nbsp;+&nbsp;T1<b>&nbsp;(14)</b>&nbsp;+&nbsp;argB<b>&nbsp;(17)</b></li>
+
 
-
<li>Device 5: plasmid_fun<b>&nbsp;(28)</b>&nbsp;+&nbsp;gpdA<b>&nbsp;(26)</b>&nbsp;+&nbsp;GFP<b>&nbsp;(24)</b>&nbsp;+&nbsp;trpC<b>&nbsp;(13)</b>&nbsp;+&nbsp;hygR<b>&nbsp;(12)</b></li>
+
<li><b>Device 3</b>: <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + PGK (<a href="http://partsregistry.org/Part:BBa_K678004">BBa_K678004</a>) + eGFP_module (<a href="http://partsregistry.org/Part:BBa_K678006">BBa_K678006</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Neomycin (<a href="http://partsregistry.org/Part:BBa_K678022">BBa_K678022</a>)</li>
-
<li>Device 6: plasmid_fun<b>&nbsp;(28)</b>&nbsp;+&nbsp;gpdA<b>&nbsp;(26)</b>&nbsp;+&nbsp;RFP<b>&nbsp;(21)</b>&nbsp;+&nbsp;trpC<b>&nbsp;(13)</b>&nbsp;+&nbsp;hygR<b>&nbsp;(12)</b></li>
+
 
-
<li>Device 7: plasmid_mam<b>&nbsp;(30)</b>&nbsp;+&nbsp;pIPTG<b>&nbsp;(C2)</b>&nbsp;+&nbsp;CYP79B2<b>&nbsp;(C5)</b>&nbsp;+&nbsp;Terminator<b>&nbsp;(C6)</b>&nbsp;+&nbsp;amp<b>&nbsp;(C1)</b></li>
+
<li><b>Device <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678058">BBa_K678058</a></b>:<br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + pAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>) + T1 (<a href="http://partsregistry.org/Part:BBa_K678037">aBBa_K678037</a>) + argB (<a href="http://partsregistry.org/Part:BBa_K678043">BBa_K678043</a>)</li>
-
<li>Device 8: plasmid_mam<b>&nbsp;(30)</b>&nbsp;+&nbsp;pIPTG<b>&nbsp;(C2)</b>&nbsp;+&nbsp;CYP79A2<b>&nbsp;(C4)</b>&nbsp;+&nbsp;Terminator<b>&nbsp;(C6)</b>&nbsp;+&nbsp;amp<b>&nbsp;(C1)</b></li>
+
 
 +
<li><b>Device 5</b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + GFP_module (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>) + trpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
 +
 
 +
<li><b>Device 6</b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + RFP_module (<a href="http://partsregistry.org/Part:BBa_K678030">BBa_K678030</a>) + trpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
 +
 
 +
<li>Device 7 (Copenhagen): <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + CYP79B2 (<a href="http://partsregistry.org/Part:BBa_K527002">BBa_K527002</a>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
 +
 
 +
<li>Device 8 (Copenhagen): <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + CYP79A2 (<a href="http://partsregistry.org/Part:BBa_K527001">BBa_K527001</a>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
</ul>
</ul>
Line 288: Line 307:
<ul>
<ul>
-
<li>eGFP+k_GOI&nbsp;<b>(6)</b></li>
+
<li>eGFP+k_GOI (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
-
<li>eGFP_TS&nbsp;<b>(31)</b></li>
+
<li>eGFP_TS (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
-
<li>yA&nbsp;<b>(27)</b></li>
+
<li>yA (<a href="http://partsregistry.org/Part:BBa_K678035">BBa_K678035</a>)</li>
-
<li>GFP_TS&nbsp;<b>(25)</b></li>
+
<li>GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>)</li>
-
<li>RFP_TS&nbsp;<b>(22)</b></li>
+
<li>RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>)</li>
-
<li>RFP_GOI&nbsp;<b>(20)</b> </li>
+
<li>RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>) </li>
-
<li>MTS&nbsp;<b>(34)</b> </li>
+
<li>MTS (<a href="http://partsregistry.org/Part:BBa_K678025">BBa_K678025</a>) </li>
-
<li>GFP_PTS1_fun&nbsp;<b>(35)</b> </li>
+
<li>GFP_PTS1_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>) </li>
-
<li>GFP_PTS1_mam&nbsp;<b>(36)</b> </li>
+
<li>GFP_PTS1_mam (<a href="http://partsregistry.org/Part:BBa_K678017">BBa_K678017</a> )</li>
-
<li>RFP_NLS_module&nbsp;<b>(37)</b>  </li>
+
<li>RFP_NLS_module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>) </li>
-
<li>pyrG&nbsp;<b>(10)</b> </li>
+
<li>pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a> ) </li>
-
<li>pyrG-DR&nbsp;<b>(11)</b>  </li>
+
<li>pyrG-DR (<a href="http://partsregistry.org/Part:BBa_K678041">BBa_K678041</a>) </li>
</ul>
</ul>
Line 307: Line 326:
<br>
<br>
<br>
<br>
-
After new DNA template we finally succeed in getting biobricks pyrG and pyrG-DR.   
+
After recieving the new DNA template we finally succeeded in getting biobricks pyrG and pyrG-DR.   
<br>
<br>
The U2OS cell line was splitted and passed on to new culture flasks. They looked really lovely in the microscope, which bodes well for future confocal microscopy. They seem to thrive in the mammalian cell lab, and they are passed on every second or third day, depending on the initial cell concentration.
The U2OS cell line was splitted and passed on to new culture flasks. They looked really lovely in the microscope, which bodes well for future confocal microscopy. They seem to thrive in the mammalian cell lab, and they are passed on every second or third day, depending on the initial cell concentration.
Line 314: Line 333:
<a name="Tuesday 09.08.2011"></a><h4>Tuesday</h4>
<a name="Tuesday 09.08.2011"></a><h4>Tuesday</h4>
-
We inoculated E.coli with the plasmides done by USER cloning yesterday.  
+
<i>E.coli </i> was inoculated with the plasmides done by USER cloning yesterday.  
<br>
<br>
<br>
<br>
Line 320: Line 339:
<ul>
<ul>
-
<li> RFP_TS</li>
+
<li> RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>)</li>
-
<li>RFP_GOI</li>
+
<li>RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>)</li>
-
<li>RFP_NLS_Module</li>
+
<li>RFP_NLS_Module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>)</li>
-
<li>nr.37</li>
+
 
</ul>
</ul>
Line 336: Line 355:
<br>
<br>
<br>
<br>
-
<b>Purification with GFX kit on the following biobricks:</b>
+
<b>Purification with GFX kit of the following biobricks:</b>
<br>
<br>
 +
<ul>
<ul>
-
<li> nr.6</li>
+
<li>eGFP+k_GOI (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
-
<li>nr. 31</li>
+
<li>eGFP_TS (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>)</li>
-
<li>nr.27</li>
+
<li>yA (<a href="http://partsregistry.org/Part:BBa_K678035">BBa_K678035</a>)</li>
-
<li>nr.25</li>
+
<li>GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>)</li>
-
<li>nr.22</li>
+
<li>RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>)</li>
-
<li>nr.34</li>
+
<li>RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>) </li>
-
<li>nr.35</li>
+
<li>MTS (<a href="http://partsregistry.org/Part:BBa_K678025">BBa_K678025</a>) </li>
-
<li>nr.36</li>
+
<li>GFP_PTS1_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>) </li>
-
<li>nr.20</li>
+
<li>GFP_PTS1_mam (<a href="http://partsregistry.org/Part:BBa_K678017">BBa_K678017</a>)</li>
-
<li>nr.37</li>
+
<li>RFP_NLS_module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>)  </li>
-
<li>nr.10</li>
+
<li>pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>) </li>
-
<li>nr.11</li>
+
<li>pyrG-DR (<a href="http://partsregistry.org/Part:BBa_K678041">BBa_K678041</a>)  </li>
</ul>
</ul>
<br>
<br>
-
 
<b>USER cloning done today:</b>
<b>USER cloning done today:</b>
<br>
<br>
<ul>
<ul>
-
<li>Device 9: plasmid_fun<b>&nbsp;(28)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(26)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(34)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(25)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(13)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(12)</b></li>
+
<li><b>Device <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K678065">BBa_K678065</a> </b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + MTS (<a href="http://partsregistry.org/Part:BBa_K678025">BBa_K678025</a>) +  GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
-
<li>Device 10: plasmid_fun<b>&nbsp;(28)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(26)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(35)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(13)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(12)</b></li>
+
<li><b>Device 10</b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + GFP_PTS1_module_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
-
<li>Device 11: plasmid_fun<b>&nbsp;(28)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(26)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(37)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(13)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(12)</b></li>
+
<li><b>Device 11</b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + RFP_NLS_module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + hygR (<a href="http://partsregistry.org/Part:BBa_K678042">BBa_K678042</a>)</li>
-
<li>Device 12: plasmid_mam<b>&nbsp;(30)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(3)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(36)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(5)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(18)</b></li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678050">BBa_K678050</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + GFP_PST1_module_mam (<a href="http://partsregistry.org/Part:BBa_K678017">BBa_K678017</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
<li>Device 13: plasmid_mam<b>&nbsp;(30)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(3)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(32)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(32)</b>&nbsp;+&nbsp;xxx<b>&nbsp;(18)</b></li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678049">BBa_K678049</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + eGFP_module (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
</ul>
</ul>
 +
<br>
<br>
<a name="Wednesday 10.08.2011"></a><h4>Wednesday</h4>
<a name="Wednesday 10.08.2011"></a><h4>Wednesday</h4>
-
The U2OS cells are checked out in the microscope. They had a confluency of about 90%, which means that they are ready to pass on to new culture flasks. This was done according to the protocol (see protocol for passing and maintenance of mammalian cells")
+
The U2OS cells was investigated in the microscope. They had a confluency of about 90%, an therefore ready to pass on to new culture flasks. This was done according to the protocol (see protocol for passing and maintenance of mammalian cells")
<br>
<br>
Line 375: Line 395:
<a name="Friday 12.08.2011"></a><h4>Friday</h4>
<a name="Friday 12.08.2011"></a><h4>Friday</h4>
-
Today we did 13 PCR reaction, some of them includes parts to characterise our two promoters pAlc and DMKP-P6 and some parts for iGEM Copenhagen team.  
+
Today 13 PCR reaction were carried out, some parts were for characterisation of two promoters: pAlc and DMKP-P6, and the rest were parts for the Copenhagen iGEM team.  
<br>
<br>
<br>
<br>
<ul>
<ul>
-
<li> GFP_GOI&nbsp;(<b>23</b>) </li>
+
<li>GFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678028">BBa_K678028</a>) </li>
-
<li>GFP_PTS_fun&nbsp;(<b>35</b>)</li>
+
<li>GFP_PTS_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>)</li>
-
<li>GFP_PTS_mam&nbsp;(<b>36</b>)</li>
+
<li>GFP_PTS_mam (<a href="http://partsregistry.org/Part:BBa_K678017">BBa_K678017</a>)</li>
-
<li>RFP_NLS&nbsp;(<b>37</b>)</li>
+
<li>RFP_NLS (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>)</li>
-
<li>DMKP_P6&nbsp;(<b>38</b>)</li>
+
<li>DMKP_P6 (<a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a>)</li>
-
<li>ptrA&nbsp;(<b>40</b>)</li>
+
<li>ptrA (<a href="http://partsregistry.org/Part:BBa_K678045">BBa_K678045</a>)</li>
-
<li>1A2&nbsp;(<b>C7</b>)</li>
+
<li>1A2 (<b>C7</b>)</li>
-
<li>2C9&nbsp;(<b>C8</b>)</li>
+
<li>2C9 (<b>C8</b>)</li>
-
<li>pAlc-L&nbsp;(<b>B1</b>)</li>
+
<li>pAlc-L (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>)</li>
-
<li>DMKP_P6-L&nbsp;(<b>B3</b>)</li>
+
<li>DMKP_P6-L (<a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a>)</li>
-
<li>RFP_TS&nbsp;(<b>22</b>)</li>
+
<li>RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>)</li>
-
<li>RFP_GOI&nbsp;(<b>20</b>)</li>
+
<li>RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>)</li>
</ul>   
</ul>   
<br>
<br>
<br>
<br>
-
<img src="https://static.igem.org/mediawiki/2011/b/b0/12.08.2011.png"height="270px" alt="""/>
+
<img src="https://static.igem.org/mediawiki/2011/b/b0/12.08.2011.png"height="250px" alt="""/>
<br>
<br>
<br>
<br>
-
The U2OS cells are splitted and passed on to two new 75 cm2 culture flasks. Hopefully, they will be ready to pass on to cover slips on monday.
+
The U2OS cells were splitted and passed on to two new 75 cm2 culture flasks. Hopefully, they will be ready to pass on to cover slips on monday.
<br>
<br>
<a name="Week 7"></a><h3><b>Week 7: 15.08.2011 - 21.08.2011</b></h3>
<a name="Week 7"></a><h3><b>Week 7: 15.08.2011 - 21.08.2011</b></h3>
<br>
<br>
<a name="Monday 15.08.2011"></a><h4>Monday</h4>
<a name="Monday 15.08.2011"></a><h4>Monday</h4>
-
The U2OS cells were splitted and passed on to a new 75 cm2 culture flask and to coverslips placed in a 6-well plate. They are placed in the incubator overnight - and tomorrow they will be ready for transfection with plasmides.
+
The U2OS cells were splitted and passed on to a new 75 cm2 culture flask and to coverslips placed in a 6-well plate. They were placed in the incubator overnight - and will be ready for transfection with plasmides tomorrow.
<br>
<br>
-
Preparing for characterization of two promoters with β-galactosidase assay were the plasmid p68 cut with restriction enzyme.  
+
In preparation for characterization of two promoters with β-galactosidase assay the plasmid p68 cut with restriction enzyme.  
<br>
<br>
<br>
<br>
-
Purification of PCR products done last Friday were done with GFX kit.  
+
Purification of PCR products with GFX kit.  
<br>
<br>
<br>   
<br>   
Line 416: Line 436:
<br>
<br>
<ul>
<ul>
-
<li> eYFP_module&nbsp;(<b>41</b>) </li>
+
<li> eYFP_module (<a href="http://partsregistry.org/Part:BBa_K678009">BBa_K678009</a>) </li>
-
<li>eYFP_GOI&nbsp;(<b>42</b>)</li>
+
<li>eYFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678010">BBa_K678010</a>)</li>
-
<li>eYFP_TS&nbsp;(<b>43</b>)</li>
+
<li>eYFP_TS (<a href="http://partsregistry.org/Part:BBa_K678011">BBa_K678011</a>)</li>
-
<li>eCFP_module&nbsp;(<b>47</b>)</li>
+
<li>eCFP_module (<a href="http://partsregistry.org/Part:BBa_K678015">BBa_K678015</a>)</li>
-
<li>eCFP_GOI&nbsp;(<b>48</b>)</li>
+
<li>eCFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678048">BBa_K678048</a>)</li>
-
<li>eCFP_TS&nbsp;(<b>49</b>)</li>
+
<li>eCFP_TS (<a href="http://partsregistry.org/Part:BBa_K678016">BBa_K678016</a>)</li>
-
<li>mCheryy_module&nbsp;(<b>44</b>)</li>
+
<li>mCheryy_module (<a href="http://partsregistry.org/Part:BBa_K678020">BBa_K678020</a>)</li>
-
<li>mCherry_GOI&nbsp;(<b>45</b>)</li>
+
<li>mCherry_GOI (<a href="http://partsregistry.org/Part:BBa_K678013">BBa_K678013</a>)</li>
-
<li>mCHerry_TS&nbsp;(<b>46</b>)</li>
+
<li>mCHerry_TS (<a href="http://partsregistry.org/Part:BBa_K678014">BBa_K678014</a>)</li>
</ul>   
</ul>   
<br>
<br>
Line 437: Line 457:
<br>
<br>
<br>
<br>
-
<b>USER cloning done today and transformed in <i>E.coli </i> today: </b>
+
<b>USER cloning and transformation in <i>E.coli </i> today: </b>
<br>
<br>
<ul>
<ul>
-
<li>Device 21: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>7</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>8</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>14</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>40</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678059">BBa_K678059</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + PAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>) + T1 (<a href="http://partsregistry.org/Part:BBa_K678037">BBa_K678037</a>) + ptrA (<a href="http://partsregistry.org/Part:BBa_K678045">BBa_K678045</a>)</li>
-
<li>Device 22: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>26</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>24</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>10</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a></b>:</br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + GFP_module (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
<li>Device 23: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>26</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>35</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>10</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678061">BBa_K678061</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + GFP_PTS1_module_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
<li>Device 24: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>26</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>21</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>10</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678062">BBa_K678062</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + RFP_module (<a href="http://partsregistry.org/Part:BBa_K678030">BBa_K678030</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
<li>Device 25: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>26</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>37</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>10</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678063">BBa_K678063</a></b>:<br> plasmid_fun (<a href="://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + RFP_NLS_module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
<li>Device 26: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>26</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>34</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>25</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>10</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678064">BBa_K678064</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + MTS (<a href="http://partsregistry.org/Part:BBa_K678025">BBa_K678025</a>) + GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
<li>Device 28: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>38</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>23</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>22</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>15</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>29</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678066">BBa_K678066</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + DMKP_P6 (<a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a>) + GFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678028">BBa_K678028</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + T2 (<a href="http://partsregistry.org/Part:BBa_K678038">BBa_K678038</a>) + bleR (<a href="http://partsregistry.org/Part:BBa_K678044">BBa_K678044</a>)</li>
-
<li>Device 29: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>7</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>20</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>22</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>16</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>11</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678067">BBa_K678067</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + pAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + T3 (<a href="http://partsregistry.org/Part:BBa_K678039">BBa_K678039</a>) + pyrG_DR (<a href="http://partsregistry.org/Part:BBa_K678041">BBa_K678041</a>)</li>
-
<li>Device 13: plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>3</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>32</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>5</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>18</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678049">BBa_K678049</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + eGFP_Module (<a href="http://partsregistry.org/Part:BBa_K678006">BBa_K678006</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</b>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
<li>Device 12: plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>3</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>36</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>5</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>18</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678050">BBa_K678050</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + GFP_PTS1_module_mam (<a href="http://partsregistry.org/Part:BBa_K678017">BBa_K678017</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromycin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
<li>Device 17: plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>1</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>6</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>31</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>33</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>19</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678054">BBa_K678054</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + PGK (<a href="http://partsregistry.org/Part:BBa_K678004">BBa_K678004</a>) + eGFP+K_GOI (<a href="http://partsregistry.org/Part:BBa_K678007">BBa_K678007</a>) + eGFP_TS (<a href="http://partsregistry.org/Part:BBa_K678008">BBa_K678008</a>) + hGH_polyA (<a href="http://partsregistry.org/Part:BBa_K678018">BBa_K678018</a>) + Neomycin (<a href="http://partsregistry.org/Part:BBa_K678022">BBa_K678022</a>)</li>
</ul>
</ul>
Line 468: Line 488:
<a name="Tuesday 16.08.2011"></a><h4>Tuesday</h4>
<a name="Tuesday 16.08.2011"></a><h4>Tuesday</h4>
-
We transfected U2OS cells with plasmides containing GFP and placed them in the incubator.  
+
We transfected U2OS cells with plasmides containing GFP and placed them in the incubator overnight.  
<br>
<br>
<br>
<br>
-
<b>USER cloning done today and transformed in <i>E.coli </i> today: </b>
+
<b>USER cloning and transformation in <i>E.coli </i> today: </b>
<br>
<br>
<ul>
<ul>
-
<li>Device 14: plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>3</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>41</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>5</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>18</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678051">BBa_K678051</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<ahref="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + eYFP_module (<a href="http://partsregistry.org/Part:BBa_K678009">BBa_K678009</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromysin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
<li>Device 15: plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>3</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>44</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>5</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>18</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678052">BBa_K678052</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + mCherry_module (<a href="http://partsregistry.org/Part:BBa_K678020">BBa_K678020</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromysin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
<li>Device 16:  plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>3</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>47</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>5</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>18</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678053">BBa_K678053</a></b>:  <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + CMV (<a href="http://partsregistry.org/Part:BBa_J52034">BBa_J52034</a>) + eCFP_module (<a href="http://partsregistry.org/Part:BBa_K678015">BBa_K678015</a>) + BGH pA (<a href="http://partsregistry.org/Part:BBa_K678019">BBa_K678019</a>) + Hygromysin (<a href="http://partsregistry.org/Part:BBa_K678021">BBa_K678021</a>)</li>
-
<li>Device 18:  plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>1</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>42</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>43</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>33</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>19</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678055">BBa_K678055</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + PGK (<a href="http://partsregistry.org/Part:BBa_K678004">BBa_K678004</a>) + eYFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678010">BBa_K678010</a>) + eYFP_TS (<a href="http://partsregistry.org/Part:BBa_K678011">BBa_K678011</a>) + hGH_polyA (<a href="http://partsregistry.org/Part:BBa_K678018">BBa_K678018</a>) + Neomycin (<a href="http://partsregistry.org/Part:BBa_K678022">BBa_K678022</a>)</li>
-
<li>Device 19:  plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>2</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>45</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>46</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>4</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>19</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678056">BBa_K678056</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + SV40+ori (<a href="http://partsregistry.org/Part:BBa_K678005">BBa_K678005</a>) + mCherry_GOI (<a href="http://partsregistry.org/Part:BBa_K678013">BBa_K678013</a>) + mCherry_TS (<a href="http://partsregistry.org/Part:BBa_K678014">BBa_K678014</a>) + SV40 pA (<a href="http://partsregistry.org/Part:BBa_K678012">BBa_K678012</a>) + Neomycin (<a href="http://partsregistry.org/Part:BBa_K678022">BBa_K678022</a>)</li>
-
<li>Device 20:  plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>2</b>&nbsp;+&nbsp;xxx&nbsp;(<b>48</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>49</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>4</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>19</b>))</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678057">BBa_K678057</a></b>:<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + Sv40+ori (<a href="http://partsregistry.org/Part:BBa_K678005">BBa_K678005</a>) + eCFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678048">BBa_K678048</a>) + eCFP_TS (<a href="http://partsregistry.org/Part:BBa_K678016">BBa_K678016</a>) + SV40 pA (<a href="http://partsregistry.org/Part:BBa_K678012">BBa_K678012</a>) + Neomycin (<a href="http://partsregistry.org/Part:BBa_K678022">BBa_K678022</a>)</li>
-
<li>Device 21:  plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>7</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>8</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>14</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>40</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678059">BBa_K678059</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + Palc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>) + T1 (<a href="http://partsregistry.org/Part:BBa_K678037">BBa_K678037</a>) + ptrA (<a hrf="http://partsregistry.org/Part:BBa_K678045">BBa_K678045</a>)</li>
</ul>
</ul>
Line 492: Line 512:
<a name="Wednesday 17.08.2011"></a><h4>Wednesday</h4>
<a name="Wednesday 17.08.2011"></a><h4>Wednesday</h4>
<br>
<br>
-
Inoculations of <i>E.coli </i> transformants with USER devices done the day before.  
+
Inoculations of <i>E.coli </i> transformants with USER devices cloned yesterday.  
<br>
<br>
<br>
<br>
-
Restriction analysis of Device 4, 7, 8 and 27
+
Restriction analysis of device 4, 7, 8, and 27.
-
<br>
+
-
<br>
+
-
Insert of gel-photo 17.08.11 09:35
+
<br>
<br>
 +
<br>
<br>
-
<b>USER cloning done today and transformed in <i>E.coli </i> today: </b>
+
<b>USER cloning and transformation in <i>E.coli </i> today: </b>
<ul>
<ul>
-
<li>Device 4: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>7</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>8</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>14</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>17</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678058">BBa_K678058</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + pAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>) + T1 (<a href="http://partsregistry.org/Part:BBa_K678037">aBBa_K678037</a>) + argB (<a href="http://partsregistry.org/Part:BBa_K678043">BBa_K678043</a>)</li>
-
<li>Device 21: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>7</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>8</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>14</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>40</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678059">BBa_K678059</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + PAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + LacZ (<a href="http://partsregistry.org/Part:BBa_K678026">BBa_K678026</a>) + T1 (<a href="http://partsregistry.org/Part:BBa_K678037">BBa_K678037</a>) + ptrA (<a href="http://partsregistry.org/Part:BBa_K678045">BBa_K678045</a></li>
-
<li>Device 22: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>26</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>24</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>10</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a></b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + GFP_module (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
<li>Device 23: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>26</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>35</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>10</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678061">BBa_K678061</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + GFP_PTS1_module_fun (<a href="http://partsregistry.org/Part:BBa_K678033">BBa_K678033</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
<li>Device 24: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>26</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>21</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>10</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678062">BBa_K678062</a></b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + RFP_module (<a href="http://partsregistry.org/Part:BBa_K678030">BBa_K678030</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
<li>Device 25: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>26</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>37</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>10</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678063">BBa_K678063</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + RFP_NLS_module (<a href="http://partsregistry.org/Part:BBa_K678034">BBa_K678034</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
<li>Device 26: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>26</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>34</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>25</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>10</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678064">BBa_K678064</a></b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + gpdA (<a href="http://partsregistry.org/Part:BBa_K6780242">BBa_K6780242</a>) + MTS (<a href="http://partsregistry.org/Part:BBa_K678025">BBa_K678025</a>) + GFP_TS (<a href="http://partsregistry.org/Part:BBa_K678029">BBa_K678029</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
<li>Device 28: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>38</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>23</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>22</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>15</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>29</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678066">BBa_K678066</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + DMKP_P6 (<a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a>) + GFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678028">BBa_K678028</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + T2 (<a href="http://partsregistry.org/Part:BBa_K678038">BBa_K678038</a>) + bleR (<a href="http://partsregistry.org/Part:BBa_K678044">BBa_K678044</a>)</li>
-
<li>Device 29: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>7</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>20</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>22</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>16</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>11</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678067">BBa_K678067</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + pAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + RFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678031">BBa_K678031</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + T3 (<a href="http://partsregistry.org/Part:BBa_K678039">BBa_K678039</a>) + pyrG_DR (<a href="http://partsregistry.org/Part:BBa_K678041">BBa_K678041</a>)</li>
-
<li>Device 31: plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C2</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C7</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C6</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C1</b>)</li>
+
<li>Device 31(Copenhagen): <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + 1A2 (<b>C7</b>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp_cas (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
-
<li>Device 32: plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C2</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C8</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C6</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C1</b>))</li>
+
<li>Device 32 (Copenhagen): <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + 2C9 (<b>C8</b>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp_cas (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
-
<li>Device 7: plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C2</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C5</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C6</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C1</b>)</li>
+
<li>Device 7 (Copenhagen):<br> plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + CYP79A2 (<a href="http://partsregistry.org/Part:BBa_K527001">BBa_K527001</a>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp_cas (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
-
<li>Device 8: plasmid_mam&nbsp;(<b>30</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C2</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C4</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C6</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C1</b>)</li>
+
<li>Device 8 (Copenhagen): <br>plasmid_mam (<a href="http://partsregistry.org/Part:BBa_K678023">BBa_K678023</a>) + pIPTG (<a href="http://partsregistry.org/Part:BBa_R0010">BBa_R0010</a>) + CYP79B2 (<a href="http://partsregistry.org/Part:BBa_K527002">BBa_K527002</a>) + Terminator (<a href="http://partsregistry.org/Part:BBa_B0015">BBa_B0015</a>) + amp_cas (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
</ul>
</ul>
<br>
<br>
<br>
<br>
-
Purifications of plasmid from transformed <i>E.coli </i> with mini-prep and restriction analysis of the plasmids.  
+
Mini-prep purifications and restriction analysis of plasmids from transformed <i>E.coli </i>.  
<a name="Thursday 18.08.2011"></a><h4>Thursday</h4>
<a name="Thursday 18.08.2011"></a><h4>Thursday</h4>
The coverslips covered with transfected U2OS cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish (very high fashion). We took them on a little journey to building 301 to get confocal pictures of them - and they looked great and green.
The coverslips covered with transfected U2OS cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish (very high fashion). We took them on a little journey to building 301 to get confocal pictures of them - and they looked great and green.
 +
<br>
 +
<br>
 +
Transformation of <i>Aspergillus nidulans</i> for promotor characterization.
<br>
<br>
<br>
<br>
<a name="Friday 19.08.2011"></a><h4>Friday</h4>
<a name="Friday 19.08.2011"></a><h4>Friday</h4>
<br>
<br>
-
<b>USER cloning done today and transformed in <i>E.coli </i> today: </b>
+
<b>USER cloning and transformation in <i>E.coli </i> today: </b>
<br>
<br>
<ul>
<ul>
-
<li>Device 28: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>38</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>23</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>22</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>15</b>&nbsp;+&nbsp;xxx&nbsp;(<b>29</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678066">BBa_K678066</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + DMKP_P6 (<a href="http://partsregistry.org/Part:BBa_K678000">BBa_K678000</a>) + GFP_GOI (<a href="http://partsregistry.org/Part:BBa_K678028">BBa_K678028</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + T2 (<a href="http://partsregistry.org/Part:BBa_K678038">BBa_K678038</a>) + bleR (<a href="http://partsregistry.org/Part:BBa_K678044">BBa_K678044</a>)</li>
-
<li>Device 22: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>26</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>24</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>10</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678060">BBa_K678060</a></b>: <br>plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + pgdA (<a href="http://partsregistry.org/Part:BBa_K678024">BBa_K678024</a>) + GFP_module (<a href="http://partsregistry.org/Part:BBa_K678027">BBa_K678027</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + pyrG (<a href="http://partsregistry.org/Part:BBa_K678040">BBa_K678040</a>)</li>
-
<li>Device 30: plasmid_fun&nbsp;(<b>28</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>7</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>27</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>22</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>13</b>)&nbsp;+&nbsp;xxx&nbsp;(<b>C1</b>)</li>
+
<li><b>Device <a href="http://partsregistry.org/Part:BBa_K678068">BBa_K678068</a></b>:<br> plasmid_fun (<a href="http://partsregistry.org/Part:BBa_K678046">BBa_K678046</a>) + PAlc (<a href="http://partsregistry.org/Part:BBa_K678001">BBa_K678001</a>) + yA (<a href="http://partsregistry.org/Part:BBa_K678035">BBa_K678035</a>) + RFP_TS (<a href="http://partsregistry.org/Part:BBa_K678032">BBa_K678032</a>) + TtrpC (<a href="http://partsregistry.org/Part:BBa_K678036">BBa_K678036</a>) + amp_cas (<a href="http://partsregistry.org/Part:BBa_K678047">BBa_K678047</a>)</li>
<br>
<br>
<br>
<br>
Line 555: Line 576:
<br>
<br>
<a name="Monday 22.08.2011"></a><h4>Monday</h4>
<a name="Monday 22.08.2011"></a><h4>Monday</h4>
-
The U2OS cells are splitted and passed on to a new culture flask and to coverslips placed in the bottom of 2 6-well-plates.
+
The U2OS cells were splitted and passed on to a new culture flask and to coverslips placed in the bottom of 2 6-well-plates.
<br>
<br>
<br>
<br>
<a name="Tuesday 23.08.2011"></a><h4>Tuesday</h4>
<a name="Tuesday 23.08.2011"></a><h4>Tuesday</h4>
-
The U2OS cells are transfected with all the mammalian plasmides. So hopefully we will get some really cool pictures soon with cells expressing RFP, CFP, YFP, GFP, and GFP targeted to the peroxisomes.
+
The U2OS cells were transfected with all the mammalian plasmides.Hopefully, we will get some really cool pictures soon with cells expressing RFP, CFP, YFP, GFP, and GFP targeted to the peroxisomes.
<br>
<br>
<br>
<br>
-
Measurements of plasmids with our device for sequencing. The measurement were done with Qubit Fluorometer from Invitrogen.  
+
Measurements of plasmids containing our devices before sending them to DNA Technology for sequencing. The measurements were done with Qubit Fluorometer from Invitrogen.  
<br>
<br>
<br>
<br>
-
And more restriction analysis of plasmids were done.
+
More restriction analysis of plasmids were done.
<br>
<br>
<br>   
<br>   
<a name="Wednesday 24.08.2011"></a><h4>Wednesday</h4>
<a name="Wednesday 24.08.2011"></a><h4>Wednesday</h4>
-
The coverslips covered with transfected cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish. We took them to building 301 for confocal microscopy. The cells looked great - everything worked = we are done with the mammalian cells. Hip hip hurra!
+
The coverslips covered with transfected cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish. We took them to building 301 for confocal microscopy. The cells looked great -everything worked = we are done with the mammalian cells. Hip hip hurra!
 +
<br>
 +
<br>
 +
Transformation of <i>Aspergillus nidulans</i> for proof of concept.
 +
<br>
<br>
<br>
-
 
<a name="September"></a><h2><b>September</b></h2>
<a name="September"></a><h2><b>September</b></h2>
<a name="Week 9"></a><h3><b>Week 9: 29.08.2011 - 04.09.2011</b></h3>
<a name="Week 9"></a><h3><b>Week 9: 29.08.2011 - 04.09.2011</b></h3>
<a name="Tuesday 30.08.2011"></a><h4>Tuesday</h4>
<a name="Tuesday 30.08.2011"></a><h4>Tuesday</h4>
-
All plasmids and primeres were sent to sequencing.  
+
Plasmids and primers were sent to DNA Technology for sequencing.  
<br>
<br>
<br>
<br>
   
   
<a name="Wednesday 31.08.2011"></a><h4>Wednesday</h4>
<a name="Wednesday 31.08.2011"></a><h4>Wednesday</h4>
-
PCR reaction of the shipping plasmid were done. The shipping plasmid had to modified with our USER tails so we quickly could clone our 2 biobricks and one device to send.  
+
PCR reaction of the shipping plasmid were done. USER tails were applied to the shipping plasmid, so it can be easy and quick assembled with the 2 BioBricks and the device.  
<br>
<br>
<br>
<br>
Line 594: Line 618:
<br>
<br>
<br>  
<br>  
-
Due to the extensive use of biobrick plasmid_fun(<b>28</b>) and plasmid_mam(<b>30</b>) we did a PCR reaction again so it can be sent to iGEM.  
+
Due to the extensive use of biobrick plasmid_fun(<b>28</b>) and plasmid_mam(<b>30</b>) we made some more.
<a name="Week 10"></a><h3><b>Week 10: 05.09.2011 - 11.09.2011</b></h3>
<a name="Week 10"></a><h3><b>Week 10: 05.09.2011 - 11.09.2011</b></h3>
<a name="Monday 05.09.2011"></a><h4> Monday </h4>
<a name="Monday 05.09.2011"></a><h4> Monday </h4>
-
Restriction analysis of shipping plasmid, to check if the USER cloning were done correct. Shipping plasmid were sent to sequencing.   
+
Restriction analysis of shipping plasmid was made to ensure that the USER cloning were done correctly. The shipping plasmid was sent to sequencing.   
<br>
<br>
<br>
<br>
-
Inoculation of fungi for β-galactosidase and Bradford assay Wednesday and Fluorescence microscope Tuesday.   
+
Inoculation of fungi for β-galactosidase, Bradford assay and Fluorescence microscope.   
<br>
<br>
<br>
<br>
<a name="Tuesday 06.09.2011"></a><h4> Tuesday</h4>
<a name="Tuesday 06.09.2011"></a><h4> Tuesday</h4>
-
Today we looked at our fungi in fluorescence microscope, and what a beautiful sight it was! We have now prooven our system works in fungi as well as mammalian cells.  
+
Today we checked out our fungi in fluorescence microscope, a beautiful sight it was! We have now proven that our system works in fungi as well as in mammalian cells.  
<br>
<br>
<br>
<br>
Line 611: Line 635:
<a name="Wednesday 07.09.2011"></a><h4>Wednesday </h4>
<a name="Wednesday 07.09.2011"></a><h4>Wednesday </h4>
-
Today we spent all day in the lab with β-galactosidase and Bradford assay for charactisation of our two promoters.
+
A whole day was spent in the lab doing the β-galactosidase and Bradford assay for characterisation of our two promoters.
<br>
<br>
<br>
<br>
<a name="Friday 09.09.2011"></a><h4>Friday</h4>
<a name="Friday 09.09.2011"></a><h4>Friday</h4>
-
Hopefully this day will be one of the last in lab. We are measuring the DNA concentration of our biobricks so they can be sent to MIT next week.  
+
This might be one of the last iGEM days in lab. We are measuring the DNA concentration of the biobricks, so they can be sent to MIT next week.  
<br>
<br>
<br>
<br>
Line 623: Line 647:
</ul>
</ul>
</td>
</td>
-
</th>
+
</tr>
</table>
</table>
 +
</div>

Latest revision as of 16:49, 21 September 2011




Lab notes

July



Week 1: 04.07.2011 - 10.07.2011

First week! Time was spent on designing the Plug 'n' Play assembly system, figuring out what 48 biobricks to construct, and searching for appropriate DNA templates.

Week 2: 11.07.2011 - 17.07.2011

Wednesday

The first batch of primers have been ordered, so we are waiting in excitement for them to arrive, so we can start our work in the lab.

Week 3: 18.07.2011 - 24.07.2011

Tuesday

First day in the lab and 48 biobricks to go. This is exciting!
We plan to construct 48 new BioBricks. We have now established 17 of them and ordered primers for them with USER tails. We started the day with 11 PCR reactions. 6 of them were correct, and they were purified with a purification Kit (GE Healthcare). Only 42 biobricks to go!

Biobricks in the bag:


Thursday

The failed PCR reactions from tuesday were repeated. This time we used a different annealing temperature, and....IT WORKED!

New biobricks in the bag:

The fragments will be frozen and later purified.

We still have not succeed to make BioBricks pyrG and pyrG-DR (used as a marker in fungi), despite different PCR attempts with a high annealing (59°) and low (56°) temperature.

Work for iGEM Copenhagen team
We have established collaboration with the iGEM team from Copenhagen University. We will costumize a number of their BioBricks to use with our Plug 'n' Play system. This is a great way of showing how easy it is to costumize the Plug 'n' Play system for any application, and the Copenhagen team will save a lot of time. Today we transformed competent E.coli cells with some of the standard biological parts from iGEM, BBa_R0010, Bba_B0015 and BBa_J52034, and later we will make them fit the Plug 'n' Play with DNA system.

Friday

We made a PCR of a troublesome BioBrick again - but this time with a small modification. We made a PCR mix with and without MgCl2 50mM in the reactions. And...IT WORKED with MgCl2 50mM!

Biobrick obtained:

The fragments will be frozen and later purified .

Week 4: 25.07.2011 - 31.07.2011


Monday

We got a new delivery of primers from IDT today. They are to be diluted and stocked, before use. We have had some difficulty with obtaining 5 of our new biobricks; SV40+ori, SV40 pA, LacZ, pyrG and pyrG-DR. We ran a touch PCR program, where the annealing temperature ran from 55° to 65° for each sample in each cycle.

The PCR reactions were successful, and following biobricks are ready for Plug'n'Play:

Tuesday

The primers recieved yesterday will be used for amplification of many many many new biobricks today.

Out of 15 PCR reactions 5 succeeded:


Wednesday

Today we have run 8 reactions, 3 standard and 5 touch PCR reactions on some of the biobricks that we still have difficulties obtaining.

One succeeded:

August


Week 5: 01.08.2011 - 07.08.2011

Monday

New week, new energy, and lots of team spirit! Today 15 PCR reactions were conducted, including parts for iGEM Copenhagen team. 14 succeeded. The annealing temperature was increased to 63°, and it worked like magic!

New biobricks in the bag:

Wednesday

There are still 2 BioBricks that we haven't been able to obtain with PCR: pyrG and pyrG-DR.
Today we tried again with gradient PCR to find the optimal melting temperature, but no magic happened.

Thursday

22 BioBricks, which have been obtained with PCR during the last week of laboratory work, was purified today. The purification of DNA was done by using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare). The procedure was carried out according to the manufactures protocol for purification of DNA from TAE and TBE agarose gels, see protocol section for more information.

Despite several attempts we have not been able to obtain BioBrick pyrG and pyrG-DR. Therefore we made a test with well-known primers without USER-tails (sometimes these USER-tails can be tricky). This was done to verify if something was wrong with the DNA template. The gel electrophoresis did not show any bands, and thereby it revealed that the DNA template was the source of error. Problem solved!

We also started cultivation of the mammalian cell line, U2OS, which is derived from a patient with osteosarcoma (bone cancer). They were defrosted and cultivated in DMEM medium containing serum, penicillin and streptomycin. The cell line was then transferred to a 25 cm2 culture flask, see protocol section.

Week 5: 08.08.2011 - 14.08.2011


Monday

Today we made the first USER cloning, we are very excited to see if the system works the way, we want it to!
All our biobricks are divided into devices, see protocol for USER MIX.
Device 1,2,3,5 and 6 was not registrated later.
The following devices were cloned today:

PCR reactions done today:


After recieving the new DNA template we finally succeeded in getting biobricks pyrG and pyrG-DR.
The U2OS cell line was splitted and passed on to new culture flasks. They looked really lovely in the microscope, which bodes well for future confocal microscopy. They seem to thrive in the mammalian cell lab, and they are passed on every second or third day, depending on the initial cell concentration.

Tuesday

E.coli was inoculated with the plasmides done by USER cloning yesterday.

PCR done today:


Purification with GFX kit of the following biobricks:

USER cloning done today:

Wednesday

The U2OS cells was investigated in the microscope. They had a confluency of about 90%, an therefore ready to pass on to new culture flasks. This was done according to the protocol (see protocol for passing and maintenance of mammalian cells")

Friday

Today 13 PCR reaction were carried out, some parts were for characterisation of two promoters: pAlc and DMKP-P6, and the rest were parts for the Copenhagen iGEM team.





The U2OS cells were splitted and passed on to two new 75 cm2 culture flasks. Hopefully, they will be ready to pass on to cover slips on monday.

Week 7: 15.08.2011 - 21.08.2011


Monday

The U2OS cells were splitted and passed on to a new 75 cm2 culture flask and to coverslips placed in a 6-well plate. They were placed in the incubator overnight - and will be ready for transfection with plasmides tomorrow.
In preparation for characterization of two promoters with β-galactosidase assay the plasmid p68 cut with restriction enzyme.

Purification of PCR products with GFX kit.

9 PCR reactions were done today




USER cloning and transformation in E.coli today:

Tuesday

We transfected U2OS cells with plasmides containing GFP and placed them in the incubator overnight.

USER cloning and transformation in E.coli today:

Wednesday


Inoculations of E.coli transformants with USER devices cloned yesterday.

Restriction analysis of device 4, 7, 8, and 27.

USER cloning and transformation in E.coli today:

Mini-prep purifications and restriction analysis of plasmids from transformed E.coli .

Thursday

The coverslips covered with transfected U2OS cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish (very high fashion). We took them on a little journey to building 301 to get confocal pictures of them - and they looked great and green.

Transformation of Aspergillus nidulans for promotor characterization.

Friday


USER cloning and transformation in E.coli today:
  • Device BBa_K678066:
    plasmid_fun (BBa_K678046) + DMKP_P6 (BBa_K678000) + GFP_GOI (BBa_K678028) + RFP_TS (BBa_K678032) + T2 (BBa_K678038) + bleR (BBa_K678044)
  • Device BBa_K678060:
    plasmid_fun (BBa_K678046) + pgdA (BBa_K678024) + GFP_module (BBa_K678027) + TtrpC (BBa_K678036) + pyrG (BBa_K678040)
  • Device BBa_K678068:
    plasmid_fun (BBa_K678046) + PAlc (BBa_K678001) + yA (BBa_K678035) + RFP_TS (BBa_K678032) + TtrpC (BBa_K678036) + amp_cas (BBa_K678047)


    • Week 8: 22.08.2011 - 28.08.2011


    Monday

    The U2OS cells were splitted and passed on to a new culture flask and to coverslips placed in the bottom of 2 6-well-plates.

    Tuesday

    The U2OS cells were transfected with all the mammalian plasmides.Hopefully, we will get some really cool pictures soon with cells expressing RFP, CFP, YFP, GFP, and GFP targeted to the peroxisomes.

    Measurements of plasmids containing our devices before sending them to DNA Technology for sequencing. The measurements were done with Qubit Fluorometer from Invitrogen.

    More restriction analysis of plasmids were done.

    Wednesday

    The coverslips covered with transfected cells were washed and fixated. We put the coverslips upside down on a glass slide and fixated them with nailpolish. We took them to building 301 for confocal microscopy. The cells looked great -everything worked = we are done with the mammalian cells. Hip hip hurra!

    Transformation of Aspergillus nidulans for proof of concept.

    September

    Week 9: 29.08.2011 - 04.09.2011

    Tuesday

    Plasmids and primers were sent to DNA Technology for sequencing.

    Wednesday

    PCR reaction of the shipping plasmid were done. USER tails were applied to the shipping plasmid, so it can be easy and quick assembled with the 2 BioBricks and the device.



    Thursday

    USER cloning of the two promoters and device 12 with the shipping plasmid were done.

    Due to the extensive use of biobrick plasmid_fun(28) and plasmid_mam(30) we made some more.

    Week 10: 05.09.2011 - 11.09.2011

    Monday

    Restriction analysis of shipping plasmid was made to ensure that the USER cloning were done correctly. The shipping plasmid was sent to sequencing.

    Inoculation of fungi for β-galactosidase, Bradford assay and Fluorescence microscope.

    Tuesday

    Today we checked out our fungi in fluorescence microscope, a beautiful sight it was! We have now proven that our system works in fungi as well as in mammalian cells.

    Wednesday

    A whole day was spent in the lab doing the β-galactosidase and Bradford assay for characterisation of our two promoters.

    Friday

    This might be one of the last iGEM days in lab. We are measuring the DNA concentration of the biobricks, so they can be sent to MIT next week.

    October

    November

Retrieved from "http://2011.igem.org/Team:DTU-Denmark-2/Notebook/Lab_notes"