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2024-03-29T02:09:53Z
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http://2013.igem.org/Team:TU_Darmstadt/materials/Stock_solutions
Team:TU Darmstadt/materials/Stock solutions
2013-10-05T03:52:12Z
<p>Schiefie: </p>
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<!-- central main menu --><br />
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<br><br />
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<!-- Taskbar --><br />
<div id="taskbar"><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
<br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/2/2c/Darmstadt_green_Result.jpg" width="80" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<br />
<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Stock solutions</font></h2><br />
</center><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
</centre><br />
</body><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<body><br />
</centre><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
</centre><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Antibiotics<br />
</p></font><br />
<ul><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Ampicillin<br />
</p></font><br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>4 g ampicillin (100 mg/mL)</li><br />
<li>ad. 40 mL dist. water</li><br />
</ul><br />
2.sterile filtration<br><br />
3. aliquot in 1 mL stocks and store at -20°C<br><br />
4. use 1µL per 1mL medium<br><br />
</ul><br />
<br><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Chloramphenicol<br />
</p></font><br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>1g chloramphenicol</li><br />
<li>ad. 40mL ethanol</li><br />
</ul><br />
2.aliquot in 1mL stocks and store at -20°C<br><br />
3. use 1µL per 1mL medium<br><br><br />
</ul><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Kanamycin<br />
</p></font><br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>3 g kanamycin (75 mg/mL) </li><br />
<li>ad. 40mL dist. water</li><br />
</ul><br />
2.sterile filtration<br><br />
3.aliquot in 1 mL stocks and store at -20°C<br><br />
3. use 1µL per 1mL medium<br><br><br />
</ul><br />
<br><br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Induction chemicals<br />
</p></font><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
IPTG (Isopropyl-beta-D-thiogalactopyranoside)<br />
</p></font> <br />
<ul><br />
1. Dissolve 238 mg IPTG in 10 mL water<br><br />
2. store in 1mL aliquots at -20°C<br />
</ul><br />
<br><br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Loading dye<br />
</p></font><br />
<a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/6x-tritrack-dna-loading-dye/"><font size="3" color="#F0F8FF" face="Arial regular"><b>tritrack loading dye</b></font></a><br />
<br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/Stock_solutions
Team:TU Darmstadt/materials/Stock solutions
2013-10-05T03:50:28Z
<p>Schiefie: </p>
<hr />
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<br />
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<br />
<br />
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{<br />
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<br />
<br />
</style><br />
<br />
<br />
<center><br />
<!-- central main menu --><br />
<br />
<br><br />
<br><br />
<br />
<br />
<!-- Taskbar --><br />
<div id="taskbar"><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
<br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/2/2c/Darmstadt_green_Result.jpg" width="80" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<br />
<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Stock solutions</font></h2><br />
</center><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
</centre><br />
</body><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<body><br />
</centre><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
</centre><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Antibiotics<br />
</p></font><br />
<ul><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Ampicillin<br />
</p></font><br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>4 g ampicillin (100 mg/mL)</li><br />
<li>ad. 40 mL dist. water</li><br />
</ul><br />
2.sterile filtration<br><br />
3. aliquot in 1 mL stocks and store at -20°C<br><br />
4. use 1µL per 1mL medium<br><br />
</ul><br />
<br><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Chloramphenicol<br />
</p></font><br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>1g chloramphenicol</li><br />
<li>ad. 40mL ethanol</li><br />
</ul><br />
2.aliquot in 1mL stocks and store at -20°C<br><br />
3. use 1µL per 1mL medium<br><br><br />
</ul><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Kanamycin<br />
</p></font><br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>3 g kanamycin (75 mg/mL) </li><br />
<li>ad. 40mL dist. water</li><br />
</ul><br />
2.sterile filtration<br><br />
3.aliquot in 1 mL stocks and store at -20°C<br><br />
3. use 1µL per 1mL medium<br><br><br />
</ul><br />
<br><br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Induction chemicals<br />
</p></font><br><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
IPTG (Isopropyl-beta-D-thiogalactopyranoside)<br />
</p></font> <br />
<ul><br />
1. Dissolve 238 mg IPTG in 10 mL water<br><br />
2. store in 1mL aliquots at -20°C<br />
</ul><br />
<br><br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Loading dye<br />
</p></font><br><br />
<a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/6x-tritrack-dna-loading-dye/"><font size="3" color="#F0F8FF" face="Arial regular"><b>tritrack loading dye</b></font></a><br />
<br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/Stock_solutions
Team:TU Darmstadt/materials/Stock solutions
2013-10-05T03:47:56Z
<p>Schiefie: </p>
<hr />
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<br />
<style type="text/css"><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
<br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/2/2c/Darmstadt_green_Result.jpg" width="80" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<br />
<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Stock solutions</font></h2><br />
</center><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
</centre><br />
</body><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<body><br />
</centre><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
</centre><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Antibiotics<br />
</p></font><br />
<ul><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Ampicillin<br />
</p></font><br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>4 g ampicillin (100 mg/mL)</li><br />
<li>ad. 40 mL dist. water</li><br />
</ul><br />
2.sterile filtration<br><br />
3. aliquot in 1 mL stocks and store at -20°C<br><br />
4. use 1µL per 1mL medium<br><br />
</ul><br />
<br><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Chloramphenicol<br />
</p></font><br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>1g chloramphenicol</li><br />
<li>ad. 40mL ethanol</li><br />
</ul><br />
2.aliquot in 1mL stocks and store at -20°C<br><br />
3. use 1µL per 1mL medium<br><br><br />
</ul><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Kanamycin<br />
</p></font><br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>3 g kanamycin (75 mg/mL) </li><br />
<li>ad. 40mL dist. water</li><br />
</ul><br />
2.sterile filtration<br><br />
3.aliquot in 1 mL stocks and store at -20°C<br><br />
3. use 1µL per 1mL medium<br><br><br />
</ul><br />
<br><br />
IPTG Isopropyl-beta-D-thiogalactopyranoside <br />
<ul><br />
1. Dissolve 238 mg IPTG in 10 mL DW<br><br />
2. store in 1mL aliquots at -20°C<br />
</ul><br />
<br><br />
<a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/6x-tritrack-dna-loading-dye/"><font size="3" color="#F0F8FF" face="Arial regular"><b>tritrack loading dye</b></font></a><br />
<br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/Stock_solutions
Team:TU Darmstadt/materials/Stock solutions
2013-10-05T03:46:31Z
<p>Schiefie: </p>
<hr />
<div><html><br />
<br />
<style type="text/css"><br />
body <br />
{<br />
margin:0; <br />
padding:15px 0 0;<br />
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background-repeat:repeat;<br />
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<br />
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<br />
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<br />
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<br />
<br />
<br />
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{<br />
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<br />
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<center><br />
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<br><br />
<br />
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<div id="taskbar"><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
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<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/2/2c/Darmstadt_green_Result.jpg" width="80" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<br />
<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Stock solutions</font></h2><br />
</center><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
</centre><br />
</body><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<body><br />
</centre><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
</centre><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Antibiotics<br />
</p></font><br />
<ul><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Ampicillin<br />
</p></font><br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>4 g ampicillin (100 mg/mL)</li><br />
<li>ad. 40 mL dist. water</li><br />
</ul><br />
2.sterile filtration<br><br />
3. aliquot in 1 mL stocks and store at -20°C<br><br />
4. use 1µL per 1mL medium<br><br />
</ul><br />
<br><br />
<font size="4" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Chloramphenicol<br />
</p></font><br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>1g chloramphenicol</li><br />
<li>ad. 40mL ethanol</li><br />
</ul><br />
2.aliquot in 1mL stocks and store at -20°C<br><br />
3. use 1µL per 1mL medium<br><br><br />
</ul><br />
<li>Kanamycin</li> <br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>3 g kanamycin (75 mg/mL) </li><br />
<li>ad. 40mL dist. water</li><br />
</ul><br />
2.sterile filtration<br><br />
3.aliquot in 1 mL stocks and store at -20°C<br />
</ul><br />
<br><br />
IPTG Isopropyl-beta-D-thiogalactopyranoside <br />
<ul><br />
1. Dissolve 238 mg IPTG in 10 mL DW<br><br />
2. store in 1mL aliquots at -20°C<br />
</ul><br />
<br><br />
<a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/6x-tritrack-dna-loading-dye/"><font size="3" color="#F0F8FF" face="Arial regular"><b>tritrack loading dye</b></font></a><br />
<br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/Stock_solutions
Team:TU Darmstadt/materials/Stock solutions
2013-10-05T03:43:38Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
<br />
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<br />
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<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
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<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
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<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<br />
<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Stock solutions</font></h2><br />
</center><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
</centre><br />
</body><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<body><br />
</centre><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
</centre><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Antibiotics<br />
</p></font><br><br />
<ul><br />
Ampicillin<br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>4 g ampicillin (100 mg/mL)</li><br />
<li>ad. 40 mL dist. water</li><br />
</ul><br />
2.sterile filtration<br><br />
3. aliquot in 1 mL stocks and store at -20°C<br><br />
4. use 1µL per 1mL medium<br><br />
</ul><br />
<br><br />
<li>Chloramphenicol</li> <br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>1g chloramphenicol</li><br />
<li>ad. 40mL ethanol</li><br />
</ul><br />
2.aliquot in 1mL stocks and store at -20°C<br><br />
<br><br />
</ul><br />
<li>Kanamycin</li> <br />
<ul><br />
1.Mix<br><br />
<ul><br />
<li>3 g kanamycin (75 mg/mL) </li><br />
<li>ad. 40mL dist. water</li><br />
</ul><br />
2.sterile filtration<br><br />
3.aliquot in 1 mL stocks and store at -20°C<br />
</ul><br />
<br><br />
IPTG Isopropyl-beta-D-thiogalactopyranoside <br />
<ul><br />
1. Dissolve 238 mg IPTG in 10 mL DW<br><br />
2. store in 1mL aliquots at -20°C<br />
</ul><br />
<br><br />
<a href="http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/6x-tritrack-dna-loading-dye/"><font size="3" color="#F0F8FF" face="Arial regular"><b>tritrack loading dye</b></font></a><br />
<br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/gBlocks
Team:TU Darmstadt/materials/gBlocks
2013-10-05T03:40:08Z
<p>Schiefie: </p>
<hr />
<div><html><br />
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<style type="text/css"><br />
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<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
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<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
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<br />
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<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Labbook |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<br />
<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">gBlocks</font></h2><br />
<br />
<font size="5" color="#F0F8FF" face="Arial regular"><p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
CMK<br />
</p></font><br />
<br />
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<div align="left" style="margin-left:30px; margin-right:50px"><br />
<ol><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/6/68/Darmstadt13_mat_gbl_B_01_166474000Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>B_01:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GTTGTTGAATTCGCGGCCGCTTCTAGAGGAAAGAGGGGACAAATGTCAAAAATCCGTGTCCTTAGCGTGGATGATTCTGCTTTGATGCGCCAGATCATGACTGAAATTATCAACTCACACAGTGATATGGAAATGGTGGCAACCGCACCGGATCCCCTGGTTGCGCGCGACCTTATTAAGAAATTTAACCCTGATGTCTTAACCCTTGACGTAGAAATGCCGCGTATGGATGGCCTGGACTTCCTGGAAAAACTGATGCGCCTGCGTCCCATGCCGGTGGTGATGGTCTCCTCACTGACGGGAAAGGGCTCCGAGGTGACGCTGCGTGCTCTGGAACTGGGGGCCATCGATTTCGTTACTAAACCGCAACTTGGTATCCGCGAGGGTATGCTGGCGTACAACGAAATGATTGCAGAAAAAGTACGTACGGCAGCGAAAGCGTCGTTAGCGGCGCACAAACCACTCAGCGCCCCAACAACGCTGAAAGCTGGCCCATTATT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/b/b1/Darmstadt13_mat_gbl_B_02_166486021Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>B_02:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GCTGAAAGCTGGCCCATTATTAAGTAGCGAAAAACTCATTGCAATTGGCGCGTGCACCGGGGGCACGGAAGCGATCCGTCATGTATTACAACCGCTGCCGCTCAGCTCACCGGCCCTTCTGATCACCCAACACATGCCACCGGGCTTTACTCGTAGTTTTGCGGATCGCCTGAATAAGTTATGCCAAATTGGGGTAAAAGAAGCGGAAGATGGCGAGCGTGTCCTGCCGGGCCATGCCTATATTGCGCCGGGCGATCGTCATATGGAGCTTTCACGGAGTGGCGCAAATTATCAGATTAAAATCCATGACGGTCCGGCTGTGAATCGGCATCGGCCGTCCGTGGACGTCCTGTTCCATTCCGTGGCGAAACAGGCCGGCCGCAATGCTGTCGGCGTCATTCTGACAGGCATGGGGAATGATGGCGCGGCCGGTATGCTGGCAATGCGCCAAGCTGGCGCGTGGACCCTGGCACAAAATGAGGCATCCTGCGTAGTGTTT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/1/1f/Darmstadt13_mat_gbl_B_03_166489532Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>B_03:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GGCATCCTGCGTAGTGTTTGGGATGCCTCGTGAAGCGATTAATATGGGAGGTGTATGTGAGGTGGTGGACCTTTCGCAGGTCAGTCAGCAGATGTTAGCCAAAATTAGCGCGGGACAGGCTATCCGTATTGGGGTAAGCAAAGGTGAAGAGTTAATCAAGGAGAACATGCACATGAAATTGTATATGGAAGGTACGGTGAATAATCACCATTTCAAATGCACAAGTGAAGGCGAAGGGAAGCCGTACGAAGGCACGCAGACGATGCGCATTAAGGTAGTGGAAGGCGGTCCTTTACCGTTCGCTTTTGATATCCTGGCCACCTCCTTCATGTATGGGTCCAAGACCTTTATCAACCATACCCAGGGCATTCCGGACTTTTTTAAACAGTCTTTTCCAGAAGGTTTCACTTGGGAGCGTGTAACGACCTACGAGGATGGTGGCGTGCTGACGGCAACCCAGGACACGAGCCTTCAGGATGGATGTCTTATTTACAACGT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/1/15/Darmstadt13_mat_gbl_B_04_166470013Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>B_04:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GGATGGATGTCTTATTTACAACGTGAAAATTCGCGGAGTGAACTTTCCGAGCAACGGTCCAGTTATGCAGAAAAAAACCCTGGGGTGGGAGGCCTCAACCGAAATGCTGTACCCAGCTGATGGTGGTCTGGAAGGTCGCTCGGACATGGCGCTGAAGCTGGTCGGTGGCGGCCATCTTATTTGTAACCTTAAAACGACCTACCGTAGCAAAAAGCCTGCGAAAAACCTGAAAATGCCCGGTGTCTACTACGTTGACCGGCGTCTGGAACGTATTAAGGAAGCCGACAAAGAGACATATGTGGAGCAGCACGAAGTTGCCGTTGCACGCTATTGCGATCTCCCGAGCAACCTGGGTCATAAACTGAATTAATAATACTAGTAGCGGCCGCTGCAGTACTAC</li><br />
</ol></div></font></p><br />
<br><br />
<br><br />
<font size="5" color="#F0F8FF" face="Arial regular"><p text-aligne:left style="margin-left:15px; margin-right:50px"><br />
Safety<br />
</p></font><br />
<br />
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<div align="left" style="margin-left:30px; margin-right:50px"><br />
<ol><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f3/Darmstadt13_mat_gbl_C1_166475119Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>C1:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> TTTCTGGAATTCGCGGCCGCTTCTAGAATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGGGTCTCTCTAGTTATTACTCGAGCGCTGCTGCCACCTGCAACATCTCCTTCTCTACCTGACTCCACTCCCCAAAGAATAACTCTTGAAGAACATCTGCTGCTGAAGTTGTATTTTCTTTTGAATCATATACACAACTTCTATCTCGTTGGTAAATTTGGATTCTTTCAAAGATAGCTAGTTCTTCCAATTTTCGTGTGTTATCAACTAGATGATTTACAATGAAATCATGATGTTCTTTTGGAGTTGCGCGTGCTTGATTTGGATTGATAATGTACAGTTCTTCATAACGGATAAGAGTACTTAGATACGACAATTCAGGCTTTGTCGCAATTAAGGCCAATT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/e/e4/Darmstadt13_mat_gbl_C2_166473991Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>C2:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GGCTTTGTCGCAATTAAGGCCAATTGTACTTCATATCCCTTATTTTTCAAGAGTTGTGCCGTTTTCTTTGGAACATCAATTGTTCGTAAAGTTCCCTCGATCAAAAGATTGTATCCCAAACTACTCAATTTTGTTACTAAAGACTCTACCATTTTTCCTGCAAAATCTTTGGTATATTCTACACTGTCTTTGCCATATTCTTGCTGTAGTTCTAAATAGTGTGGATGCTGAGAACGAAAACTATCACCATCTATGATAACAATATTTCCTTGAAATTCTTTCTGTTTAATACGATGAATTGTAGTCTTACCGGCACCACTTTGACCTCCAAGCAAAATCGCTATAGGTTGCTTACTGGACTTTTTTCCTCTTGTCAGTGAACGAAGATTCCTTGCTAAAGCATGTTTGAACTCACTATCAGTATAATCTTGGATTTCCATAATTTGAGACCCTCCTTCTTAAAGTTAAACAAAATTATTTCTTGAGCAACCATTATCACC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_mat_gbl_C3_166482414Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>C3:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
TATTTCTTGAGCAACCATTATCACCGCCAGAGGTAAAATAGTCAACACGCACGGTGTTACTCTAGTATATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACTCTAGTAGAGAGCGTTCACCGACAAACAACAGATAGCACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCTCTAGTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCGCCAAACGTCTCTTCAGGCCACTGACTAGCGATAACTTTCCCCACAACGGAACAACTCTCATTGCATGGGATCATTGGGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCTATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCAAGTCTGGCTATGCAGAAATCACCTGGCTCAACAGCCTGCTCAGGGTCAACGAGAATTAACATTCCGTCAGGAAAG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/6/63/Darmstadt13_mat_gbl_C4_166486022Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>C4:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
AGAATTAACATTCCGTCAGGAAAGCTCGGCTTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTCAACCTCAAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTGCTTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGCTCAGGTGAGAACATCCCTGCCTGAACATGAGAAAAAACAGGGTACTCATACTCACTTCTAAGTGACGGCTGCATACTAACCGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAGGGCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGCAAGCAATGCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAATAAAGCACCAACGCCTGACTGCCCCATCCCCATCTTGTCTGCGACAGATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTTTTCATAAATTGCTTTAAGGCGACGTGCGTCCTCAAGCTGCTCTTGTGTTAATGGTTTCTTTTTTGTGC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/2/23/Darmstadt13_mat_gbl_C5_166474049Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>C5:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GTGTTAATGGTTTCTTTTTTGTGCTCATCTAGTATTTCTCCTCTTTTCTTGACTCCGTTGTGATGACGCATTGGTACGCGGTATCGGGAGGTTCGAAAATTTCGAGCGATATCTTAAGAGGGGTGCCTTACGTAGAACCCCGTAGGTCATGCCCGAGGCCGGTCCTGGATGGCGCGGCGGATACGCTTGAGCAGGTTTTCGTCGAGAAGCGGCTTCAAAACCACGTCTTTTACGCCGGCCTCGGCGGCCCGGGTCGAGATGTTTTCGTCCGGATAGCCGGTGATCAGGATCACGGGCGTAGATCTCGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCTCCACAGGTGCGGTTGCTGGCACCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCACCATCTCCTTGCA</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/b/b3/Darmstadt13_mat_gbl_C6_166475120Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>C6:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGATCCCGGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTGAGGGTGGTGAATGTGGCTAGTTTTCAATCATTTGGGATACCAGGACAGCTGGAAGTCATCAAAAAAGCACTTGATCACGTGCGAGTCGGTGTGGTAATTACAGATCCCGCACTTGAAGATAATCCTATTGTCTACGTAAATCAAGGCTTTGTTCAAATGACCGGCTACGAGACGGAGGAAATTTTAGGAAAGAACTGTCGCTTCTTACAGGGGAAACACACAGATCCTGCTGAAGTGGACAACATCAGAACCGCTTTACAAAATAAAGAACCGGTCACCGTTCAGATCCAAAACTACAAAAAAGACGGAA</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_mat_gbl_C7_166473992Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>C7:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
TCCAAAACTACAAAAAAGACGGAACGATGTTCTGGAATGAATTAAATATTGATCCAATGGAAATAGAGGATAAAACGTATTTTGTCGGTATTCAGAATGATATCACCGAGCACCAGCAGACCCAGGCACGCCTCCAGGAACTGCAATCCGAGCTCGTCCACGTCTCCAGGCTGAGCGCCATGGGCGAAATGGCGTCCGCTCTCGCGCACGAGCTCAACCAGCCGCTGGCGGCGATCAGCAACTACATGAAGGGCTCGCGGCGGCTGCTTGCCGGCAGCAGTGATCCGAACACACCGAAGGTCGAAAGCGCCCTGGACCGCGCCGCCGAGCAGGCACTACGCGCAGGACAGATCATCCGACGACTGCGAGACTTCGTTGCCCGCGGCGAATCGGAGAAGCGGGTCGAGAGTCTCTCCAAGCTGATCGAGGAGGCAGGAGCACTCGGGCTTGCAGGAGCTCGCGAGCAGAACGTGCAGCTCCGTTTCAGTCTCGATCCAGGC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_mat_gbl_C8_166473991Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>C8:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GCTCCGCTTCAGTCTCGATCCGGGCGCCGATCTCGTTCTCGCCGACCGGGTGCAGATCCAGCAGGTCCTGGTCAACCTGTTCCGCAACGCGCTGGAAGCGATGGCTCAGTCGCAGCGACGCGAGCTCGTCGTCACCAACACCCCCGCCGCCGACGACATGATCGAGGTCGAAGTGTCCGACACCGGCAGCGGTTTCCAGGACGACGTCATTCCGAACCTGTTTCAGACTTTCTTCACCACCAAGGACACCGGCATGGGCGTGGGACTGTCCATCAGCCGCTCGATCATCGAAGCTCACGGCGGGCGCATGTGGGCCGAGAGCAACGCATCGGGCGGGGCGACCTTCCGCTTCACCCTCCCGGCAGCCGACGAGATGATAGGAGGTCTAGCATGACGACCAAGGGACATATCTACGTCATCGACGACGACGCGGCGATGCGGGATTCGCTGAATTTCCTGCTGGATTCTGCCGGCTTCGGCGTCACGCTGTTTGACGACG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f3/Darmstadt13_mat_gbl_C9_166482415Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>C9:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
TCGGCGTCACGCTGTTTGACGACGCGCAAGCCTTTCTTGACGCCCTGCCGGGACTCTCCTTCGGCTGTGTCGTCTCCGACGTGCGCATGCCGGGCCTTGACGGCATCGAGCTGTTGAAGCGGATGAAGGCGCAGCAAAGCCCCTTTCCGATCCTCATCATGACCGGTCACGGCGACGTGCCGCTCGCGGTAGAGGCGATGAAGTTAGGGGCGGTGGACTTTCTGGAAAAGCCTTTCGAGGACGACCGCCTCACCGCCATGATCGAATCGGCGATCCGCCAGGCCGAGCCGGCCGCCAAGAGCGAGGCCGTCGCGCAGGATATCGCCGCCCGCGTCGCCTCTTTGAGCCCCAGGGAGCGCCAGGTCATGGAAGGGCTGATCGCCGGCCTTTCCAACAAGCTGATCGCCCGCGAGTACGACATCAGCCCGCGCACCATCGAGGTGTATCGGGCCAACGTCATGACCAAGATGCAGGCCAACAGCCTTTCGGAGCTGGTTCGC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/d/df/Darmstadt13_mat_gbl_C10_166470014Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>C10:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
CAACAGCCTTTCGGAGCTGGTTCGCCTCGCGATGCGCGCCGGCATGCTCAACGATTGACAATTGATGTAAGTTAGCTCACTCATTAGGCACCGGGATCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGTACTAGTAGCGGCCGCTGCAGTCCGGC</li><br />
</ol></div></font></p><br />
<br><br />
<br><br />
<font size="5" color="#F0F8FF" face="Arial regular"><p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
TLO<br />
</p></font><br />
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<div align="left" style="margin-left:30px; margin-right:50px"><br />
<ol><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f2/Darmstadt13_mat_gbl_A_01_166474041Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>A_01:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GTTGTTGAATTCGCGGCCGCTTCTAGAGGAAAGAGGGGACAAATGATTAACCGTATTCGTGTCGTGACGTTGCTGGTCATGGTTCTGGGTGTTTTCGCGCTCCTCCAGCTGATCTCCGGCTCGCTTTTTTTTTCGTCGCTCCATCATTCCCAGAAATCTTTTGTGGTGAGTAATCAACTGCGGGAACAGCAGGGTGAGCTCACCAGCACCTGGGATTTAATGCTCCAGACGCGCATCAATCTGAGCCGCTCGGCCGTCCGTATGATGATGGATAGTAGCAACCAGCAGTCCAACGCCAAAGTTGAGTTGCTGGATTCAGCCCGTAAAACCCTTGCCCAGGCAGCCACCCATTACAAAAAATTCAAGTCTATGGCGCCGCTGCCCGAGATGGTAGCGACGTCGCGGAATATTGACGAGAAGTATAAAAATTATTACACCGCTCTGACTGAACTGATTGATTATTTGGACTATGGCAACACC</li><br />
<br><br />
</li><li class=list1><a href="https://static.igem.org/mediawiki/2013/c/cf/Darmstadt13_mat_gbl_A_02_166474053Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>A_02:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GTTGTTGAATTCGCGGCCGCTTCTAGAGGAAAGAGGGGACAAATGATTAACCGTATTCGTGTCGTGACGTTGCTGGTCATGGTTCTGGGTGTTTTCGCGCTCCTCCAGCTGATCTCCGGCTCGCTTTTTTTTTCGTCGCTCCATCATTCCCAGAAATCTTTTGTGGTGAGTAATCAACTGCGGGAACAGCAGGGTGAGCTCACCAGCACCTGGGATTTAATGCTCCAGACGCGCATCAATCTGAGCCGCTCGGCCGTCCGTATGATGATGGATAGTAGCAACCAGCAGTCCAACGCCAAAGTTGAGTTGCTGGATTCAGCCCGTAAAACCCTTGCCCAGGCAGCCACCCATTACAAAAAATTCAAGTCTATGGCGCCGCTGCCCGAGATGGTAGCGACGTCGCGGAATATTGACGAGAAGTATAAAAATTATTACACCGCTCTGACTGAACTGATTGATTATTTGGACTATGGCAACACC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/5/59/Darmstadt13_mat_gbl_A_03_166475118Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>A_03:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;">AGCCCTGGAAGAAACCGCAGCGTCTATGGAACAGTTAACGGCAACTGTTAAGCAGAACGCGGATAATGCGCGTCAGGCCTCTCAGCTTGCACAGAGTGCGTCTGATACGGCCCAGCACGGTGGAAAAGTCGTCGACGGAGTGGTTAAAACGATGCATGAAATTGCCGATTCGTCCAAGAAAATCGCTGATATTATTAGCGTCATCGACGGTATTGCATTTCAGACCAATATTCTGGCTCTGAACGCGGCGGTAGAAGCAGCGCGTGCGGGGGAACAGGGCCGTGGCTTTGCGGTAGTTGCCGGTGAAGTGCGTAACCTGGCGTCTCGTAGCGCCCAGGCGGCAAAAGAGATTAAGGCACTGATCGAAGACAGTGTGTCGCGGGTCGATACGGGAAGCGTGTTAGTGGAATCAGCAGGCGAAACAATGAATAATATCGTGAATGCTGTGACTCGCGTGACCGACATTATGGGCGAGATCGCTAGCGCCTCTGATGAACAG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/4/4e/Darmstadt13_mat_gbl_A_04_166474048Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>A_04:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;">CTAGCGCCTCTGATGAACAGTCCCGTGGTATTGATCAAGTGGCTCTTGCGGTTAGCGAGATGGATCGTGTTACGCAGCAGAACGCCAGCTTGGTCCAGGAATCTGCGGCAGCGGCTGCGGCCCTGGAAGAACAAGCTTCTCGTCTGACGCAAGCAGTCAGCGCCTTTCGCCTGGCAGCGAGCCCACTTACAAACAAACCGCAGACGCCGAGTCGTCCAGCATCAGAACAGCCGCCGGCCCAACCTCGCCTGCGGATCGCTGAACAGGACCCCAACTGGGAAACCTTTGGGGTGAGCAAAGGCGAAGAAAATAACATGGCTATTATCAAGGAATTTATGCGCTTTAAAGTGCGTATGGAAGGGAGTGTGAACGGTCATGAATTTGAAATTGAAGGCGAGGGTGAAGGCCGTCCTTATGAAGGGTTTCAGACCGTGAAACTGAAGGTGACCAAAGGTGGTCCCCTTCCCTTCGCCTGGGATATTCTGTCGCCGCAGTT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/8/8f/Darmstadt13_mat_gbl_A_05_166469986Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>A_05:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;">GATATTCTGTCGCCGCAGTTCACCTATGGCAGCAAGGCATATGTGAAGCATCCCGCCGATATCCCCGATTATTTGAAACTGTCGTTTCCTGAGGGCTTTAAGTGGGAACGTGTGATGAATTTCGAAGATGGCGGGGTCGTGACGGTAACTCAAGACAGTTCGCTTCAGGATGGCGAGTTTATTTATAAGGTGAAGTTGCGTGGTACCAATTTCCCAAGCGACGGTCCGGTGATGCAAAAAAAAACAATGG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/b/b8/Darmstadt13_mat_gbl_A_06_166469994Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>A_06:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
CGGTGATGCAAAAAAAAACAATGGGTATGGAAGCGTCGTCGGAACGTATGTATCCAGAAGACGGTGCACTGAAAGGCGAAGACAAATTACGTCTGAAACTGAAGGACGGTGGCCATTACACGAGCGAAGTAAAAACCACCTATAAGGCGAAAAAACCGGTGCAACTTCCAGGTGCATACATCGTTGACATTAAGTTGGATATTACGAGCCATAACGAGGATTACACCATTGTTGAGCAGTATGAGCGTGCCGAGGGGCGTCATAGCACCGGCGGCATGGACGAGCTGTACAAATGATAATACTAGTAGCGGCCGCTGCAGTACTAC</li><br />
</ol></div></font></p><br />
<br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/gBlocks
Team:TU Darmstadt/materials/gBlocks
2013-10-05T03:35:47Z
<p>Schiefie: </p>
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<div id="taskbar"><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
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<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/2/2c/Darmstadt_green_Result.jpg" width="80" height="30"></a><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Labbook |</font><br />
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</center><br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">gBlocks</font></h2><br />
<br />
<font size="5" color="#F0F8FF" face="Arial regular"><p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
CMK<br />
</p></font><br />
<br />
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<div align="left" style="margin-left:30px; margin-right:50px"><br />
<ol><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/6/68/Darmstadt13_mat_gbl_B_01_166474000Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>B_01:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GTTGTTGAATTCGCGGCCGCTTCTAGAGGAAAGAGGGGACAAATGTCAAAAATCCGTGTCCTTAGCGTGGATGATTCTGCTTTGATGCGCCAGATCATGACTGAAATTATCAACTCACACAGTGATATGGAAATGGTGGCAACCGCACCGGATCCCCTGGTTGCGCGCGACCTTATTAAGAAATTTAACCCTGATGTCTTAACCCTTGACGTAGAAATGCCGCGTATGGATGGCCTGGACTTCCTGGAAAAACTGATGCGCCTGCGTCCCATGCCGGTGGTGATGGTCTCCTCACTGACGGGAAAGGGCTCCGAGGTGACGCTGCGTGCTCTGGAACTGGGGGCCATCGATTTCGTTACTAAACCGCAACTTGGTATCCGCGAGGGTATGCTGGCGTACAACGAAATGATTGCAGAAAAAGTACGTACGGCAGCGAAAGCGTCGTTAGCGGCGCACAAACCACTCAGCGCCCCAACAACGCTGAAAGCTGGCCCATTATT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/b/b1/Darmstadt13_mat_gbl_B_02_166486021Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>B_02:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GCTGAAAGCTGGCCCATTATTAAGTAGCGAAAAACTCATTGCAATTGGCGCGTGCACCGGGGGCACGGAAGCGATCCGTCATGTATTACAACCGCTGCCGCTCAGCTCACCGGCCCTTCTGATCACCCAACACATGCCACCGGGCTTTACTCGTAGTTTTGCGGATCGCCTGAATAAGTTATGCCAAATTGGGGTAAAAGAAGCGGAAGATGGCGAGCGTGTCCTGCCGGGCCATGCCTATATTGCGCCGGGCGATCGTCATATGGAGCTTTCACGGAGTGGCGCAAATTATCAGATTAAAATCCATGACGGTCCGGCTGTGAATCGGCATCGGCCGTCCGTGGACGTCCTGTTCCATTCCGTGGCGAAACAGGCCGGCCGCAATGCTGTCGGCGTCATTCTGACAGGCATGGGGAATGATGGCGCGGCCGGTATGCTGGCAATGCGCCAAGCTGGCGCGTGGACCCTGGCACAAAATGAGGCATCCTGCGTAGTGTTT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/1/1f/Darmstadt13_mat_gbl_B_03_166489532Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>B_03:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GGCATCCTGCGTAGTGTTTGGGATGCCTCGTGAAGCGATTAATATGGGAGGTGTATGTGAGGTGGTGGACCTTTCGCAGGTCAGTCAGCAGATGTTAGCCAAAATTAGCGCGGGACAGGCTATCCGTATTGGGGTAAGCAAAGGTGAAGAGTTAATCAAGGAGAACATGCACATGAAATTGTATATGGAAGGTACGGTGAATAATCACCATTTCAAATGCACAAGTGAAGGCGAAGGGAAGCCGTACGAAGGCACGCAGACGATGCGCATTAAGGTAGTGGAAGGCGGTCCTTTACCGTTCGCTTTTGATATCCTGGCCACCTCCTTCATGTATGGGTCCAAGACCTTTATCAACCATACCCAGGGCATTCCGGACTTTTTTAAACAGTCTTTTCCAGAAGGTTTCACTTGGGAGCGTGTAACGACCTACGAGGATGGTGGCGTGCTGACGGCAACCCAGGACACGAGCCTTCAGGATGGATGTCTTATTTACAACGT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/1/15/Darmstadt13_mat_gbl_B_04_166470013Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>B_04:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GGATGGATGTCTTATTTACAACGTGAAAATTCGCGGAGTGAACTTTCCGAGCAACGGTCCAGTTATGCAGAAAAAAACCCTGGGGTGGGAGGCCTCAACCGAAATGCTGTACCCAGCTGATGGTGGTCTGGAAGGTCGCTCGGACATGGCGCTGAAGCTGGTCGGTGGCGGCCATCTTATTTGTAACCTTAAAACGACCTACCGTAGCAAAAAGCCTGCGAAAAACCTGAAAATGCCCGGTGTCTACTACGTTGACCGGCGTCTGGAACGTATTAAGGAAGCCGACAAAGAGACATATGTGGAGCAGCACGAAGTTGCCGTTGCACGCTATTGCGATCTCCCGAGCAACCTGGGTCATAAACTGAATTAATAATACTAGTAGCGGCCGCTGCAGTACTAC</li><br />
</ol></div></font></p><br />
<br><br />
<br><br />
<font size="5" color="#F0F8FF" face="Arial regular"><p text-aligne:left style="margin-left:15px; margin-right:50px"><br />
Safety<br />
</p></font><br />
<br />
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<div align="left" style="margin-left:30px; margin-right:50px"><br />
<ol><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f3/Darmstadt13_mat_gbl_C1_166475119Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular"><font size="3" color="#F0F8FF" face="Arial regular"><b>C1:</b></font></a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> TTTCTGGAATTCGCGGCCGCTTCTAGAATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGGGTCTCTCTAGTTATTACTCGAGCGCTGCTGCCACCTGCAACATCTCCTTCTCTACCTGACTCCACTCCCCAAAGAATAACTCTTGAAGAACATCTGCTGCTGAAGTTGTATTTTCTTTTGAATCATATACACAACTTCTATCTCGTTGGTAAATTTGGATTCTTTCAAAGATAGCTAGTTCTTCCAATTTTCGTGTGTTATCAACTAGATGATTTACAATGAAATCATGATGTTCTTTTGGAGTTGCGCGTGCTTGATTTGGATTGATAATGTACAGTTCTTCATAACGGATAAGAGTACTTAGATACGACAATTCAGGCTTTGTCGCAATTAAGGCCAATT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/e/e4/Darmstadt13_mat_gbl_C2_166473991Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C2:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GGCTTTGTCGCAATTAAGGCCAATTGTACTTCATATCCCTTATTTTTCAAGAGTTGTGCCGTTTTCTTTGGAACATCAATTGTTCGTAAAGTTCCCTCGATCAAAAGATTGTATCCCAAACTACTCAATTTTGTTACTAAAGACTCTACCATTTTTCCTGCAAAATCTTTGGTATATTCTACACTGTCTTTGCCATATTCTTGCTGTAGTTCTAAATAGTGTGGATGCTGAGAACGAAAACTATCACCATCTATGATAACAATATTTCCTTGAAATTCTTTCTGTTTAATACGATGAATTGTAGTCTTACCGGCACCACTTTGACCTCCAAGCAAAATCGCTATAGGTTGCTTACTGGACTTTTTTCCTCTTGTCAGTGAACGAAGATTCCTTGCTAAAGCATGTTTGAACTCACTATCAGTATAATCTTGGATTTCCATAATTTGAGACCCTCCTTCTTAAAGTTAAACAAAATTATTTCTTGAGCAACCATTATCACC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_mat_gbl_C3_166482414Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C3:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
TATTTCTTGAGCAACCATTATCACCGCCAGAGGTAAAATAGTCAACACGCACGGTGTTACTCTAGTATATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACTCTAGTAGAGAGCGTTCACCGACAAACAACAGATAGCACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCTCTAGTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCGCCAAACGTCTCTTCAGGCCACTGACTAGCGATAACTTTCCCCACAACGGAACAACTCTCATTGCATGGGATCATTGGGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCTATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCAAGTCTGGCTATGCAGAAATCACCTGGCTCAACAGCCTGCTCAGGGTCAACGAGAATTAACATTCCGTCAGGAAAG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/6/63/Darmstadt13_mat_gbl_C4_166486022Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C4:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
AGAATTAACATTCCGTCAGGAAAGCTCGGCTTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTCAACCTCAAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTGCTTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGCTCAGGTGAGAACATCCCTGCCTGAACATGAGAAAAAACAGGGTACTCATACTCACTTCTAAGTGACGGCTGCATACTAACCGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAGGGCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGCAAGCAATGCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAATAAAGCACCAACGCCTGACTGCCCCATCCCCATCTTGTCTGCGACAGATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTTTTCATAAATTGCTTTAAGGCGACGTGCGTCCTCAAGCTGCTCTTGTGTTAATGGTTTCTTTTTTGTGC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/2/23/Darmstadt13_mat_gbl_C5_166474049Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C5:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GTGTTAATGGTTTCTTTTTTGTGCTCATCTAGTATTTCTCCTCTTTTCTTGACTCCGTTGTGATGACGCATTGGTACGCGGTATCGGGAGGTTCGAAAATTTCGAGCGATATCTTAAGAGGGGTGCCTTACGTAGAACCCCGTAGGTCATGCCCGAGGCCGGTCCTGGATGGCGCGGCGGATACGCTTGAGCAGGTTTTCGTCGAGAAGCGGCTTCAAAACCACGTCTTTTACGCCGGCCTCGGCGGCCCGGGTCGAGATGTTTTCGTCCGGATAGCCGGTGATCAGGATCACGGGCGTAGATCTCGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCTCCACAGGTGCGGTTGCTGGCACCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCACCATCTCCTTGCA</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/b/b3/Darmstadt13_mat_gbl_C6_166475120Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C6:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGATCCCGGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTGAGGGTGGTGAATGTGGCTAGTTTTCAATCATTTGGGATACCAGGACAGCTGGAAGTCATCAAAAAAGCACTTGATCACGTGCGAGTCGGTGTGGTAATTACAGATCCCGCACTTGAAGATAATCCTATTGTCTACGTAAATCAAGGCTTTGTTCAAATGACCGGCTACGAGACGGAGGAAATTTTAGGAAAGAACTGTCGCTTCTTACAGGGGAAACACACAGATCCTGCTGAAGTGGACAACATCAGAACCGCTTTACAAAATAAAGAACCGGTCACCGTTCAGATCCAAAACTACAAAAAAGACGGAA</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_mat_gbl_C7_166473992Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C7:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
TCCAAAACTACAAAAAAGACGGAACGATGTTCTGGAATGAATTAAATATTGATCCAATGGAAATAGAGGATAAAACGTATTTTGTCGGTATTCAGAATGATATCACCGAGCACCAGCAGACCCAGGCACGCCTCCAGGAACTGCAATCCGAGCTCGTCCACGTCTCCAGGCTGAGCGCCATGGGCGAAATGGCGTCCGCTCTCGCGCACGAGCTCAACCAGCCGCTGGCGGCGATCAGCAACTACATGAAGGGCTCGCGGCGGCTGCTTGCCGGCAGCAGTGATCCGAACACACCGAAGGTCGAAAGCGCCCTGGACCGCGCCGCCGAGCAGGCACTACGCGCAGGACAGATCATCCGACGACTGCGAGACTTCGTTGCCCGCGGCGAATCGGAGAAGCGGGTCGAGAGTCTCTCCAAGCTGATCGAGGAGGCAGGAGCACTCGGGCTTGCAGGAGCTCGCGAGCAGAACGTGCAGCTCCGTTTCAGTCTCGATCCAGGC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_mat_gbl_C8_166473991Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C8:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GCTCCGCTTCAGTCTCGATCCGGGCGCCGATCTCGTTCTCGCCGACCGGGTGCAGATCCAGCAGGTCCTGGTCAACCTGTTCCGCAACGCGCTGGAAGCGATGGCTCAGTCGCAGCGACGCGAGCTCGTCGTCACCAACACCCCCGCCGCCGACGACATGATCGAGGTCGAAGTGTCCGACACCGGCAGCGGTTTCCAGGACGACGTCATTCCGAACCTGTTTCAGACTTTCTTCACCACCAAGGACACCGGCATGGGCGTGGGACTGTCCATCAGCCGCTCGATCATCGAAGCTCACGGCGGGCGCATGTGGGCCGAGAGCAACGCATCGGGCGGGGCGACCTTCCGCTTCACCCTCCCGGCAGCCGACGAGATGATAGGAGGTCTAGCATGACGACCAAGGGACATATCTACGTCATCGACGACGACGCGGCGATGCGGGATTCGCTGAATTTCCTGCTGGATTCTGCCGGCTTCGGCGTCACGCTGTTTGACGACG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f3/Darmstadt13_mat_gbl_C9_166482415Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C9:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
TCGGCGTCACGCTGTTTGACGACGCGCAAGCCTTTCTTGACGCCCTGCCGGGACTCTCCTTCGGCTGTGTCGTCTCCGACGTGCGCATGCCGGGCCTTGACGGCATCGAGCTGTTGAAGCGGATGAAGGCGCAGCAAAGCCCCTTTCCGATCCTCATCATGACCGGTCACGGCGACGTGCCGCTCGCGGTAGAGGCGATGAAGTTAGGGGCGGTGGACTTTCTGGAAAAGCCTTTCGAGGACGACCGCCTCACCGCCATGATCGAATCGGCGATCCGCCAGGCCGAGCCGGCCGCCAAGAGCGAGGCCGTCGCGCAGGATATCGCCGCCCGCGTCGCCTCTTTGAGCCCCAGGGAGCGCCAGGTCATGGAAGGGCTGATCGCCGGCCTTTCCAACAAGCTGATCGCCCGCGAGTACGACATCAGCCCGCGCACCATCGAGGTGTATCGGGCCAACGTCATGACCAAGATGCAGGCCAACAGCCTTTCGGAGCTGGTTCGC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/d/df/Darmstadt13_mat_gbl_C10_166470014Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C10:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
CAACAGCCTTTCGGAGCTGGTTCGCCTCGCGATGCGCGCCGGCATGCTCAACGATTGACAATTGATGTAAGTTAGCTCACTCATTAGGCACCGGGATCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGTACTAGTAGCGGCCGCTGCAGTCCGGC</li><br />
</ol></div></font></p><br />
<br><br />
<br><br />
<font size="5" color="#F0F8FF" face="Arial regular"><p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
TLO<br />
</p></font><br />
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<div align="left" style="margin-left:30px; margin-right:50px"><br />
<ol><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f2/Darmstadt13_mat_gbl_A_01_166474041Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_01:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GTTGTTGAATTCGCGGCCGCTTCTAGAGGAAAGAGGGGACAAATGATTAACCGTATTCGTGTCGTGACGTTGCTGGTCATGGTTCTGGGTGTTTTCGCGCTCCTCCAGCTGATCTCCGGCTCGCTTTTTTTTTCGTCGCTCCATCATTCCCAGAAATCTTTTGTGGTGAGTAATCAACTGCGGGAACAGCAGGGTGAGCTCACCAGCACCTGGGATTTAATGCTCCAGACGCGCATCAATCTGAGCCGCTCGGCCGTCCGTATGATGATGGATAGTAGCAACCAGCAGTCCAACGCCAAAGTTGAGTTGCTGGATTCAGCCCGTAAAACCCTTGCCCAGGCAGCCACCCATTACAAAAAATTCAAGTCTATGGCGCCGCTGCCCGAGATGGTAGCGACGTCGCGGAATATTGACGAGAAGTATAAAAATTATTACACCGCTCTGACTGAACTGATTGATTATTTGGACTATGGCAACACC</li><br />
<br><br />
</li><li class=list1><a href="https://static.igem.org/mediawiki/2013/c/cf/Darmstadt13_mat_gbl_A_02_166474053Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_02:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GTTGTTGAATTCGCGGCCGCTTCTAGAGGAAAGAGGGGACAAATGATTAACCGTATTCGTGTCGTGACGTTGCTGGTCATGGTTCTGGGTGTTTTCGCGCTCCTCCAGCTGATCTCCGGCTCGCTTTTTTTTTCGTCGCTCCATCATTCCCAGAAATCTTTTGTGGTGAGTAATCAACTGCGGGAACAGCAGGGTGAGCTCACCAGCACCTGGGATTTAATGCTCCAGACGCGCATCAATCTGAGCCGCTCGGCCGTCCGTATGATGATGGATAGTAGCAACCAGCAGTCCAACGCCAAAGTTGAGTTGCTGGATTCAGCCCGTAAAACCCTTGCCCAGGCAGCCACCCATTACAAAAAATTCAAGTCTATGGCGCCGCTGCCCGAGATGGTAGCGACGTCGCGGAATATTGACGAGAAGTATAAAAATTATTACACCGCTCTGACTGAACTGATTGATTATTTGGACTATGGCAACACC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/5/59/Darmstadt13_mat_gbl_A_03_166475118Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_03:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;">AGCCCTGGAAGAAACCGCAGCGTCTATGGAACAGTTAACGGCAACTGTTAAGCAGAACGCGGATAATGCGCGTCAGGCCTCTCAGCTTGCACAGAGTGCGTCTGATACGGCCCAGCACGGTGGAAAAGTCGTCGACGGAGTGGTTAAAACGATGCATGAAATTGCCGATTCGTCCAAGAAAATCGCTGATATTATTAGCGTCATCGACGGTATTGCATTTCAGACCAATATTCTGGCTCTGAACGCGGCGGTAGAAGCAGCGCGTGCGGGGGAACAGGGCCGTGGCTTTGCGGTAGTTGCCGGTGAAGTGCGTAACCTGGCGTCTCGTAGCGCCCAGGCGGCAAAAGAGATTAAGGCACTGATCGAAGACAGTGTGTCGCGGGTCGATACGGGAAGCGTGTTAGTGGAATCAGCAGGCGAAACAATGAATAATATCGTGAATGCTGTGACTCGCGTGACCGACATTATGGGCGAGATCGCTAGCGCCTCTGATGAACAG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/4/4e/Darmstadt13_mat_gbl_A_04_166474048Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_04:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;">CTAGCGCCTCTGATGAACAGTCCCGTGGTATTGATCAAGTGGCTCTTGCGGTTAGCGAGATGGATCGTGTTACGCAGCAGAACGCCAGCTTGGTCCAGGAATCTGCGGCAGCGGCTGCGGCCCTGGAAGAACAAGCTTCTCGTCTGACGCAAGCAGTCAGCGCCTTTCGCCTGGCAGCGAGCCCACTTACAAACAAACCGCAGACGCCGAGTCGTCCAGCATCAGAACAGCCGCCGGCCCAACCTCGCCTGCGGATCGCTGAACAGGACCCCAACTGGGAAACCTTTGGGGTGAGCAAAGGCGAAGAAAATAACATGGCTATTATCAAGGAATTTATGCGCTTTAAAGTGCGTATGGAAGGGAGTGTGAACGGTCATGAATTTGAAATTGAAGGCGAGGGTGAAGGCCGTCCTTATGAAGGGTTTCAGACCGTGAAACTGAAGGTGACCAAAGGTGGTCCCCTTCCCTTCGCCTGGGATATTCTGTCGCCGCAGTT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/8/8f/Darmstadt13_mat_gbl_A_05_166469986Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_05:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;">GATATTCTGTCGCCGCAGTTCACCTATGGCAGCAAGGCATATGTGAAGCATCCCGCCGATATCCCCGATTATTTGAAACTGTCGTTTCCTGAGGGCTTTAAGTGGGAACGTGTGATGAATTTCGAAGATGGCGGGGTCGTGACGGTAACTCAAGACAGTTCGCTTCAGGATGGCGAGTTTATTTATAAGGTGAAGTTGCGTGGTACCAATTTCCCAAGCGACGGTCCGGTGATGCAAAAAAAAACAATGG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/b/b8/Darmstadt13_mat_gbl_A_06_166469994Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_06:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
CGGTGATGCAAAAAAAAACAATGGGTATGGAAGCGTCGTCGGAACGTATGTATCCAGAAGACGGTGCACTGAAAGGCGAAGACAAATTACGTCTGAAACTGAAGGACGGTGGCCATTACACGAGCGAAGTAAAAACCACCTATAAGGCGAAAAAACCGGTGCAACTTCCAGGTGCATACATCGTTGACATTAAGTTGGATATTACGAGCCATAACGAGGATTACACCATTGTTGAGCAGTATGAGCGTGCCGAGGGGCGTCATAGCACCGGCGGCATGGACGAGCTGTACAAATGATAATACTAGTAGCGGCCGCTGCAGTACTAC</li><br />
</ol></div></font></p><br />
<br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials
Team:TU Darmstadt/materials
2013-10-05T03:30:56Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
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<br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Materials</font></h2><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
This page contains detailed instructions on the preparation of all employed materials including suppliers and working conditions. <br />
<br />
<br />
<ul style="margin-left:50px; margin-right:50px; text-align:justify; "><br />
<li> <a href="https://2013.igem.org/Team:TU_Darmstadt/materials/Strains "><font size="3" color="#F0F8FF" face="Arial regular"><b> Strains </b></font></a></li><br />
<li> <a href=" https://2013.igem.org/Team:TU_Darmstadt/materials/Media "><font size="3" color="#F0F8FF" face="Arial regular"><b> Media </b></font></a></li><br />
<li> <a href=" https://2013.igem.org/Team:TU_Darmstadt/materials/Enzymes "><font size="3" color="#F0F8FF" face="Arial regular"><b> Enzymes </b></font></a></li><br />
<li> <a href=" https://2013.igem.org/Team:TU_Darmstadt/materials/Buffers "><font size="3" color="#F0F8FF" face="Arial regular"><b> Buffers </b></font></a></li><br />
<li> <a href=" https://2013.igem.org/Team:TU_Darmstadt/materials/Stock_solutions "><font size="3" color="#F0F8FF" face="Arial regular"><b> Stock solutions </b></font></a></li><br />
<li> <a href=" https://2013.igem.org/Team:TU_Darmstadt/materials/Primers "><font size="3" color="#F0F8FF" face="Arial regular"><b> Primers </b></font></a></li><br />
<li> <a href=" https://2013.igem.org/Team:TU_Darmstadt/materials/gBlocks "><font size="3" color="#F0F8FF" face="Arial regular"><b> gBlocks </b></font></a></li><br />
<li> <a href=" https://2013.igem.org/Team:TU_Darmstadt/materials/Plasmids "><font size="3" color="#F0F8FF" face="Arial regular"><b> Plasmids </b></font></a></li><br />
<li> <a href=" https://2013.igem.org/Team:TU_Darmstadt/materials/Equipment "><font size="3" color="#F0F8FF" face="Arial regular"><b> Equipment </b></font></a></li><br />
</u><br />
<br />
</front><br />
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</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/Buffers
Team:TU Darmstadt/materials/Buffers
2013-10-05T03:28:30Z
<p>Schiefie: </p>
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<br><br />
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<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<br />
<br />
<br />
<body><br />
</center><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Buffers</font></h2><br />
<br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<br />
</body><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
Tris (1M, pH 7,5)<br />
</p></font><br />
<ul><br />
<li>60,5g Tris base</li><br />
<li>adjust pH to 7,5 using 5M HCL</li><br />
<li>add to 500 mL><br />
<br><br />
</ul><br />
<br><br />
<body><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
1x PBS<br />
</p></font><br />
<ul><br />
<li>8.18 g NaCl (140 mM)</li><br />
<li>0.2 g KCl (2.7 mM)</li><br />
<li>1.77 g Na<sub>2</sub>HPO<sub>4</sub> (10 mM)</li><br />
<li>0.24 g KH<sub>2</sub>PO<sub>4</sub> (1.8 mM)</li><br />
<li>Add. 1L dH<sub>2</sub>O</li><br />
<li>Adjust to pH 7.4</li><br />
</ul><br />
<br><br />
<body><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
TE buffer<br />
</p></font><br />
<ul><br />
<li>10 mM Tris, bring to pH 8.0 with HCl</li><br />
<li>1 mM EDTA (Ethylenediaminetetraacetic acid)</li><br />
</body><br />
</ul><br />
<body><br />
<br><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
<font size="5" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
50x TAE<br />
</p></font><br />
<ul><br />
<li> 242g Tris base in water</li><br />
<li> add 57.1mL glacial acetic acid</li><br />
<li>100mL of 500mM EDTA (pH 8.0) solution</li><br />
<li>add up to 1 litre of water</li><br />
</body><br />
</ul><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook/Biobricks
Team:TU Darmstadt/labbook/Biobricks
2013-10-05T03:16:34Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
<br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/2/2c/Darmstadt_green_Result.jpg" width="80" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
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<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
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<br />
<br />
</div><br />
<br />
<br />
<br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br />
<br><br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">BioBricks mKate and LssmOrange</font></h2> <br><br />
<br />
<html><br />
<head><br />
<title></title><br />
<br />
<br />
</head><br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<br />
<br />
<table border="1" rules="rows" style="margin-left:50px; margin-right:50px"><br />
<tr><br />
<td>01.08</td><br />
<td>gBLOCK assembly of CMK and TLO</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>PCR mixture <ul><br />
<li>1 µL of each gBLOCK<ul><br />
<li>CMK: B_01 - B_04</li><br />
<li>TLO: A_01 - A_06</li><br />
</ul></li><br />
<li>10 µL 5x Q5 Reaction Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>1 µL Q5 Hot Start Polymerase</li><br />
<li>10 µL 5x Q5 High GC Enhancer</li><br />
<li>1 µL primer suffix-R (10 mM)</li><br />
<li>1 µL primer prefix_R (10 mM)</li><br />
</ul></li> <br><br />
<li>PCR program (40 cycles)<ul><br />
<li>initial denaturation 94°C, 100s</li><br />
<li>denaturation 94°C, 55s </li><br />
<li>annealing 64°C, 55s</li><br />
<li>elongation 72°C, 120s </li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays bands of expected size ' assembly was successful</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>01.08</td><br />
<td>Purification</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 7,7 ng/µL</li><br />
<li>CMK c = 7,1 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>02.08</td><br />
<td>Isolation of LssmOrange and mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<br />
<td>&nbsp;</td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume) <ul><br />
<li>1.5 µl DMSO </li><br />
<li>1 µL dNTPs</li><br />
<li>4 µL Pfu Polymerase</li><br />
<li>10 µL 5x Pfu buffer Buffer with MgSO4 </li><br />
<li>1 µL reverse primer </li><br />
<li>1 µL forward primer</li><br />
<li> 5 µl template</li><br />
<li>add 50 µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>LssmOrange: TLO (c = 7,7 ng/µL, 01.08) + LO-pre-F + LO-suf-R</li><br />
<li>mKate: CMK (c = 7,1 ng/µL, 01.08) + mKate-suf-R + mKate-pre-ATG-F</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 95°C, 300s</li><br />
<li>denaturation 95°C, 30s</li><br />
<li>annealing 55°C, 55s</li><br />
<li>elongation 72°C, 2 min</li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>PCR batches show the expected bands ' isolation was successful</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/f7/Darmstadt13_biobricks_IsolationPCR_LssmOrange_mKate.png" alt=""></td><br />
</tr><br />
<br />
<tr><br />
<td>02.08</td><br />
<td>Purification</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>LssmOrange1 c = 78,5 ng/µL 260/280 = 1,87 230/260 = 1,19</li><br />
<li>mKate1 c = 45,8 ng/µL 260/280 = 1,78 230/260 = 0,49</li><br />
<li>mKate2 c = 44,9 ng/µL 260/280 = 1,89 230/260 = 0,47</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>02.08</td><br />
<td> Restriction of LssmOrange and mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>double digest with PstI and EcoRI for 1 hour at 37°C</li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>03.08</td><br />
<td>Purification</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li> using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>LssmOrange1 c = 51,6 ng/µL 260/280 = 1,82 230/260 = 0,63</li><br />
<li> mKate1 c = 45,4 ng/µL 260/280 = 1,9 230/260 = 0,48</li><br />
<li>mKate2 c = 17,0 ng/µL 260/280 = 1,87 230/260 = 0,12</li><br />
<br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>03.08</td><br />
<td> Ligation of pSB1C3 with LssmOrange and pSB1C3 with mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>ligation mixture<ul><br />
<li>120 ng pSB1C3 cut with EcoRI and PstI</li><br />
<li> 124 ng insert cut with EcoRI and PstI</li><br />
<li>1 µL T4 DNA ligase (NEB)</li><br />
<li>2 µL 10x t4 DNA ligase buffer (NEB)</li><br />
<li>add to 20 µL</li><br />
</ul></li> <br><br />
<li> incubation for 30 minutes at room temperature</li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>03.08</td><br />
<td>Transformation</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>2 µL of every ligation batch were transformed into E.coli Top10 according to heat shock protocol<ul><br />
<li>only few colonies grew on LB-Cam plates</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>05.08</td><br />
<td>Colony PCR of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li> 1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl bacterial culture in LB-Cam medium </li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Program<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li> elongation 60s 72°C</li><br />
<li>final elongation 300s 72°C</li><br />
<br />
</ul></li> <br><br />
<li> analytical 1% agarose gel<ul><br />
<li>no colony yielded a band of approximately 800 bp, which would account for a positive result</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>09.08</td><br />
<td>Ligation of pSB1C3 with LssmOrange and pSB1C3 with mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>ligation mixture <ul><br />
<li>120 ng pSB1C3 cut with EcoRI and PstI</li><br />
<li> 124 ng insert cut with EcoRI and PstI</li><br />
<li>1 µL T4 DNA ligase (NEB)</li><br />
<li>2 µL 10x t4 DNA ligase buffer (NEB)</li><br />
<li>add to 20 µL</li><br />
</ul></li><br />
<li>incubation for 2 hours at 37°C and additionally incubation over 24 hours at 8°C </li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>10.08</td><br />
<td>Transformation</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>2 µL of every ligation batch were transformed into E.coli Top10 according to heat shock protocol<ul><br />
<li>a lot of colonies grew on LB-Cam plates</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>12.08</td><br />
<td>Colony PCR of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume)<ul><br />
<li>1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl bacterial culture in LB-Cam medium</li><br />
<li>2µl MgCl2 </li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Program<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li> annealing 30s 55°C</li><br />
<li>elongation 60s 72°C</li><br />
<li>final elongation 300s 72°C </li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>a lot clones gave a positive result</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>13.08</td><br />
<td>Sequencing of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>results show that all analyzed clones contain the expected sequence</li><br />
<li>one pSB1C3[LssmOrange] clone and one pSB1C3[mKate] clone were chosen </li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>26.08</td><br />
<td>Plasmid prep of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using PureYield Plasmid Miniprep System (Promega)<ul><br />
<li>pSB1C3[LssmOrange] c = 96,4 ng/µL 260/280 = 1,68 230/260 = 1,46</li><br />
<li>pSB1C3[mKate] c = 74,8 ng/µL 260/280 = 1,86 230/260 = 1,82</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>13.09</td><br />
<td>Inoculation of DH5? pPR-IBA2 and subsequent plasmid prep</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>6 mL LB-Amp were inoculated with 50 µL of DH5? pPR-IBA2 <ul><br />
<li>using PureYield Plasmid Miniprep System (Promega)</li><br />
<li>pPR-IBA2 c = 71,6 ng/µL 260/280 = 1,82 260/230 = 1,76</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>13.09</td><br />
<td>Isolation PCR of LssmOrange and mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li> 5x Q5 Reaction Buffer</li><br />
<li>5x Q5 GC Enhancer</li><br />
<li>1 µL dNTPs (10mM)</li><br />
<li> 2,5 µL forward primer (10 mM)</li><br />
<li>2,5 µL reverse primer (10 mM)</li><br />
<li>1 µL template</li><br />
<li>0,5 µL Q5 Polymerase </li><br />
<li>22,5 µL nucleasefree water </li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>LssmOrange: pSB1C3[LssmOrange ] (c = 96,4 ng/µL, 1:100 dilution) + lssmorange pprf + lssmorange ppr</li><br />
<li>mKate: pSB1C3[mKate] (c = 74,8 ng/µL, 1:80 dilution) + mkate pprf + mkate ppr</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles) <ul><br />
<li>initial denaturation, 60 s, 98°C</li><br />
<li>denaturation, 10 s, 98°C</li><br />
<li>annealing, 30 s, 65°C</li><br />
<li>elongation, 60 s, 72°C</li><br />
<li>final elongation, 120 s, 72°C </li><br />
<br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>each PCR batch displays the expected band of approximately 700 bp</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>13.09</td><br />
<td>Purification</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>LssmOrange1 c = 51,4 ng/µL 260/280 = 1,69 230/260 = 1,59</li><br />
<li>LssmOrange1 c = 51,6 ng/µL 260/280 = 1,66 230/260 = 1,55</li><br />
<li>mKate1 c = 38,4 ng/µL 260/280 = 1,64 230/260 = 1,43</li><br />
<li>mKate2 c = 37,8 ng/µL 260/280 = 1,59 230/260 = 1,42</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>13.09</td><br />
<td>Restriction of pPR-IBA2, LssmOrange and mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>double digest with NheI and PstI at 37°C for 4 hours</li> <br><br />
<li>preparative 1% agarose gel <ul><br />
<li>LssmOrange and mKate show a band of the expected size </li><br />
<li>pPR-IBA2 shows intense band at 3000bp, weak band at 2250bp and weak band below 100bp<ul><br />
<li>we were unsure which band the correct one is, and cut out the 3000band as well as 2250band</li><br />
</ul></li><br />
<br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>14.09</td><br />
<td>Purification of pPR-IBA2, LssmOrange and mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pPR-IBA2 (3000band) c = 31,2 ng/µL 260/280 = 1,88 230/260 = 1,00</li><br />
<li>pPR-IBA2 (2250band) c = 9,5 ng/µL 260/280 = 1,39 230/260 = 0,38 (neglected due to low purity)</li><br />
<li>LssmOrange c = 57,3 ng/µL 260/280 = 1,74 230/260 = 1,53</li><br />
<li>mKate c = 22,0 ng/µL 260/280 = 2,04 230/260 = 1,16</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>14.09</td><br />
<td>Ligation</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>ligation batch of pPR-IBA2[LssmOrange]<ul><br />
<li>3 µL pPR-IBA2 3000 (93 ng) </li><br />
<li>1,5 µL LssmOrange (72 ng)</li><br />
<li>2 µL 10x T4 Ligase Buffer (NEB)</li><br />
<li>1 µL T4 Ligase (NEB) </li><br />
<li>12,5 µL nucleasefree water</li><br />
</ul></li> <br><br />
<li>ligation batch of pPR-IBA2[mKate]<ul><br />
<li>3 µL pPR-IBA2 3000 (93 ng)</li><br />
<li>3,5 µL mKate (72 ng)</li><br />
<li> 2 µL 10x T4 Ligase Buffer (NEB)</li><br />
<li> 1 µL T4 Ligase (NEB)</li><br />
<li>10,5 µL nucleasefree water</li><br />
</ul></li> <br><br />
<li>incubation at room temperature for 30 minutes</li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>15.09</td><br />
<td>Transformation of pPR-IBA2[LssmOrange] and pPR-IBA2[mKate]</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>2 µL of ligation batch were transformed into E.coli DH5? according to heat shock protocol <ul><br />
<li>a lot of colonies grew on LB-Amp plates</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>16.09</td><br />
<td>Colony PCR of pPR-IBA2[LssmOrange] and pPR-IBA2[mKate]</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL forward primer</li><br />
<li>1 µL reverse primer</li><br />
<li>colony</li><br />
<li>2 µl MgCl2</li><br />
<li>1 µl dNTP Mix</li><br />
<li>1 µl DMSO</li><br />
<li>5 µl 10x Taq buffer</li><br />
<li>1 µl Taq-Polymerase </li><br />
<li>38 µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pSB1C3[LssmOrange]: lssmorange pprf + lssmorange ppr </li><br />
<li>pSB1C3[mKate]: mkate pprf + mkate ppr</li><br />
</ul></li> <br><br />
<li>PCR Program<ul><br />
<li>initial Denaturation 300s 95°C</li><br />
<li>Denaturation 30s 95°C</li><br />
<li>Annealing 30s 65°C</li><br />
<li>Elongation 60s 72°C </li><br />
<li>Final Elongation 300s 72°C </li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>2 positive clones for pPR-IBA2-LssmOrange</li><br />
<li>7 positive clones for pPR-IBA2-mKate</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/e/ef/Darmstadt13_biobricks_colonyPCR.png" alt="xxx"></td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>19.09</td><br />
<td>Plasmid prep</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using PureYield Plasmid Miniprep System (Promega)<ul><br />
<li>pPR-IBA2[LssmOrange] clone 5 c = 51,2 ng/µL</li><br />
<li>pPR-IBA2[LssmOrange] clone 10 c = 55,5 ng/µL</li><br />
<li>pPR-IBA2[mKate] clone 2 c = 32,3 ng/µL</li><br />
<li>pPR-IBA2[mKate] clone 3 c = 35,2 ng/µL</li><br />
<li>pPR-IBA2[mKate] clone 4 c = 29,1 ng/µL</li><br />
<li>pPR-IBA2[mKate] clone 8 c = 37,6 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>19.09</td><br />
<td>Transformation in BL21DE3</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>1 µL of plasmid was transformed into E.coli DH5? according to heat shock protocol<ul><br />
<li>biofilm grew on LB-Amp plates </li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>21.09</td><br />
<td>Induction with IPTG</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>pPR-IBA2[LssmOrange] and pPR-IBA2[mKate] clones were cultivated in SOC-Amp medium at 37°C and 150 rpm to an OD600 = 0,6</li><br />
<li>subsequent induction with IPTG (2 mM end concentration) and cultivation overnight at 30°C and 150 rpm<ul><br />
<li>induction worked for pPR-IBA2[LssmOrange] clone 10 and pPR-IBA2[mKate] clone 3 and clone 4</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>23.09</td><br />
<td>Spectral analysis of induced clones</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>cells and cell lysate was analyzed using an fluorospectrometer</li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>27.09</td><br />
<td>SDS-PAGE</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>Gel of Biobrick producing cells<br><br />
M.Marker NEB Color Prestaind Broadrange<br><br />
2.LssmOrange supernatant<br><br />
3.mKate cells<br><br />
4.Control (no visible band)<br><br />
</li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/c/cf/Darmstadt13_biobrick_Sdspage_29_09_13.png" width="50%" alt="xxx"></td><br />
</tr><br />
<br />
</table><br />
<br />
<br />
<br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook
Team:TU Darmstadt/labbook
2013-10-05T03:13:29Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
<br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/2/2c/Darmstadt_green_Result.jpg" width="80" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
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<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
</div><br />
<br />
<br />
<br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br />
<br />
<br><br><br />
<br />
<br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Detection construct</font></h2> <br><br />
<br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
The assembly of our detection construct proved to be tricky. Not only the traditional cloning approach failed but also the assembly using synthesized gBlocks delivered no results. We finally turned to an In-Fusion cloning approach to assemble our construct. Visit our lab book to read more about our work flow.<br />
<br />
</p></font><br />
<br />
<br><br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct"><br />
<img alt="Labbookdetection" border="5" width="57,5" height="50" src="/wiki/images/8/81/Book2.gif"><br />
<font size="6" color="#F0F8FF" face="Arial regular">Visit our lab book for the detection construct</font><br><br />
</a><br />
<br />
<br />
<br><br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Fluorescence proteins</font></h2> <br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
The genes for our FRET pair, mKate and LssmOrange, were isolated by PCR, cloned into the biobrick vector and sequenced afterwards. The genes were also cloned into a commercially available expression vector in order to characterize the fluorescent proteins. Visit our lab book to read more about our work flow.<br />
<center><br />
<body><br />
<br><br />
<br><br />
<br />
<br />
<img alt="mkate" src="/wiki/images/3/3a/Mkate-active_light1.png" width="50%" height="50%" align="right"><br />
<br />
<img alt="mkateactive" src="/wiki/images/5/58/Mkate-active_surface_activesite4_light1.png" width="50%" height="50%" align="right"><br />
<br />
<br><br />
<br><br />
</p></font><br />
<br />
<br><br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook/Biobricks"><br />
<img alt="Labbookbiobricks" border="5" width="57,5" height="50" src="/wiki/images/8/81/Book2.gif"><br />
<font size="6" color="#F0F8FF" face="Arial regular">Visit our lab book for the fluorescence proteins</font><br><br />
</a><br />
<br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Safety</font></h2> <br><br />
<br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
The assembly of the blue-light sensitive pDawn construct from gBlocks failed. Although the construction of the pezT construct from gBlocks was successful, multiple transformations of pSB1C3-pezT failed. Visit our lab book to read more about our work flow.<br />
<br />
</p></font><br />
<br><br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety/Labjournal"><br />
<img alt="Labbookbiobricks" border="5" width="57,5" height="50" src="/wiki/images/8/81/Book2.gif"><br />
<font size="6" color="#F0F8FF" face="Arial regular">Visit our lab book for the safety construct</font><br /><br />
</a><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;<br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Cutinase</font></h2> <br><br />
<br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
Here, we present the improvement approach of the <i>Fusarium Solani Cutinase</i> (FsC, PID: BBa K808025).<br />
The FsC was one of the PET cleavage enzymes from the iGEM Team TU-Darmstadt 2012. We improved this part <br />
with a pelB leader and a reporter(mRFP1-His6) system with a TEV cleavage site. This construct is regulated by pBAD <br />
(PID: BBa I0500) and cloned into the pSB1C3 backbone. <br />
<br />
</p></font><br />
<center><br />
<img alt="fsc" src="/wiki/images/0/0d/Fscpet2.png" width="50%" height="50%"><br />
<br><br><br />
<br />
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Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook
Team:TU Darmstadt/labbook
2013-10-05T03:10:12Z
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<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Detection construct</font></h2> <br><br />
<br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
The assembly of our detection construct proved to be tricky. Not only the traditional cloning approach failed but also the assembly using synthesized gBlocks delivered no results. We finally turned to an In-Fusion cloning approach to assemble our construct. Visit our lab book to read more about our work flow.<br />
<br />
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<br><br><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct"><br />
<img alt="Labbookdetection" border="5" width="57,5" height="50" src="/wiki/images/8/81/Book2.gif"><br />
<font size="6" color="#F0F8FF" face="Arial regular">Visit our lab book for the detection construct</font><br /><br />
</a><br />
<br />
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<br><br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Fluorescence proteins</font></h2> <br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
The genes for our FRET pair, mKate and LssmOrange, were isolated by PCR, cloned into the biobrick vector and sequenced afterwards. The genes were also cloned into a commercially available expression vector in order to characterize the fluorescent proteins. Visit our lab book to read more about our work flow.<br />
<center><br />
<body><br />
<br><br />
<br><br />
<br />
<br />
<img alt="mkate" src="/wiki/images/3/3a/Mkate-active_light1.png" width="50%" height="50%" align="right"><br />
<br />
<img alt="mkateactive" src="/wiki/images/5/58/Mkate-active_surface_activesite4_light1.png" width="50%" height="50%" align="right"><br />
<br />
<br><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook/Biobricks"><br />
<img alt="Labbookbiobricks" border="5" width="57,5" height="50" src="/wiki/images/8/81/Book2.gif"><br />
<font size="6" color="#F0F8FF" face="Arial regular">Visit our lab book for the fluorescence proteins</font>< br/><br />
</a><br />
<br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Safety</font></h2> <br><br />
<br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
The assembly of the blue-light sensitive pDawn construct from gBlocks failed. Although the construction of the pezT construct from gBlocks was successful, multiple transformations of pSB1C3-pezT failed. Visit our lab book to read more about our work flow.<br />
<br />
</p></font><br />
<br><br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety/Labjournal"><br />
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</a><br />
&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;<br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Cutinase</font></h2> <br><br />
<br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
Here, we present the improvement approach of the <i>Fusarium Solani Cutinase</i> (FsC, PID: BBa K808025).<br />
The FsC was one of the PET cleavage enzymes from the iGEM Team TU-Darmstadt 2012. We improved this part <br />
with a pelB leader and a reporter(mRFP1-His6) system with a TEV cleavage site. This construct is regulated by pBAD <br />
(PID: BBa I0500) and cloned into the pSB1C3 backbone. <br />
<br />
</p></font><br />
<center><br />
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Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct
Team:TU Darmstadt/labbook/DetectionConstruct
2013-10-05T03:04:46Z
<p>Schiefie: </p>
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<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
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<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
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<br />
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<br><br />
<br><br />
<br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Construction of the Detection Construct</font></h2><br />
<br />
<br><br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
The detection construct contains both fusionproteins, tar-LssmOrange and CheB-mKate, which are needed to put the mycotxin mediated FRET system to effect in an bacterial cell. Thus it comprises the following parts.<br />
<center><br />
<img src="https://static.igem.org/mediawiki/2013/e/ef/Darmstadt13_labbook_detec_Psb1c3_detection.png" width="450" aligne="center" alt=""><br />
<br><br><br />
</center><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Traditional cloning approach</font></h2><br />
<br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<br><br />
We worked on this approach from the 10th of may till the 10th of june. It was abandoned due to major difficulties during restriction, ligation and transformation.<br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">gBLOCK approach</font></h2><br />
<br><br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br><br />
<br />
We worked on this approach from the 11th of june till the 12th of august. As before it was abandoned due to major difficulties during restriction, ligation and transformation.<br />
<br />
<br><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">InFusion approach</font></h2><br />
<br />
<br><br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<br />
<br />
<table border="1" rules="rows" style="margin-left:50px; margin-right:50px"><br />
<br />
<br />
<tr><br />
<td >13.08</td><br />
<td>gBLOCK assembly of CMK and TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>PCR mixture<br />
<ul><li>1 µL of each gBLOCK<br />
<ul><br />
<li>CMK: B_01 - B_04</li><br />
<li>TLO: A_01 - A_06</li><br />
</ul></li><br />
<li>10 µL 5x Q5 Reaction Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>1 µL Q5 Hot Start Polymerase</li><br />
<li>10 µL 5x Q5 High GC Enhancer</li><br />
<li>1 µL primer suffix-R (10 mM)</li><br />
<li>1 µL primer prefix_R (10 mM)</li><br />
</ul></ul></li><br><br />
<ul><li>PCR program (40 cycles)<br />
<ul><li>initial denaturation 94°C, 100s</li><br />
<li>denaturation 94°C, 55s</li><br />
<li>annealing 64°C, 55s</li><br />
<li>elongation 72°C, 120s</li><br />
<li>final elongation 72°C, 300s</li><br />
</li></ul></ul><br><br />
<ul><li>preparative 1% agarose gel<br />
<ul><li>gel displays bands of expected size, therefore assembly was successful</li><br />
</ul></li></ul><br />
</td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/6c/Darmstadt13_InFusion_130813.png" alt=""></td><br />
</tr><br />
<tr><br />
<td>13.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>using Wizard SV Gel and PCR Clean-Up System (Promega)<br />
<ul><br />
<li>TLO c = 34,4 ng/µL</li><br />
<li>CMK c = 35,6 ng/µL</li><br />
</ul><br />
</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 19.8. --><br />
<tr><br />
<td>19.08</td><br />
<td>Overlap extension PCR</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<br />
<td><ul><br />
<li>reaction mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev</li><br />
<li>CMK: CMK gBLOCK assembly + frag2_for + frag2_rev</li><br />
<li>terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev</li><br />
<li>pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev</li><br />
<li>TLO: TLO gBLOCK assembly + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3[fsC] + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 60°C, 40s</li><br />
<li>elongation: 72°C, 90s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays byproducts<ul><br />
<li>subsequent purification and amplification of desired bands (framed)</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/56/Darmstadt13_infusion_130819_InFusion_overlap_extension_PCR.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 10 ng/µL</li><br />
<li>CMK c = 30 ng/µL </li><br />
<li>terminator c = 7 ng/µL</li><br />
<li>pBAD4 c = 11 ng/µL</li><br />
<li>TLO c = 22 ng/µL</li><br />
<li> pSB1C3 c = 16 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>19.08 </td><br />
<td>Amplification PCR of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>9,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li> pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev </li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 70°C, 30s</li><br />
<li>elongation: 72°C, 30s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul> </li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature </li><br />
<li>terminator and pBAD4 show no bands</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Gradient PCR of the amplification of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>4 µL template</li><br />
<li> 1 µL forward primer (10 mM) </li><br />
<li> 1 µL reverse primer (10 mM)</li><br />
<li>6,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev</li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s</li><br />
<li>annealing 55°C/57°C/61°C/65°C, 30s</li><br />
<li>elongation 72°C, 30s</li><br />
<li> final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>optimal annealing temperature for terminator is 55°C</li><br />
<li>optimal annealing temperature for pBAD4 is 55°C</li><br />
</ul></li><br />
<br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_infusion_130820_InFusion_Gradient_PCR.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 20.8. --><br />
<br />
<tr><br />
<td>20.08</td><br />
<td>Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 45 ng/µL</li><br />
<li>terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73</li><br />
<li>pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 21.8. --><br />
<tr><br />
<td>21.08 </td><br />
<td>Amplification PCR of CMK, TLO and pSB1C3</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer</li><br />
<li>1 µL reverse primer</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev</li><br />
<li>TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s </li><br />
<li>annealing 55°C, 40s</li><br />
<li>elongation 72°C, 90s </li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>gel displays critical amount of byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 21.8 --><br />
<tr><br />
<td>22.08</td><br />
<td>Purification of CMK, TLO and pSB1C3 from preparative gel</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88</li><br />
<li>TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60</li><br />
<li> pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 22.8. --><br />
<tr><br />
<td>23.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<br />
<!--23.8.--><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs </li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/66/Darmstadt13_infusion_130823_InFusion_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 24.8.--><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O </li><br />
<br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li> annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands (just like on 23.08)<ul><br />
<li>probably the template is poorly </li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL) </li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO </li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li> 2 µL MgCl2 (50 mM) </li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s</li><br />
<li>annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
<br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/39/Darmstadt13_infusion_130824_2_PCR_TLO.png" width="75%" height="75%" alt="x"></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s</li><br />
<li> elongation 72°C, 300s </li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (71 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li> 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li>1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li>3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>18µl 5x InFusion Pfu MasterMix</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li><br />
<br><br><br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>no colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li> 27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/3b/Darmstadt13_infusion_130826_PCR_TLOamplification_AS.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>26.08 </td><br />
<td>Purification of TLO</td><br />
<td></td><br />
<br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19,7 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO (c = 19,7 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li> 1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles) <ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_infusion_130828_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>27.08</td><br />
<td> Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (1:10)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>25,5 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs (10 mM)</li><br />
<li>2,5 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s </li><br />
<li>elongation 72°C, 480s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel <ul><br />
<li>no distinctive bands (just like on 26.08)</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O </li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul> </li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>no distinctive bands (just like on 26.08)<ul><br />
<li>probably the template is poorly</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O</li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li> gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/ff/Darmstadt_infusion_130829_PCR_TLOamplification.png" width="75%" height="75%" alt="x"></td><br />
</tr><br />
<br />
<tr><br />
<td>29.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega) <ul><br />
<li> TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>31.08 </td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (20 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li>1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li> 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li> 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>4µl 5x InFusion Pfu MasterMix</li><br />
<li>1,5µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li> <br> <br><br />
<br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>2 colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>02.09 </td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>Colony PCR<ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl Colony LB Medium (Colony 1/Colony 2)</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Programm <ul><br />
<li> initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li> final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<br />
</ul><br />
</li><br />
<li>Purification using Pure Yield Plasmid Miniprep System (Promega)<ul><br />
<li>Colonie 1 = pSB1C3-TLO-CMK 1<ul><br />
<li>c = 245,5 ng/ul </li><br />
<li>260/280 = 1,87</li><br />
<li>260/230 = 2,23</li><br />
</ul></li> <br><br />
<li>Colonie 2 = pSB1C3-TLO-CMK 2<ul><br />
<li>c = 107,7 ng/ul</li><br />
<li>260/280 = 1,95</li><br />
<li>260/230 = 2,28 </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>digestion with EcoRI and double digest with EcoRI and PstI</li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pattern is as expected</li><br />
<li>colony PCR product is off by 2000 bp</li><br />
<li>linearized plasmid is off by 1000 bp</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/b/ba/Darmstadt13_infusion_130902_1%282%29.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl forward primer</li><br />
<li>1µl reverse primer</li><br />
<li>1µl template</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO </li><br />
<li>5µl 10x Taq-Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>37µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li>PCR tube 4: frag4_for + frag4_rev</li><br />
<li>PCR tube 5: frag5_for + frag5_rev</li><br />
</ul></li> <br><br />
<li>template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li> PCR tube 4: frag4_for + frag4_rev </li><br />
<li>PCR tube 5: frag5_for + frag5_rev </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li>final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>besides many byproducts all fragments could be amplified from templates (framed bands)<ul><br />
<li>clones are most likely correct</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_infusion_130904_pSB1C3-TLO-CMK.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Induction of pSB1C3-TLO-CMK clones with L-Arabinose</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>induction with L-arabinose (0,02% w/v) at OD600 = 0,6<ul><br />
<li>induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>03.09 </td><br />
<td>Sequencing of pSB1C3-TLO-CMK clones</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>were not able to sequence the whole insert, as pimers did not anneal sufficiently</li><br />
<li>results indicate that all five fragments were successfully ligated in the correct order</li><br />
<li>results show that gBLOCK assembly of TLO and CMK did not work correctly</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
</table><br />
</front><br />
</p><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct
Team:TU Darmstadt/labbook/DetectionConstruct
2013-10-05T03:03:24Z
<p>Schiefie: </p>
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<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Construction of the Detection Construct</font></h2><br />
<br />
<br><br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
The detection construct contains both fusionproteins, tar-LssmOrange and CheB-mKate, which are needed to put the mycotxin mediated FRET system to effect in an bacterial cell. Thus it comprises the following parts.<br />
<br />
<img src="https://static.igem.org/mediawiki/2013/e/ef/Darmstadt13_labbook_detec_Psb1c3_detection.png" width="450" aligne="center" alt=""><br />
<br><br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Traditional cloning approach</font></h2><br />
<br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<br><br />
We worked on this approach from the 10th of may till the 10th of june. It was abandoned due to major difficulties during restriction, ligation and transformation.<br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">gBLOCK approach</font></h2><br />
<br><br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br><br />
<br />
We worked on this approach from the 11th of june till the 12th of august. As before it was abandoned due to major difficulties during restriction, ligation and transformation.<br />
<br />
<br><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">InFusion approach</font></h2><br />
<br />
<br><br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<br />
<br />
<table border="1" rules="rows" style="margin-left:50px; margin-right:50px"><br />
<br />
<br />
<tr><br />
<td >13.08</td><br />
<td>gBLOCK assembly of CMK and TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>PCR mixture<br />
<ul><li>1 µL of each gBLOCK<br />
<ul><br />
<li>CMK: B_01 - B_04</li><br />
<li>TLO: A_01 - A_06</li><br />
</ul></li><br />
<li>10 µL 5x Q5 Reaction Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>1 µL Q5 Hot Start Polymerase</li><br />
<li>10 µL 5x Q5 High GC Enhancer</li><br />
<li>1 µL primer suffix-R (10 mM)</li><br />
<li>1 µL primer prefix_R (10 mM)</li><br />
</ul></ul></li><br><br />
<ul><li>PCR program (40 cycles)<br />
<ul><li>initial denaturation 94°C, 100s</li><br />
<li>denaturation 94°C, 55s</li><br />
<li>annealing 64°C, 55s</li><br />
<li>elongation 72°C, 120s</li><br />
<li>final elongation 72°C, 300s</li><br />
</li></ul></ul><br><br />
<ul><li>preparative 1% agarose gel<br />
<ul><li>gel displays bands of expected size, therefore assembly was successful</li><br />
</ul></li></ul><br />
</td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/6c/Darmstadt13_InFusion_130813.png" alt=""></td><br />
</tr><br />
<tr><br />
<td>13.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>using Wizard SV Gel and PCR Clean-Up System (Promega)<br />
<ul><br />
<li>TLO c = 34,4 ng/µL</li><br />
<li>CMK c = 35,6 ng/µL</li><br />
</ul><br />
</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 19.8. --><br />
<tr><br />
<td>19.08</td><br />
<td>Overlap extension PCR</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<br />
<td><ul><br />
<li>reaction mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev</li><br />
<li>CMK: CMK gBLOCK assembly + frag2_for + frag2_rev</li><br />
<li>terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev</li><br />
<li>pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev</li><br />
<li>TLO: TLO gBLOCK assembly + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3[fsC] + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 60°C, 40s</li><br />
<li>elongation: 72°C, 90s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays byproducts<ul><br />
<li>subsequent purification and amplification of desired bands (framed)</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/56/Darmstadt13_infusion_130819_InFusion_overlap_extension_PCR.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 10 ng/µL</li><br />
<li>CMK c = 30 ng/µL </li><br />
<li>terminator c = 7 ng/µL</li><br />
<li>pBAD4 c = 11 ng/µL</li><br />
<li>TLO c = 22 ng/µL</li><br />
<li> pSB1C3 c = 16 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>19.08 </td><br />
<td>Amplification PCR of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>9,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li> pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev </li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 70°C, 30s</li><br />
<li>elongation: 72°C, 30s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul> </li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature </li><br />
<li>terminator and pBAD4 show no bands</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Gradient PCR of the amplification of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>4 µL template</li><br />
<li> 1 µL forward primer (10 mM) </li><br />
<li> 1 µL reverse primer (10 mM)</li><br />
<li>6,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev</li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s</li><br />
<li>annealing 55°C/57°C/61°C/65°C, 30s</li><br />
<li>elongation 72°C, 30s</li><br />
<li> final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>optimal annealing temperature for terminator is 55°C</li><br />
<li>optimal annealing temperature for pBAD4 is 55°C</li><br />
</ul></li><br />
<br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_infusion_130820_InFusion_Gradient_PCR.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 20.8. --><br />
<br />
<tr><br />
<td>20.08</td><br />
<td>Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 45 ng/µL</li><br />
<li>terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73</li><br />
<li>pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 21.8. --><br />
<tr><br />
<td>21.08 </td><br />
<td>Amplification PCR of CMK, TLO and pSB1C3</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer</li><br />
<li>1 µL reverse primer</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev</li><br />
<li>TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s </li><br />
<li>annealing 55°C, 40s</li><br />
<li>elongation 72°C, 90s </li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>gel displays critical amount of byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 21.8 --><br />
<tr><br />
<td>22.08</td><br />
<td>Purification of CMK, TLO and pSB1C3 from preparative gel</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88</li><br />
<li>TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60</li><br />
<li> pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 22.8. --><br />
<tr><br />
<td>23.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<br />
<!--23.8.--><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs </li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/66/Darmstadt13_infusion_130823_InFusion_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 24.8.--><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O </li><br />
<br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li> annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands (just like on 23.08)<ul><br />
<li>probably the template is poorly </li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL) </li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO </li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li> 2 µL MgCl2 (50 mM) </li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s</li><br />
<li>annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
<br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/39/Darmstadt13_infusion_130824_2_PCR_TLO.png" width="75%" height="75%" alt="x"></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s</li><br />
<li> elongation 72°C, 300s </li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (71 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li> 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li>1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li>3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>18µl 5x InFusion Pfu MasterMix</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li><br />
<br><br><br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>no colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li> 27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/3b/Darmstadt13_infusion_130826_PCR_TLOamplification_AS.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>26.08 </td><br />
<td>Purification of TLO</td><br />
<td></td><br />
<br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19,7 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO (c = 19,7 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li> 1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles) <ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_infusion_130828_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>27.08</td><br />
<td> Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (1:10)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>25,5 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs (10 mM)</li><br />
<li>2,5 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s </li><br />
<li>elongation 72°C, 480s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel <ul><br />
<li>no distinctive bands (just like on 26.08)</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O </li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul> </li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>no distinctive bands (just like on 26.08)<ul><br />
<li>probably the template is poorly</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O</li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li> gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/ff/Darmstadt_infusion_130829_PCR_TLOamplification.png" width="75%" height="75%" alt="x"></td><br />
</tr><br />
<br />
<tr><br />
<td>29.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega) <ul><br />
<li> TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>31.08 </td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (20 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li>1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li> 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li> 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>4µl 5x InFusion Pfu MasterMix</li><br />
<li>1,5µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li> <br> <br><br />
<br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>2 colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>02.09 </td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>Colony PCR<ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl Colony LB Medium (Colony 1/Colony 2)</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Programm <ul><br />
<li> initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li> final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<br />
</ul><br />
</li><br />
<li>Purification using Pure Yield Plasmid Miniprep System (Promega)<ul><br />
<li>Colonie 1 = pSB1C3-TLO-CMK 1<ul><br />
<li>c = 245,5 ng/ul </li><br />
<li>260/280 = 1,87</li><br />
<li>260/230 = 2,23</li><br />
</ul></li> <br><br />
<li>Colonie 2 = pSB1C3-TLO-CMK 2<ul><br />
<li>c = 107,7 ng/ul</li><br />
<li>260/280 = 1,95</li><br />
<li>260/230 = 2,28 </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>digestion with EcoRI and double digest with EcoRI and PstI</li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pattern is as expected</li><br />
<li>colony PCR product is off by 2000 bp</li><br />
<li>linearized plasmid is off by 1000 bp</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/b/ba/Darmstadt13_infusion_130902_1%282%29.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl forward primer</li><br />
<li>1µl reverse primer</li><br />
<li>1µl template</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO </li><br />
<li>5µl 10x Taq-Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>37µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li>PCR tube 4: frag4_for + frag4_rev</li><br />
<li>PCR tube 5: frag5_for + frag5_rev</li><br />
</ul></li> <br><br />
<li>template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li> PCR tube 4: frag4_for + frag4_rev </li><br />
<li>PCR tube 5: frag5_for + frag5_rev </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li>final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>besides many byproducts all fragments could be amplified from templates (framed bands)<ul><br />
<li>clones are most likely correct</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_infusion_130904_pSB1C3-TLO-CMK.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Induction of pSB1C3-TLO-CMK clones with L-Arabinose</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>induction with L-arabinose (0,02% w/v) at OD600 = 0,6<ul><br />
<li>induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>03.09 </td><br />
<td>Sequencing of pSB1C3-TLO-CMK clones</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>were not able to sequence the whole insert, as pimers did not anneal sufficiently</li><br />
<li>results indicate that all five fragments were successfully ligated in the correct order</li><br />
<li>results show that gBLOCK assembly of TLO and CMK did not work correctly</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
</table><br />
</front><br />
</p><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct
Team:TU Darmstadt/labbook/DetectionConstruct
2013-10-05T03:02:19Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Construction of the Detection Construct</font></h2><br />
<br />
<br><br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
The detection construct contains both fusionproteins, tar-LssmOrange and CheB-mKate, which are needed to put the mycotxin mediated FRET system to effect in an bacterial cell. Thus it comprises the following parts.<br />
<br />
<img src="https://static.igem.org/mediawiki/2013/e/ef/Darmstadt13_labbook_detec_Psb1c3_detection.png" width="450" align="center" alt=""><br />
<br><br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Traditional cloning approach</font></h2><br />
<br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<br><br />
We worked on this approach from the 10th of may till the 10th of june. It was abandoned due to major difficulties during restriction, ligation and transformation.<br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">gBLOCK approach</font></h2><br />
<br><br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br><br />
<br />
We worked on this approach from the 11th of june till the 12th of august. As before it was abandoned due to major difficulties during restriction, ligation and transformation.<br />
<br />
<br><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">InFusion approach</font></h2><br />
<br />
<br><br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<br />
<br />
<table border="1" rules="rows" style="margin-left:50px; margin-right:50px"><br />
<br />
<br />
<tr><br />
<td >13.08</td><br />
<td>gBLOCK assembly of CMK and TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>PCR mixture<br />
<ul><li>1 µL of each gBLOCK<br />
<ul><br />
<li>CMK: B_01 - B_04</li><br />
<li>TLO: A_01 - A_06</li><br />
</ul></li><br />
<li>10 µL 5x Q5 Reaction Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>1 µL Q5 Hot Start Polymerase</li><br />
<li>10 µL 5x Q5 High GC Enhancer</li><br />
<li>1 µL primer suffix-R (10 mM)</li><br />
<li>1 µL primer prefix_R (10 mM)</li><br />
</ul></ul></li><br><br />
<ul><li>PCR program (40 cycles)<br />
<ul><li>initial denaturation 94°C, 100s</li><br />
<li>denaturation 94°C, 55s</li><br />
<li>annealing 64°C, 55s</li><br />
<li>elongation 72°C, 120s</li><br />
<li>final elongation 72°C, 300s</li><br />
</li></ul></ul><br><br />
<ul><li>preparative 1% agarose gel<br />
<ul><li>gel displays bands of expected size, therefore assembly was successful</li><br />
</ul></li></ul><br />
</td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/6c/Darmstadt13_InFusion_130813.png" alt=""></td><br />
</tr><br />
<tr><br />
<td>13.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>using Wizard SV Gel and PCR Clean-Up System (Promega)<br />
<ul><br />
<li>TLO c = 34,4 ng/µL</li><br />
<li>CMK c = 35,6 ng/µL</li><br />
</ul><br />
</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 19.8. --><br />
<tr><br />
<td>19.08</td><br />
<td>Overlap extension PCR</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<br />
<td><ul><br />
<li>reaction mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev</li><br />
<li>CMK: CMK gBLOCK assembly + frag2_for + frag2_rev</li><br />
<li>terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev</li><br />
<li>pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev</li><br />
<li>TLO: TLO gBLOCK assembly + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3[fsC] + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 60°C, 40s</li><br />
<li>elongation: 72°C, 90s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays byproducts<ul><br />
<li>subsequent purification and amplification of desired bands (framed)</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/56/Darmstadt13_infusion_130819_InFusion_overlap_extension_PCR.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 10 ng/µL</li><br />
<li>CMK c = 30 ng/µL </li><br />
<li>terminator c = 7 ng/µL</li><br />
<li>pBAD4 c = 11 ng/µL</li><br />
<li>TLO c = 22 ng/µL</li><br />
<li> pSB1C3 c = 16 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>19.08 </td><br />
<td>Amplification PCR of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>9,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li> pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev </li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 70°C, 30s</li><br />
<li>elongation: 72°C, 30s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul> </li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature </li><br />
<li>terminator and pBAD4 show no bands</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Gradient PCR of the amplification of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>4 µL template</li><br />
<li> 1 µL forward primer (10 mM) </li><br />
<li> 1 µL reverse primer (10 mM)</li><br />
<li>6,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev</li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s</li><br />
<li>annealing 55°C/57°C/61°C/65°C, 30s</li><br />
<li>elongation 72°C, 30s</li><br />
<li> final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>optimal annealing temperature for terminator is 55°C</li><br />
<li>optimal annealing temperature for pBAD4 is 55°C</li><br />
</ul></li><br />
<br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_infusion_130820_InFusion_Gradient_PCR.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 20.8. --><br />
<br />
<tr><br />
<td>20.08</td><br />
<td>Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 45 ng/µL</li><br />
<li>terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73</li><br />
<li>pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 21.8. --><br />
<tr><br />
<td>21.08 </td><br />
<td>Amplification PCR of CMK, TLO and pSB1C3</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer</li><br />
<li>1 µL reverse primer</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev</li><br />
<li>TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s </li><br />
<li>annealing 55°C, 40s</li><br />
<li>elongation 72°C, 90s </li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>gel displays critical amount of byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 21.8 --><br />
<tr><br />
<td>22.08</td><br />
<td>Purification of CMK, TLO and pSB1C3 from preparative gel</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88</li><br />
<li>TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60</li><br />
<li> pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 22.8. --><br />
<tr><br />
<td>23.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<br />
<!--23.8.--><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs </li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/66/Darmstadt13_infusion_130823_InFusion_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 24.8.--><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O </li><br />
<br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li> annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands (just like on 23.08)<ul><br />
<li>probably the template is poorly </li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL) </li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO </li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li> 2 µL MgCl2 (50 mM) </li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s</li><br />
<li>annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
<br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/39/Darmstadt13_infusion_130824_2_PCR_TLO.png" width="75%" height="75%" alt="x"></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s</li><br />
<li> elongation 72°C, 300s </li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (71 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li> 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li>1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li>3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>18µl 5x InFusion Pfu MasterMix</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li><br />
<br><br><br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>no colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li> 27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/3b/Darmstadt13_infusion_130826_PCR_TLOamplification_AS.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>26.08 </td><br />
<td>Purification of TLO</td><br />
<td></td><br />
<br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19,7 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO (c = 19,7 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li> 1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles) <ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_infusion_130828_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>27.08</td><br />
<td> Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (1:10)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>25,5 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs (10 mM)</li><br />
<li>2,5 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s </li><br />
<li>elongation 72°C, 480s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel <ul><br />
<li>no distinctive bands (just like on 26.08)</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O </li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul> </li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>no distinctive bands (just like on 26.08)<ul><br />
<li>probably the template is poorly</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O</li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li> gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/ff/Darmstadt_infusion_130829_PCR_TLOamplification.png" width="75%" height="75%" alt="x"></td><br />
</tr><br />
<br />
<tr><br />
<td>29.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega) <ul><br />
<li> TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>31.08 </td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (20 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li>1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li> 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li> 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>4µl 5x InFusion Pfu MasterMix</li><br />
<li>1,5µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li> <br> <br><br />
<br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>2 colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>02.09 </td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>Colony PCR<ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl Colony LB Medium (Colony 1/Colony 2)</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Programm <ul><br />
<li> initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li> final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<br />
</ul><br />
</li><br />
<li>Purification using Pure Yield Plasmid Miniprep System (Promega)<ul><br />
<li>Colonie 1 = pSB1C3-TLO-CMK 1<ul><br />
<li>c = 245,5 ng/ul </li><br />
<li>260/280 = 1,87</li><br />
<li>260/230 = 2,23</li><br />
</ul></li> <br><br />
<li>Colonie 2 = pSB1C3-TLO-CMK 2<ul><br />
<li>c = 107,7 ng/ul</li><br />
<li>260/280 = 1,95</li><br />
<li>260/230 = 2,28 </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>digestion with EcoRI and double digest with EcoRI and PstI</li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pattern is as expected</li><br />
<li>colony PCR product is off by 2000 bp</li><br />
<li>linearized plasmid is off by 1000 bp</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/b/ba/Darmstadt13_infusion_130902_1%282%29.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl forward primer</li><br />
<li>1µl reverse primer</li><br />
<li>1µl template</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO </li><br />
<li>5µl 10x Taq-Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>37µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li>PCR tube 4: frag4_for + frag4_rev</li><br />
<li>PCR tube 5: frag5_for + frag5_rev</li><br />
</ul></li> <br><br />
<li>template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li> PCR tube 4: frag4_for + frag4_rev </li><br />
<li>PCR tube 5: frag5_for + frag5_rev </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li>final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>besides many byproducts all fragments could be amplified from templates (framed bands)<ul><br />
<li>clones are most likely correct</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_infusion_130904_pSB1C3-TLO-CMK.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Induction of pSB1C3-TLO-CMK clones with L-Arabinose</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>induction with L-arabinose (0,02% w/v) at OD600 = 0,6<ul><br />
<li>induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>03.09 </td><br />
<td>Sequencing of pSB1C3-TLO-CMK clones</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>were not able to sequence the whole insert, as pimers did not anneal sufficiently</li><br />
<li>results indicate that all five fragments were successfully ligated in the correct order</li><br />
<li>results show that gBLOCK assembly of TLO and CMK did not work correctly</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
</table><br />
</front><br />
</p><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct
Team:TU Darmstadt/labbook/DetectionConstruct
2013-10-05T03:00:48Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Construction of the Detection Construct</font></h2><br />
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<font size="3" color="#F0F8FF" face="Arial regular"><br />
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<br />
The detection construct contains both fusionproteins, tar-LssmOrange and CheB-mKate, which are needed to put the mycotxin mediated FRET system to effect in an bacterial cell. Thus it comprises the following parts.<br />
<br />
<img src="https://static.igem.org/mediawiki/2013/e/ef/Darmstadt13_labbook_detec_Psb1c3_detection.png" width="450" alt=""><br />
<br><br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Traditional cloning approach</font></h2><br />
<br><br />
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<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
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<br><br />
We worked on this approach from the 10th of may till the 10th of june. It was abandoned due to major difficulties during restriction, ligation and transformation.<br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">gBLOCK approach</font></h2><br />
<br><br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br><br />
<br />
We worked on this approach from the 11th of june till the 12th of august. As before it was abandoned due to major difficulties during restriction, ligation and transformation.<br />
<br />
<br><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">InFusion approach</font></h2><br />
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<br><br><br />
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<font size="3" color="#F0F8FF" face="Arial regular"><br />
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<table border="1" rules="rows" style="margin-left:50px; margin-right:50px"><br />
<br />
<br />
<tr><br />
<td >13.08</td><br />
<td>gBLOCK assembly of CMK and TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>PCR mixture<br />
<ul><li>1 µL of each gBLOCK<br />
<ul><br />
<li>CMK: B_01 - B_04</li><br />
<li>TLO: A_01 - A_06</li><br />
</ul></li><br />
<li>10 µL 5x Q5 Reaction Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>1 µL Q5 Hot Start Polymerase</li><br />
<li>10 µL 5x Q5 High GC Enhancer</li><br />
<li>1 µL primer suffix-R (10 mM)</li><br />
<li>1 µL primer prefix_R (10 mM)</li><br />
</ul></ul></li><br><br />
<ul><li>PCR program (40 cycles)<br />
<ul><li>initial denaturation 94°C, 100s</li><br />
<li>denaturation 94°C, 55s</li><br />
<li>annealing 64°C, 55s</li><br />
<li>elongation 72°C, 120s</li><br />
<li>final elongation 72°C, 300s</li><br />
</li></ul></ul><br><br />
<ul><li>preparative 1% agarose gel<br />
<ul><li>gel displays bands of expected size, therefore assembly was successful</li><br />
</ul></li></ul><br />
</td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/6c/Darmstadt13_InFusion_130813.png" alt=""></td><br />
</tr><br />
<tr><br />
<td>13.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>using Wizard SV Gel and PCR Clean-Up System (Promega)<br />
<ul><br />
<li>TLO c = 34,4 ng/µL</li><br />
<li>CMK c = 35,6 ng/µL</li><br />
</ul><br />
</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 19.8. --><br />
<tr><br />
<td>19.08</td><br />
<td>Overlap extension PCR</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<br />
<td><ul><br />
<li>reaction mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev</li><br />
<li>CMK: CMK gBLOCK assembly + frag2_for + frag2_rev</li><br />
<li>terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev</li><br />
<li>pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev</li><br />
<li>TLO: TLO gBLOCK assembly + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3[fsC] + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 60°C, 40s</li><br />
<li>elongation: 72°C, 90s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays byproducts<ul><br />
<li>subsequent purification and amplification of desired bands (framed)</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/56/Darmstadt13_infusion_130819_InFusion_overlap_extension_PCR.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 10 ng/µL</li><br />
<li>CMK c = 30 ng/µL </li><br />
<li>terminator c = 7 ng/µL</li><br />
<li>pBAD4 c = 11 ng/µL</li><br />
<li>TLO c = 22 ng/µL</li><br />
<li> pSB1C3 c = 16 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>19.08 </td><br />
<td>Amplification PCR of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>9,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li> pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev </li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 70°C, 30s</li><br />
<li>elongation: 72°C, 30s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul> </li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature </li><br />
<li>terminator and pBAD4 show no bands</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Gradient PCR of the amplification of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>4 µL template</li><br />
<li> 1 µL forward primer (10 mM) </li><br />
<li> 1 µL reverse primer (10 mM)</li><br />
<li>6,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev</li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s</li><br />
<li>annealing 55°C/57°C/61°C/65°C, 30s</li><br />
<li>elongation 72°C, 30s</li><br />
<li> final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>optimal annealing temperature for terminator is 55°C</li><br />
<li>optimal annealing temperature for pBAD4 is 55°C</li><br />
</ul></li><br />
<br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_infusion_130820_InFusion_Gradient_PCR.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 20.8. --><br />
<br />
<tr><br />
<td>20.08</td><br />
<td>Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 45 ng/µL</li><br />
<li>terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73</li><br />
<li>pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 21.8. --><br />
<tr><br />
<td>21.08 </td><br />
<td>Amplification PCR of CMK, TLO and pSB1C3</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer</li><br />
<li>1 µL reverse primer</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev</li><br />
<li>TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s </li><br />
<li>annealing 55°C, 40s</li><br />
<li>elongation 72°C, 90s </li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>gel displays critical amount of byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 21.8 --><br />
<tr><br />
<td>22.08</td><br />
<td>Purification of CMK, TLO and pSB1C3 from preparative gel</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88</li><br />
<li>TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60</li><br />
<li> pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 22.8. --><br />
<tr><br />
<td>23.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<br />
<!--23.8.--><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs </li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/66/Darmstadt13_infusion_130823_InFusion_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 24.8.--><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O </li><br />
<br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li> annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands (just like on 23.08)<ul><br />
<li>probably the template is poorly </li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL) </li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO </li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li> 2 µL MgCl2 (50 mM) </li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s</li><br />
<li>annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
<br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/39/Darmstadt13_infusion_130824_2_PCR_TLO.png" width="75%" height="75%" alt="x"></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s</li><br />
<li> elongation 72°C, 300s </li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (71 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li> 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li>1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li>3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>18µl 5x InFusion Pfu MasterMix</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li><br />
<br><br><br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>no colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li> 27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/3b/Darmstadt13_infusion_130826_PCR_TLOamplification_AS.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>26.08 </td><br />
<td>Purification of TLO</td><br />
<td></td><br />
<br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19,7 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO (c = 19,7 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li> 1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles) <ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_infusion_130828_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>27.08</td><br />
<td> Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (1:10)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>25,5 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs (10 mM)</li><br />
<li>2,5 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s </li><br />
<li>elongation 72°C, 480s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel <ul><br />
<li>no distinctive bands (just like on 26.08)</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O </li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul> </li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>no distinctive bands (just like on 26.08)<ul><br />
<li>probably the template is poorly</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O</li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li> gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/ff/Darmstadt_infusion_130829_PCR_TLOamplification.png" width="75%" height="75%" alt="x"></td><br />
</tr><br />
<br />
<tr><br />
<td>29.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega) <ul><br />
<li> TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>31.08 </td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (20 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li>1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li> 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li> 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>4µl 5x InFusion Pfu MasterMix</li><br />
<li>1,5µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li> <br> <br><br />
<br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>2 colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>02.09 </td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>Colony PCR<ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl Colony LB Medium (Colony 1/Colony 2)</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Programm <ul><br />
<li> initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li> final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<br />
</ul><br />
</li><br />
<li>Purification using Pure Yield Plasmid Miniprep System (Promega)<ul><br />
<li>Colonie 1 = pSB1C3-TLO-CMK 1<ul><br />
<li>c = 245,5 ng/ul </li><br />
<li>260/280 = 1,87</li><br />
<li>260/230 = 2,23</li><br />
</ul></li> <br><br />
<li>Colonie 2 = pSB1C3-TLO-CMK 2<ul><br />
<li>c = 107,7 ng/ul</li><br />
<li>260/280 = 1,95</li><br />
<li>260/230 = 2,28 </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>digestion with EcoRI and double digest with EcoRI and PstI</li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pattern is as expected</li><br />
<li>colony PCR product is off by 2000 bp</li><br />
<li>linearized plasmid is off by 1000 bp</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/b/ba/Darmstadt13_infusion_130902_1%282%29.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl forward primer</li><br />
<li>1µl reverse primer</li><br />
<li>1µl template</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO </li><br />
<li>5µl 10x Taq-Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>37µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li>PCR tube 4: frag4_for + frag4_rev</li><br />
<li>PCR tube 5: frag5_for + frag5_rev</li><br />
</ul></li> <br><br />
<li>template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li> PCR tube 4: frag4_for + frag4_rev </li><br />
<li>PCR tube 5: frag5_for + frag5_rev </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li>final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>besides many byproducts all fragments could be amplified from templates (framed bands)<ul><br />
<li>clones are most likely correct</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_infusion_130904_pSB1C3-TLO-CMK.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Induction of pSB1C3-TLO-CMK clones with L-Arabinose</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>induction with L-arabinose (0,02% w/v) at OD600 = 0,6<ul><br />
<li>induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>03.09 </td><br />
<td>Sequencing of pSB1C3-TLO-CMK clones</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>were not able to sequence the whole insert, as pimers did not anneal sufficiently</li><br />
<li>results indicate that all five fragments were successfully ligated in the correct order</li><br />
<li>results show that gBLOCK assembly of TLO and CMK did not work correctly</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
</table><br />
</front><br />
</p><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_labbook_detec_Psb1c3_detection.png
File:Darmstadt13 labbook detec Psb1c3 detection.png
2013-10-05T02:58:30Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct
Team:TU Darmstadt/labbook/DetectionConstruct
2013-10-05T02:56:56Z
<p>Schiefie: </p>
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<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
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<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Construction of the Detection Construct</font></h2><br />
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The detection construct contains both fusionproteins, tar-LssmOrange and CheB-mKate, which are needed to put the mycotxin mediated FRET system to effect in an bacterial cell. Thus it comprises the following parts.<br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Traditional cloning approach</font></h2><br />
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We worked on this approach from the 10th of may till the 10th of june. It was abandoned due to major difficulties during restriction, ligation and transformation.<br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">gBLOCK approach</font></h2><br />
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We worked on this approach from the 11th of june till the 12th of august. As before it was abandoned due to major difficulties during restriction, ligation and transformation.<br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">InFusion approach</font></h2><br />
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<table border="1" rules="rows" style="margin-left:50px; margin-right:50px"><br />
<br />
<br />
<tr><br />
<td >13.08</td><br />
<td>gBLOCK assembly of CMK and TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>PCR mixture<br />
<ul><li>1 µL of each gBLOCK<br />
<ul><br />
<li>CMK: B_01 - B_04</li><br />
<li>TLO: A_01 - A_06</li><br />
</ul></li><br />
<li>10 µL 5x Q5 Reaction Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>1 µL Q5 Hot Start Polymerase</li><br />
<li>10 µL 5x Q5 High GC Enhancer</li><br />
<li>1 µL primer suffix-R (10 mM)</li><br />
<li>1 µL primer prefix_R (10 mM)</li><br />
</ul></ul></li><br><br />
<ul><li>PCR program (40 cycles)<br />
<ul><li>initial denaturation 94°C, 100s</li><br />
<li>denaturation 94°C, 55s</li><br />
<li>annealing 64°C, 55s</li><br />
<li>elongation 72°C, 120s</li><br />
<li>final elongation 72°C, 300s</li><br />
</li></ul></ul><br><br />
<ul><li>preparative 1% agarose gel<br />
<ul><li>gel displays bands of expected size, therefore assembly was successful</li><br />
</ul></li></ul><br />
</td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/6c/Darmstadt13_InFusion_130813.png" alt=""></td><br />
</tr><br />
<tr><br />
<td>13.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>using Wizard SV Gel and PCR Clean-Up System (Promega)<br />
<ul><br />
<li>TLO c = 34,4 ng/µL</li><br />
<li>CMK c = 35,6 ng/µL</li><br />
</ul><br />
</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 19.8. --><br />
<tr><br />
<td>19.08</td><br />
<td>Overlap extension PCR</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<br />
<td><ul><br />
<li>reaction mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev</li><br />
<li>CMK: CMK gBLOCK assembly + frag2_for + frag2_rev</li><br />
<li>terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev</li><br />
<li>pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev</li><br />
<li>TLO: TLO gBLOCK assembly + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3[fsC] + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 60°C, 40s</li><br />
<li>elongation: 72°C, 90s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays byproducts<ul><br />
<li>subsequent purification and amplification of desired bands (framed)</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/56/Darmstadt13_infusion_130819_InFusion_overlap_extension_PCR.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 10 ng/µL</li><br />
<li>CMK c = 30 ng/µL </li><br />
<li>terminator c = 7 ng/µL</li><br />
<li>pBAD4 c = 11 ng/µL</li><br />
<li>TLO c = 22 ng/µL</li><br />
<li> pSB1C3 c = 16 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>19.08 </td><br />
<td>Amplification PCR of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>9,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li> pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev </li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 70°C, 30s</li><br />
<li>elongation: 72°C, 30s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul> </li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature </li><br />
<li>terminator and pBAD4 show no bands</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Gradient PCR of the amplification of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>4 µL template</li><br />
<li> 1 µL forward primer (10 mM) </li><br />
<li> 1 µL reverse primer (10 mM)</li><br />
<li>6,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev</li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s</li><br />
<li>annealing 55°C/57°C/61°C/65°C, 30s</li><br />
<li>elongation 72°C, 30s</li><br />
<li> final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>optimal annealing temperature for terminator is 55°C</li><br />
<li>optimal annealing temperature for pBAD4 is 55°C</li><br />
</ul></li><br />
<br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_infusion_130820_InFusion_Gradient_PCR.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 20.8. --><br />
<br />
<tr><br />
<td>20.08</td><br />
<td>Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 45 ng/µL</li><br />
<li>terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73</li><br />
<li>pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 21.8. --><br />
<tr><br />
<td>21.08 </td><br />
<td>Amplification PCR of CMK, TLO and pSB1C3</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer</li><br />
<li>1 µL reverse primer</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev</li><br />
<li>TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s </li><br />
<li>annealing 55°C, 40s</li><br />
<li>elongation 72°C, 90s </li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>gel displays critical amount of byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 21.8 --><br />
<tr><br />
<td>22.08</td><br />
<td>Purification of CMK, TLO and pSB1C3 from preparative gel</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88</li><br />
<li>TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60</li><br />
<li> pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 22.8. --><br />
<tr><br />
<td>23.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<br />
<!--23.8.--><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs </li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/66/Darmstadt13_infusion_130823_InFusion_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 24.8.--><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O </li><br />
<br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li> annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands (just like on 23.08)<ul><br />
<li>probably the template is poorly </li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL) </li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO </li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li> 2 µL MgCl2 (50 mM) </li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s</li><br />
<li>annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
<br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/39/Darmstadt13_infusion_130824_2_PCR_TLO.png" width="75%" height="75%" alt="x"></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s</li><br />
<li> elongation 72°C, 300s </li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (71 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li> 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li>1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li>3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>18µl 5x InFusion Pfu MasterMix</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li><br />
<br><br><br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>no colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li> 27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/3b/Darmstadt13_infusion_130826_PCR_TLOamplification_AS.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>26.08 </td><br />
<td>Purification of TLO</td><br />
<td></td><br />
<br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19,7 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO (c = 19,7 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li> 1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles) <ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_infusion_130828_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>27.08</td><br />
<td> Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (1:10)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>25,5 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs (10 mM)</li><br />
<li>2,5 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s </li><br />
<li>elongation 72°C, 480s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel <ul><br />
<li>no distinctive bands (just like on 26.08)</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O </li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul> </li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>no distinctive bands (just like on 26.08)<ul><br />
<li>probably the template is poorly</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O</li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li> gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/ff/Darmstadt_infusion_130829_PCR_TLOamplification.png" width="75%" height="75%" alt="x"></td><br />
</tr><br />
<br />
<tr><br />
<td>29.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega) <ul><br />
<li> TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>31.08 </td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (20 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li>1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li> 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li> 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>4µl 5x InFusion Pfu MasterMix</li><br />
<li>1,5µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li> <br> <br><br />
<br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>2 colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>02.09 </td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>Colony PCR<ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl Colony LB Medium (Colony 1/Colony 2)</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Programm <ul><br />
<li> initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li> final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<br />
</ul><br />
</li><br />
<li>Purification using Pure Yield Plasmid Miniprep System (Promega)<ul><br />
<li>Colonie 1 = pSB1C3-TLO-CMK 1<ul><br />
<li>c = 245,5 ng/ul </li><br />
<li>260/280 = 1,87</li><br />
<li>260/230 = 2,23</li><br />
</ul></li> <br><br />
<li>Colonie 2 = pSB1C3-TLO-CMK 2<ul><br />
<li>c = 107,7 ng/ul</li><br />
<li>260/280 = 1,95</li><br />
<li>260/230 = 2,28 </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>digestion with EcoRI and double digest with EcoRI and PstI</li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pattern is as expected</li><br />
<li>colony PCR product is off by 2000 bp</li><br />
<li>linearized plasmid is off by 1000 bp</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/b/ba/Darmstadt13_infusion_130902_1%282%29.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl forward primer</li><br />
<li>1µl reverse primer</li><br />
<li>1µl template</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO </li><br />
<li>5µl 10x Taq-Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>37µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li>PCR tube 4: frag4_for + frag4_rev</li><br />
<li>PCR tube 5: frag5_for + frag5_rev</li><br />
</ul></li> <br><br />
<li>template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li> PCR tube 4: frag4_for + frag4_rev </li><br />
<li>PCR tube 5: frag5_for + frag5_rev </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li>final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>besides many byproducts all fragments could be amplified from templates (framed bands)<ul><br />
<li>clones are most likely correct</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_infusion_130904_pSB1C3-TLO-CMK.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Induction of pSB1C3-TLO-CMK clones with L-Arabinose</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>induction with L-arabinose (0,02% w/v) at OD600 = 0,6<ul><br />
<li>induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>03.09 </td><br />
<td>Sequencing of pSB1C3-TLO-CMK clones</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>were not able to sequence the whole insert, as pimers did not anneal sufficiently</li><br />
<li>results indicate that all five fragments were successfully ligated in the correct order</li><br />
<li>results show that gBLOCK assembly of TLO and CMK did not work correctly</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
</table><br />
</front><br />
</p><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct
Team:TU Darmstadt/labbook/DetectionConstruct
2013-10-05T02:53:27Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
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<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Construction of the Detection Construct</font></h2><br />
<br />
<br><br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
The detection construct contains both fusionproteins, tar-LssmOrange and CheB-mKate, which are needed to put the mycotxin mediated FRET system to effect in an bacterial cell. Thus it comprises the following parts.<br />
<br><br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Traditional cloning approach</font></h2><br />
<br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<br><br />
We worked on this approach from the 10th of may till the 10th of june. It was abandoned due to major difficulties during restriction, ligation and transformation.<br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">gBLOCK approach</font></h2><br />
<br><br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br><br />
<br />
We worked on this approach from the 11th of june till the 12th of august. As before it was abandoned due to major difficulties during restriction, ligation and transformation.<br />
<br />
<br><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">InFusion approach</font></h2><br />
<br />
<br><br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<br />
<br />
<table border="1" rules="rows" style="margin-left:50px; margin-right:50px"><br />
<br />
<br />
<tr><br />
<td >13.08</td><br />
<td>gBLOCK assembly of CMK and TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>PCR mixture<br />
<ul><li>1 µL of each gBLOCK<br />
<ul><br />
<li>CMK: B_01 - B_04</li><br />
<li>TLO: A_01 - A_06</li><br />
</ul></li><br />
<li>10 µL 5x Q5 Reaction Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>1 µL Q5 Hot Start Polymerase</li><br />
<li>10 µL 5x Q5 High GC Enhancer</li><br />
<li>1 µL primer suffix-R (10 mM)</li><br />
<li>1 µL primer prefix_R (10 mM)</li><br />
</ul></ul></li><br><br />
<ul><li>PCR program (40 cycles)<br />
<ul><li>initial denaturation 94°C, 100s</li><br />
<li>denaturation 94°C, 55s</li><br />
<li>annealing 64°C, 55s</li><br />
<li>elongation 72°C, 120s</li><br />
<li>final elongation 72°C, 300s</li><br />
</li></ul></ul><br><br />
<ul><li>preparative 1% agarose gel<br />
<ul><li>gel displays bands of expected size, therefore assembly was successful</li><br />
</ul></li></ul><br />
</td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/6c/Darmstadt13_InFusion_130813.png" alt=""></td><br />
</tr><br />
<tr><br />
<td>13.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>using Wizard SV Gel and PCR Clean-Up System (Promega)<br />
<ul><br />
<li>TLO c = 34,4 ng/µL</li><br />
<li>CMK c = 35,6 ng/µL</li><br />
</ul><br />
</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 19.8. --><br />
<tr><br />
<td>19.08</td><br />
<td>Overlap extension PCR</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<br />
<td><ul><br />
<li>reaction mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev</li><br />
<li>CMK: CMK gBLOCK assembly + frag2_for + frag2_rev</li><br />
<li>terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev</li><br />
<li>pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev</li><br />
<li>TLO: TLO gBLOCK assembly + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3[fsC] + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 60°C, 40s</li><br />
<li>elongation: 72°C, 90s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays byproducts<ul><br />
<li>subsequent purification and amplification of desired bands (framed)</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/56/Darmstadt13_infusion_130819_InFusion_overlap_extension_PCR.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 10 ng/µL</li><br />
<li>CMK c = 30 ng/µL </li><br />
<li>terminator c = 7 ng/µL</li><br />
<li>pBAD4 c = 11 ng/µL</li><br />
<li>TLO c = 22 ng/µL</li><br />
<li> pSB1C3 c = 16 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>19.08 </td><br />
<td>Amplification PCR of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>9,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li> pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev </li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 70°C, 30s</li><br />
<li>elongation: 72°C, 30s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul> </li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature </li><br />
<li>terminator and pBAD4 show no bands</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Gradient PCR of the amplification of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>4 µL template</li><br />
<li> 1 µL forward primer (10 mM) </li><br />
<li> 1 µL reverse primer (10 mM)</li><br />
<li>6,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev</li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s</li><br />
<li>annealing 55°C/57°C/61°C/65°C, 30s</li><br />
<li>elongation 72°C, 30s</li><br />
<li> final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>optimal annealing temperature for terminator is 55°C</li><br />
<li>optimal annealing temperature for pBAD4 is 55°C</li><br />
</ul></li><br />
<br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_infusion_130820_InFusion_Gradient_PCR.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 20.8. --><br />
<br />
<tr><br />
<td>20.08</td><br />
<td>Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 45 ng/µL</li><br />
<li>terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73</li><br />
<li>pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 21.8. --><br />
<tr><br />
<td>21.08 </td><br />
<td>Amplification PCR of CMK, TLO and pSB1C3</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer</li><br />
<li>1 µL reverse primer</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev</li><br />
<li>TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s </li><br />
<li>annealing 55°C, 40s</li><br />
<li>elongation 72°C, 90s </li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>gel displays critical amount of byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 21.8 --><br />
<tr><br />
<td>22.08</td><br />
<td>Purification of CMK, TLO and pSB1C3 from preparative gel</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88</li><br />
<li>TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60</li><br />
<li> pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 22.8. --><br />
<tr><br />
<td>23.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<br />
<!--23.8.--><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs </li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/66/Darmstadt13_infusion_130823_InFusion_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 24.8.--><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O </li><br />
<br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li> annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands (just like on 23.08)<ul><br />
<li>probably the template is poorly </li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL) </li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO </li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li> 2 µL MgCl2 (50 mM) </li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s</li><br />
<li>annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
<br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/39/Darmstadt13_infusion_130824_2_PCR_TLO.png" width="50%" height="50%" alt="x"></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s</li><br />
<li> elongation 72°C, 300s </li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (71 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li> 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li>1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li>3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>18µl 5x InFusion Pfu MasterMix</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li><br />
<br><br><br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>no colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li> 27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/3b/Darmstadt13_infusion_130826_PCR_TLOamplification_AS.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>26.08 </td><br />
<td>Purification of TLO</td><br />
<td></td><br />
<br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19,7 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO (c = 19,7 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li> 1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles) <ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_infusion_130828_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>27.08</td><br />
<td> Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (1:10)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>25,5 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs (10 mM)</li><br />
<li>2,5 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s </li><br />
<li>elongation 72°C, 480s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel <ul><br />
<li>no distinctive bands (just like on 26.08)</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O </li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul> </li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>no distinctive bands (just like on 26.08)<ul><br />
<li>probably the template is poorly</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O</li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li> gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/ff/Darmstadt_infusion_130829_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<br />
<tr><br />
<td>29.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega) <ul><br />
<li> TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>31.08 </td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (20 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li>1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li> 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li> 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>4µl 5x InFusion Pfu MasterMix</li><br />
<li>1,5µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li> <br> <br><br />
<br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>2 colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>02.09 </td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>Colony PCR<ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl Colony LB Medium (Colony 1/Colony 2)</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Programm <ul><br />
<li> initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li> final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<br />
</ul><br />
</li><br />
<li>Purification using Pure Yield Plasmid Miniprep System (Promega)<ul><br />
<li>Colonie 1 = pSB1C3-TLO-CMK 1<ul><br />
<li>c = 245,5 ng/ul </li><br />
<li>260/280 = 1,87</li><br />
<li>260/230 = 2,23</li><br />
</ul></li> <br><br />
<li>Colonie 2 = pSB1C3-TLO-CMK 2<ul><br />
<li>c = 107,7 ng/ul</li><br />
<li>260/280 = 1,95</li><br />
<li>260/230 = 2,28 </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>digestion with EcoRI and double digest with EcoRI and PstI</li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pattern is as expected</li><br />
<li>colony PCR product is off by 2000 bp</li><br />
<li>linearized plasmid is off by 1000 bp</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/b/ba/Darmstadt13_infusion_130902_1%282%29.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl forward primer</li><br />
<li>1µl reverse primer</li><br />
<li>1µl template</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO </li><br />
<li>5µl 10x Taq-Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>37µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li>PCR tube 4: frag4_for + frag4_rev</li><br />
<li>PCR tube 5: frag5_for + frag5_rev</li><br />
</ul></li> <br><br />
<li>template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li> PCR tube 4: frag4_for + frag4_rev </li><br />
<li>PCR tube 5: frag5_for + frag5_rev </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li>final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>besides many byproducts all fragments could be amplified from templates (framed bands)<ul><br />
<li>clones are most likely correct</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_infusion_130904_pSB1C3-TLO-CMK.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Induction of pSB1C3-TLO-CMK clones with L-Arabinose</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>induction with L-arabinose (0,02% w/v) at OD600 = 0,6<ul><br />
<li>induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>03.09 </td><br />
<td>Sequencing of pSB1C3-TLO-CMK clones</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>were not able to sequence the whole insert, as pimers did not anneal sufficiently</li><br />
<li>results indicate that all five fragments were successfully ligated in the correct order</li><br />
<li>results show that gBLOCK assembly of TLO and CMK did not work correctly</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
</table><br />
</front><br />
</p><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/humanpractice/Survey
Team:TU Darmstadt/humanpractice/Survey
2013-10-05T02:51:10Z
<p>Schiefie: </p>
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<font size="8" color="#F0F8FF" face="Arial regular">Survey |</font><br />
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<p><a href="http://www.umfrageonline.com/s/79f4fc0"><b><u><font size="6" color="#F0F8FF" face="Arial regular">Biotechnology in everyday life<center></u></b></a></p></font><br />
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2013-10-05T02:50:14Z
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<p><a href="http://www.umfrageonline.com/s/79f4fc0"><b><font size="6" color="#F0F8FF" face="Arial regular">Biotechnology in everyday life<center></b></a></p></font><br />
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Schiefie
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2013-10-05T02:49:47Z
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<font size="8" color="#F0F8FF" face="Arial regular">Idea |</font><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice/Survey"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Survey |</font><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice/Results"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Results </font><br />
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<br />
<br><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Survey</font></h2><br />
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<br><br />
<p><a href="http://www.umfrageonline.com/s/79f4fc0"><b><font size="6" color="#F0F8FF" face="Arial regular">Biotechnology in everyday life</b></a></p></font><br />
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Schiefie
http://2013.igem.org/Team:TU_Darmstadt/humanpractice/Survey
Team:TU Darmstadt/humanpractice/Survey
2013-10-05T02:48:35Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice/Survey"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Survey |</font><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice/Results"><br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Survey</font></h2><br />
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<br><br />
<p><a href="http://www.umfrageonline.com/s/79f4fc0"><b><font size="6" color="#F0F8FF" face="Arial regular">Biotechnology in everyday life</b></a></p><br />
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Schiefie
http://2013.igem.org/Team:TU_Darmstadt/humanpractice/Survey
Team:TU Darmstadt/humanpractice/Survey
2013-10-05T02:47:47Z
<p>Schiefie: </p>
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<br><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice/idea"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Idea |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice/Survey"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Survey |</font><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice/Results"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Results </font><br />
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<br />
<center><br />
<br><br />
<br />
<br><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Survey</font></h2><br />
<div id="all"><br />
</div><br />
<br><br />
<p><a href="http://www.umfrageonline.com/s/79f4fc0"><font size="6" color="#F0F8FF" face="Arial regular"><b>Biotechnology in everyday life</b></a></p><br />
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Schiefie
http://2013.igem.org/Team:TU_Darmstadt/humanpractice/Survey
Team:TU Darmstadt/humanpractice/Survey
2013-10-05T02:47:06Z
<p>Schiefie: </p>
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<p><a href="http://www.umfrageonline.com/s/79f4fc0"><font size="3" color="#F0F8FF" face="Arial regular">Biotechnology in everyday life</a></p><br />
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Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct
Team:TU Darmstadt/labbook/DetectionConstruct
2013-10-05T02:45:34Z
<p>Schiefie: </p>
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Construction of the Detection Construct</font></h2><br />
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The detection construct contains both fusionproteins, tar-LssmOrange and CheB-mKate, which are needed to put the mycotxin mediated FRET system to effect in an bacterial cell. Thus it comprises the following parts.<br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Traditional cloning approach</font></h2><br />
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We worked on this approach from the 10th of may till the 10th of june. It was abandoned due to major difficulties during restriction, ligation and transformation.<br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">gBLOCK approach</font></h2><br />
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We worked on this approach from the 11th of june till the 12th of august. As before it was abandoned due to major difficulties during restriction, ligation and transformation.<br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">InFusion approach</font></h2><br />
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<tr><br />
<td >13.08</td><br />
<td>gBLOCK assembly of CMK and TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>PCR mixture<br />
<ul><li>1 µL of each gBLOCK<br />
<ul><br />
<li>CMK: B_01 - B_04</li><br />
<li>TLO: A_01 - A_06</li><br />
</ul></li><br />
<li>10 µL 5x Q5 Reaction Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>1 µL Q5 Hot Start Polymerase</li><br />
<li>10 µL 5x Q5 High GC Enhancer</li><br />
<li>1 µL primer suffix-R (10 mM)</li><br />
<li>1 µL primer prefix_R (10 mM)</li><br />
</ul></ul></li><br><br />
<ul><li>PCR program (40 cycles)<br />
<ul><li>initial denaturation 94°C, 100s</li><br />
<li>denaturation 94°C, 55s</li><br />
<li>annealing 64°C, 55s</li><br />
<li>elongation 72°C, 120s</li><br />
<li>final elongation 72°C, 300s</li><br />
</li></ul></ul><br><br />
<ul><li>preparative 1% agarose gel<br />
<ul><li>gel displays bands of expected size, therefore assembly was successful</li><br />
</ul></li></ul><br />
</td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/6c/Darmstadt13_InFusion_130813.png" alt=""></td><br />
</tr><br />
<tr><br />
<td>13.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>using Wizard SV Gel and PCR Clean-Up System (Promega)<br />
<ul><br />
<li>TLO c = 34,4 ng/µL</li><br />
<li>CMK c = 35,6 ng/µL</li><br />
</ul><br />
</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 19.8. --><br />
<tr><br />
<td>19.08</td><br />
<td>Overlap extension PCR</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<br />
<td><ul><br />
<li>reaction mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev</li><br />
<li>CMK: CMK gBLOCK assembly + frag2_for + frag2_rev</li><br />
<li>terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev</li><br />
<li>pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev</li><br />
<li>TLO: TLO gBLOCK assembly + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3[fsC] + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 60°C, 40s</li><br />
<li>elongation: 72°C, 90s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays byproducts<ul><br />
<li>subsequent purification and amplification of desired bands (framed)</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/56/Darmstadt13_infusion_130819_InFusion_overlap_extension_PCR.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 10 ng/µL</li><br />
<li>CMK c = 30 ng/µL </li><br />
<li>terminator c = 7 ng/µL</li><br />
<li>pBAD4 c = 11 ng/µL</li><br />
<li>TLO c = 22 ng/µL</li><br />
<li> pSB1C3 c = 16 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>19.08 </td><br />
<td>Amplification PCR of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>9,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li> pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev </li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 70°C, 30s</li><br />
<li>elongation: 72°C, 30s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul> </li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature </li><br />
<li>terminator and pBAD4 show no bands</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Gradient PCR of the amplification of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>4 µL template</li><br />
<li> 1 µL forward primer (10 mM) </li><br />
<li> 1 µL reverse primer (10 mM)</li><br />
<li>6,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev</li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s</li><br />
<li>annealing 55°C/57°C/61°C/65°C, 30s</li><br />
<li>elongation 72°C, 30s</li><br />
<li> final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>optimal annealing temperature for terminator is 55°C</li><br />
<li>optimal annealing temperature for pBAD4 is 55°C</li><br />
</ul></li><br />
<br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_infusion_130820_InFusion_Gradient_PCR.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 20.8. --><br />
<br />
<tr><br />
<td>20.08</td><br />
<td>Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 45 ng/µL</li><br />
<li>terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73</li><br />
<li>pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 21.8. --><br />
<tr><br />
<td>21.08 </td><br />
<td>Amplification PCR of CMK, TLO and pSB1C3</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer</li><br />
<li>1 µL reverse primer</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev</li><br />
<li>TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s </li><br />
<li>annealing 55°C, 40s</li><br />
<li>elongation 72°C, 90s </li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>gel displays critical amount of byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 21.8 --><br />
<tr><br />
<td>22.08</td><br />
<td>Purification of CMK, TLO and pSB1C3 from preparative gel</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88</li><br />
<li>TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60</li><br />
<li> pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 22.8. --><br />
<tr><br />
<td>23.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<br />
<!--23.8.--><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs </li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/66/Darmstadt13_infusion_130823_InFusion_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 24.8.--><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O </li><br />
<br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li> annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands (just like on 23.08)<ul><br />
<li>probably the template is poorly </li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL) </li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO </li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li> 2 µL MgCl2 (50 mM) </li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s</li><br />
<li>annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
<br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/39/Darmstadt13_infusion_130824_2_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s</li><br />
<li> elongation 72°C, 300s </li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (71 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li> 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li>1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li>3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>18µl 5x InFusion Pfu MasterMix</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li><br />
<br><br><br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>no colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li> 27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/3b/Darmstadt13_infusion_130826_PCR_TLOamplification_AS.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>26.08 </td><br />
<td>Purification of TLO</td><br />
<td></td><br />
<br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19,7 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO (c = 19,7 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li> 1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles) <ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_infusion_130828_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>27.08</td><br />
<td> Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (1:10)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>25,5 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs (10 mM)</li><br />
<li>2,5 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s </li><br />
<li>elongation 72°C, 480s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel <ul><br />
<li>no distinctive bands (just like on 26.08)</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O </li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul> </li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>no distinctive bands (just like on 26.08)<ul><br />
<li>probably the template is poorly</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O</li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li> gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/ff/Darmstadt_infusion_130829_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<br />
<tr><br />
<td>29.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega) <ul><br />
<li> TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>31.08 </td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (20 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li>1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li> 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li> 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>4µl 5x InFusion Pfu MasterMix</li><br />
<li>1,5µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li> <br> <br><br />
<br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>2 colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>02.09 </td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>Colony PCR<ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl Colony LB Medium (Colony 1/Colony 2)</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Programm <ul><br />
<li> initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li> final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<br />
</ul><br />
</li><br />
<li>Purification using Pure Yield Plasmid Miniprep System (Promega)<ul><br />
<li>Colonie 1 = pSB1C3-TLO-CMK 1<ul><br />
<li>c = 245,5 ng/ul </li><br />
<li>260/280 = 1,87</li><br />
<li>260/230 = 2,23</li><br />
</ul></li> <br><br />
<li>Colonie 2 = pSB1C3-TLO-CMK 2<ul><br />
<li>c = 107,7 ng/ul</li><br />
<li>260/280 = 1,95</li><br />
<li>260/230 = 2,28 </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>digestion with EcoRI and double digest with EcoRI and PstI</li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pattern is as expected</li><br />
<li>colony PCR product is off by 2000 bp</li><br />
<li>linearized plasmid is off by 1000 bp</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/b/ba/Darmstadt13_infusion_130902_1%282%29.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl forward primer</li><br />
<li>1µl reverse primer</li><br />
<li>1µl template</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO </li><br />
<li>5µl 10x Taq-Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>37µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li>PCR tube 4: frag4_for + frag4_rev</li><br />
<li>PCR tube 5: frag5_for + frag5_rev</li><br />
</ul></li> <br><br />
<li>template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li> PCR tube 4: frag4_for + frag4_rev </li><br />
<li>PCR tube 5: frag5_for + frag5_rev </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li>final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>besides many byproducts all fragments could be amplified from templates (framed bands)<ul><br />
<li>clones are most likely correct</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_infusion_130904_pSB1C3-TLO-CMK.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Induction of pSB1C3-TLO-CMK clones with L-Arabinose</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>induction with L-arabinose (0,02% w/v) at OD600 = 0,6<ul><br />
<li>induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>03.09 </td><br />
<td>Sequencing of pSB1C3-TLO-CMK clones</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>were not able to sequence the whole insert, as pimers did not anneal sufficiently</li><br />
<li>results indicate that all five fragments were successfully ligated in the correct order</li><br />
<li>results show that gBLOCK assembly of TLO and CMK did not work correctly</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
</table><br />
</front><br />
</p><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct
Team:TU Darmstadt/labbook/DetectionConstruct
2013-10-05T02:43:39Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
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<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Construction of the Detection Construct</font></h2><br />
<br />
<br><br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
The detection construct contains both fusionproteins, tar-LssmOrange and CheB-mKate, which are needed to put the mycotxin mediated FRET system to effect in an bacterial cell. Thus it comprises the following parts.<br />
<br><br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Traditional cloning approach</font></h2><br />
<br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<br><br />
Was abandoned due to major difficulties during restriction, ligation and transformation<br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">gBLOCK approach</font></h2><br />
<br><br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br><br />
Was abandoned due to major difficulties during restriction, ligation and transformation<br />
<br><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">InFusion approach</font></h2><br />
<br />
<br><br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<br />
<br />
<table border="1" rules="rows" style="margin-left:50px; margin-right:50px"><br />
<br />
<br />
<tr><br />
<td >13.08</td><br />
<td>gBLOCK assembly of CMK and TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>PCR mixture<br />
<ul><li>1 µL of each gBLOCK<br />
<ul><br />
<li>CMK: B_01 - B_04</li><br />
<li>TLO: A_01 - A_06</li><br />
</ul></li><br />
<li>10 µL 5x Q5 Reaction Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>1 µL Q5 Hot Start Polymerase</li><br />
<li>10 µL 5x Q5 High GC Enhancer</li><br />
<li>1 µL primer suffix-R (10 mM)</li><br />
<li>1 µL primer prefix_R (10 mM)</li><br />
</ul></ul></li><br><br />
<ul><li>PCR program (40 cycles)<br />
<ul><li>initial denaturation 94°C, 100s</li><br />
<li>denaturation 94°C, 55s</li><br />
<li>annealing 64°C, 55s</li><br />
<li>elongation 72°C, 120s</li><br />
<li>final elongation 72°C, 300s</li><br />
</li></ul></ul><br><br />
<ul><li>preparative 1% agarose gel<br />
<ul><li>gel displays bands of expected size, therefore assembly was successful</li><br />
</ul></li></ul><br />
</td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/6c/Darmstadt13_InFusion_130813.png" alt=""></td><br />
</tr><br />
<tr><br />
<td>13.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>using Wizard SV Gel and PCR Clean-Up System (Promega)<br />
<ul><br />
<li>TLO c = 34,4 ng/µL</li><br />
<li>CMK c = 35,6 ng/µL</li><br />
</ul><br />
</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 19.8. --><br />
<tr><br />
<td>19.08</td><br />
<td>Overlap extension PCR</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<br />
<td><ul><br />
<li>reaction mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev</li><br />
<li>CMK: CMK gBLOCK assembly + frag2_for + frag2_rev</li><br />
<li>terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev</li><br />
<li>pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev</li><br />
<li>TLO: TLO gBLOCK assembly + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3[fsC] + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 60°C, 40s</li><br />
<li>elongation: 72°C, 90s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays byproducts<ul><br />
<li>subsequent purification and amplification of desired bands (framed)</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/56/Darmstadt13_infusion_130819_InFusion_overlap_extension_PCR.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 10 ng/µL</li><br />
<li>CMK c = 30 ng/µL </li><br />
<li>terminator c = 7 ng/µL</li><br />
<li>pBAD4 c = 11 ng/µL</li><br />
<li>TLO c = 22 ng/µL</li><br />
<li> pSB1C3 c = 16 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>19.08 </td><br />
<td>Amplification PCR of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>9,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li> pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev </li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 70°C, 30s</li><br />
<li>elongation: 72°C, 30s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul> </li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature </li><br />
<li>terminator and pBAD4 show no bands</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Gradient PCR of the amplification of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>4 µL template</li><br />
<li> 1 µL forward primer (10 mM) </li><br />
<li> 1 µL reverse primer (10 mM)</li><br />
<li>6,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev</li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s</li><br />
<li>annealing 55°C/57°C/61°C/65°C, 30s</li><br />
<li>elongation 72°C, 30s</li><br />
<li> final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>optimal annealing temperature for terminator is 55°C</li><br />
<li>optimal annealing temperature for pBAD4 is 55°C</li><br />
</ul></li><br />
<br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_infusion_130820_InFusion_Gradient_PCR.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 20.8. --><br />
<br />
<tr><br />
<td>20.08</td><br />
<td>Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 45 ng/µL</li><br />
<li>terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73</li><br />
<li>pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 21.8. --><br />
<tr><br />
<td>21.08 </td><br />
<td>Amplification PCR of CMK, TLO and pSB1C3</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer</li><br />
<li>1 µL reverse primer</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev</li><br />
<li>TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s </li><br />
<li>annealing 55°C, 40s</li><br />
<li>elongation 72°C, 90s </li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>gel displays critical amount of byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 21.8 --><br />
<tr><br />
<td>22.08</td><br />
<td>Purification of CMK, TLO and pSB1C3 from preparative gel</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88</li><br />
<li>TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60</li><br />
<li> pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 22.8. --><br />
<tr><br />
<td>23.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<br />
<!--23.8.--><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs </li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/66/Darmstadt13_infusion_130823_InFusion_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 24.8.--><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O </li><br />
<br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li> annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands (just like on 23.08)<ul><br />
<li>probably the template is poorly </li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL) </li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO </li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li> 2 µL MgCl2 (50 mM) </li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s</li><br />
<li>annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
<br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/39/Darmstadt13_infusion_130824_2_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s</li><br />
<li> elongation 72°C, 300s </li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (71 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li> 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li>1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li>3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>18µl 5x InFusion Pfu MasterMix</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li><br />
<br><br><br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>no colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li> 27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/3b/Darmstadt13_infusion_130826_PCR_TLOamplification_AS.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>26.08 </td><br />
<td>Purification of TLO</td><br />
<td></td><br />
<br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19,7 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO (c = 19,7 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li> 1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles) <ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_infusion_130828_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>27.08</td><br />
<td> Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (1:10)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>25,5 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs (10 mM)</li><br />
<li>2,5 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s </li><br />
<li>elongation 72°C, 480s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel <ul><br />
<li>no distinctive bands (just like on 26.08)</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O </li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul> </li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>no distinctive bands (just like on 26.08)<ul><br />
<li>probably the template is poorly</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O</li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li> gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/ff/Darmstadt_infusion_130829_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<br />
<tr><br />
<td>29.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega) <ul><br />
<li> TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>31.08 </td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (20 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li>1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li> 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li> 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>4µl 5x InFusion Pfu MasterMix</li><br />
<li>1,5µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li> <br> <br><br />
<br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>2 colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>02.09 </td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>Colony PCR<ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl Colony LB Medium (Colony 1/Colony 2)</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Programm <ul><br />
<li> initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li> final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<br />
</ul><br />
</li><br />
<li>Purification using Pure Yield Plasmid Miniprep System (Promega)<ul><br />
<li>Colonie 1 = pSB1C3-TLO-CMK 1<ul><br />
<li>c = 245,5 ng/ul </li><br />
<li>260/280 = 1,87</li><br />
<li>260/230 = 2,23</li><br />
</ul></li> <br><br />
<li>Colonie 2 = pSB1C3-TLO-CMK 2<ul><br />
<li>c = 107,7 ng/ul</li><br />
<li>260/280 = 1,95</li><br />
<li>260/230 = 2,28 </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>digestion with EcoRI and double digest with EcoRI and PstI</li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pattern is as expected</li><br />
<li>colony PCR product is off by 2000 bp</li><br />
<li>linearized plasmid is off by 1000 bp</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/b/ba/Darmstadt13_infusion_130902_1%282%29.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl forward primer</li><br />
<li>1µl reverse primer</li><br />
<li>1µl template</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO </li><br />
<li>5µl 10x Taq-Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>37µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li>PCR tube 4: frag4_for + frag4_rev</li><br />
<li>PCR tube 5: frag5_for + frag5_rev</li><br />
</ul></li> <br><br />
<li>template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li> PCR tube 4: frag4_for + frag4_rev </li><br />
<li>PCR tube 5: frag5_for + frag5_rev </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li>final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>besides many byproducts all fragments could be amplified from templates (framed bands)<ul><br />
<li>clones are most likely correct</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_infusion_130904_pSB1C3-TLO-CMK.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Induction of pSB1C3-TLO-CMK clones with L-Arabinose</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>induction with L-arabinose (0,02% w/v) at OD600 = 0,6<ul><br />
<li>induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>03.09 </td><br />
<td>Sequencing of pSB1C3-TLO-CMK clones</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>were not able to sequence the whole insert, as pimers did not anneal sufficiently</li><br />
<li>results indicate that all five fragments were successfully ligated in the correct order</li><br />
<li>results show that gBLOCK assembly of TLO and CMK did not work correctly</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
</table><br />
</front><br />
</p><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/Modelling/Statistics
Team:TU Darmstadt/Modelling/Statistics
2013-10-05T02:42:46Z
<p>Schiefie: </p>
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<font size="8" color="#F0F8FF" face="Arial regular">Statistics |</font><br />
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<br><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular"> Information Theory </font></h2><br />
<div id="all"><br />
<body><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<font size="5" color="#F0F8FF" face="Arial regular">The DKL Analysis</font><br />
<br><br />
<br><br />
<br />
<br />
In information theory the Kullback-Leibler-Divergence (DKL<sup><span style="color:blue">[1]</span></sup>) describes and quantifies the distance between<br />
two distributions P and Q. Where P denotes an experimental distribution, it is compared with Q, a reference distribution. DKL is also known as ‘relative entropy’ as well as ‘mutual information’.<br />
Although DKL is often used as a metric or distance measurement, it is not a true measurement because it is not symmetric. <br />
<br />
<br><br />
<br><br />
<br />
<center><br />
<img alt="DKL" src="/wiki/images/7/71/DKL.png" width="555" height="138"><br />
</center><br />
<br />
<br><br />
<br><br />
<br><br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
Here, P(i) and Q(i) denote the densities of P and Q at a position i.<br />
In our study, we use the DKL to describe the distances of the survey datasets from the human practice project.<br />
Therefore, we have to calculate a histogram out of the different datasets. Here, it is important to perform a constant binsize. In this approach we assume that a hypothetical distribution Q is uniformly distributed. <br />
To achieve this, we grate an appropriate test data set with the random generator runif in R. <br />
<br />
</p></font> <br />
<br><br />
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<br><br />
<br />
</body><br />
<br />
<br />
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<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Results</font></h2><br />
<br />
<br><br />
<br><br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
To validate the significance of our data we gathered in our human practice survey, we computed the DKL between our contestants and an artificial random distribution. High DKL values indicate a significant distribution.<br />
<br />
</p><br />
<br><br />
<br />
<center><br />
<br />
<img alt="Test" src="/wiki/images/e/ea/DKL2.png" width="400" height="400"><br />
<br />
<br><br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<br><br />
<br />
As can be seen in the graphic above, the DKL concerning our contestants indicate a significant data acquisition. For detailed analysis, see : <a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice/Results"><font size="3" color="#F0F8FF" face="Arial regular"><b>Human resource / results</b></a><br />
</p><br />
<br><br />
<br />
<center><br />
<br />
<img alt="Test" src="/wiki/images/c/c4/Gender_Produkts.png" width="400" height="400"><br />
</center><br />
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<br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
If we focus on the general opinion of male vs female contestants regarding basic research, we can observe a similar density distribution among the sexes. However, there seem to be more men, who tend to vote in the extreme end of the scale, with a tendency towards a positive opinion.<br />
</p><br />
<br><br />
<br />
<center><br />
<br />
<img alt="Test" src="/wiki/images/7/79/XXGender.png" width="400" height="400"><br />
<br />
</center><br />
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<br><br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
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If correlated to their age, men and women show the same density distribution. <br />
<br />
</p><br />
<br><br><br><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">References</font></h2><br />
<br />
<br><br />
<br><br />
<ol><br />
<li style="margin-left:15px; margin-right:50px; text-align:justify">Kullback, S.; Leibler, R.A. (1951) <i>On Information and Sufficiency</i> Annals of Mathematical Statistics 22 (1): 79–86. doi:10.1214/aoms/1177729694. MR 39968.</li><br />
</ol><br />
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Schiefie
http://2013.igem.org/Team:TU_Darmstadt/result
Team:TU Darmstadt/result
2013-10-05T02:26:33Z
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2013-10-05T02:17:17Z
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<tr><br />
<td>01.08</td><br />
<td>gBLOCK assembly of CMK and TLO</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>PCR mixture <ul><br />
<li>1 µL of each gBLOCK<ul><br />
<li>CMK: B_01 - B_04</li><br />
<li>TLO: A_01 - A_06</li><br />
</ul></li><br />
<li>10 µL 5x Q5 Reaction Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>1 µL Q5 Hot Start Polymerase</li><br />
<li>10 µL 5x Q5 High GC Enhancer</li><br />
<li>1 µL primer suffix-R (10 mM)</li><br />
<li>1 µL primer prefix_R (10 mM)</li><br />
</ul></li> <br><br />
<li>PCR program (40 cycles)<ul><br />
<li>initial denaturation 94°C, 100s</li><br />
<li>denaturation 94°C, 55s </li><br />
<li>annealing 64°C, 55s</li><br />
<li>elongation 72°C, 120s </li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays bands of expected size ' assembly was successful</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>01.08</td><br />
<td>Purification</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 7,7 ng/µL</li><br />
<li>CMK c = 7,1 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>02.08</td><br />
<td>Isolation of LssmOrange and mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<br />
<td>&nbsp;</td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume) <ul><br />
<li>1.5 µl DMSO </li><br />
<li>1 µL dNTPs</li><br />
<li>4 µL Pfu Polymerase</li><br />
<li>10 µL 5x Pfu buffer Buffer with MgSO4 </li><br />
<li>1 µL reverse primer </li><br />
<li>1 µL forward primer</li><br />
<li> 5 µl template</li><br />
<li>add 50 µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>LssmOrange: TLO (c = 7,7 ng/µL, 01.08) + LO-pre-F + LO-suf-R</li><br />
<li>mKate: CMK (c = 7,1 ng/µL, 01.08) + mKate-suf-R + mKate-pre-ATG-F</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 95°C, 300s</li><br />
<li>denaturation 95°C, 30s</li><br />
<li>annealing 55°C, 55s</li><br />
<li>elongation 72°C, 2 min</li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>PCR batches show the expected bands ' isolation was successful</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/f7/Darmstadt13_biobricks_IsolationPCR_LssmOrange_mKate.png" alt=""></td><br />
</tr><br />
<br />
<tr><br />
<td>02.08</td><br />
<td>Purification</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>LssmOrange1 c = 78,5 ng/µL 260/280 = 1,87 230/260 = 1,19</li><br />
<li>mKate1 c = 45,8 ng/µL 260/280 = 1,78 230/260 = 0,49</li><br />
<li>mKate2 c = 44,9 ng/µL 260/280 = 1,89 230/260 = 0,47</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>02.08</td><br />
<td> Restriction of LssmOrange and mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>double digest with PstI and EcoRI for 1 hour at 37°C</li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>03.08</td><br />
<td>Purification</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li> using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>LssmOrange1 c = 51,6 ng/µL 260/280 = 1,82 230/260 = 0,63</li><br />
<li> mKate1 c = 45,4 ng/µL 260/280 = 1,9 230/260 = 0,48</li><br />
<li>mKate2 c = 17,0 ng/µL 260/280 = 1,87 230/260 = 0,12</li><br />
<br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>03.08</td><br />
<td> Ligation of pSB1C3 with LssmOrange and pSB1C3 with mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>ligation mixture<ul><br />
<li>120 ng pSB1C3 cut with EcoRI and PstI</li><br />
<li> 124 ng insert cut with EcoRI and PstI</li><br />
<li>1 µL T4 DNA ligase (NEB)</li><br />
<li>2 µL 10x t4 DNA ligase buffer (NEB)</li><br />
<li>add to 20 µL</li><br />
</ul></li> <br><br />
<li> incubation for 30 minutes at room temperature</li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>03.08</td><br />
<td>Transformation</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>2 µL of every ligation batch were transformed into E.coli Top10 according to heat shock protocol<ul><br />
<li>only few colonies grew on LB-Cam plates</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>05.08</td><br />
<td>Colony PCR of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li> 1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl bacterial culture in LB-Cam medium </li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Program<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li> elongation 60s 72°C</li><br />
<li>final elongation 300s 72°C</li><br />
<br />
</ul></li> <br><br />
<li> analytical 1% agarose gel<ul><br />
<li>no colony yielded a band of approximately 800 bp, which would account for a positive result</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>09.08</td><br />
<td>Ligation of pSB1C3 with LssmOrange and pSB1C3 with mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>ligation mixture <ul><br />
<li>120 ng pSB1C3 cut with EcoRI and PstI</li><br />
<li> 124 ng insert cut with EcoRI and PstI</li><br />
<li>1 µL T4 DNA ligase (NEB)</li><br />
<li>2 µL 10x t4 DNA ligase buffer (NEB)</li><br />
<li>add to 20 µL</li><br />
</ul></li><br />
<li>incubation for 2 hours at 37°C and additionally incubation over 24 hours at 8°C </li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>10.08</td><br />
<td>Transformation</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>2 µL of every ligation batch were transformed into E.coli Top10 according to heat shock protocol<ul><br />
<li>a lot of colonies grew on LB-Cam plates</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>12.08</td><br />
<td>Colony PCR of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume)<ul><br />
<li>1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl bacterial culture in LB-Cam medium</li><br />
<li>2µl MgCl2 </li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Program<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li> annealing 30s 55°C</li><br />
<li>elongation 60s 72°C</li><br />
<li>final elongation 300s 72°C </li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>a lot clones gave a positive result</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>13.08</td><br />
<td>Sequencing of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>results show that all analyzed clones contain the expected sequence</li><br />
<li>one pSB1C3[LssmOrange] clone and one pSB1C3[mKate] clone were chosen </li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>26.08</td><br />
<td>Plasmid prep of pSB1C3[LssmOrange] clones and pSB1C3[mKate] clones</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using PureYield Plasmid Miniprep System (Promega)<ul><br />
<li>pSB1C3[LssmOrange] c = 96,4 ng/µL 260/280 = 1,68 230/260 = 1,46</li><br />
<li>pSB1C3[mKate] c = 74,8 ng/µL 260/280 = 1,86 230/260 = 1,82</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>13.09</td><br />
<td>Inoculation of DH5? pPR-IBA2 and subsequent plasmid prep</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>6 mL LB-Amp were inoculated with 50 µL of DH5? pPR-IBA2 <ul><br />
<li>using PureYield Plasmid Miniprep System (Promega)</li><br />
<li>pPR-IBA2 c = 71,6 ng/µL 260/280 = 1,82 260/230 = 1,76</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>13.09</td><br />
<td>Isolation PCR of LssmOrange and mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li> 5x Q5 Reaction Buffer</li><br />
<li>5x Q5 GC Enhancer</li><br />
<li>1 µL dNTPs (10mM)</li><br />
<li> 2,5 µL forward primer (10 mM)</li><br />
<li>2,5 µL reverse primer (10 mM)</li><br />
<li>1 µL template</li><br />
<li>0,5 µL Q5 Polymerase </li><br />
<li>22,5 µL nucleasefree water </li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>LssmOrange: pSB1C3[LssmOrange ] (c = 96,4 ng/µL, 1:100 dilution) + lssmorange pprf + lssmorange ppr</li><br />
<li>mKate: pSB1C3[mKate] (c = 74,8 ng/µL, 1:80 dilution) + mkate pprf + mkate ppr</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles) <ul><br />
<li>initial denaturation, 60 s, 98°C</li><br />
<li>denaturation, 10 s, 98°C</li><br />
<li>annealing, 30 s, 65°C</li><br />
<li>elongation, 60 s, 72°C</li><br />
<li>final elongation, 120 s, 72°C </li><br />
<br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>each PCR batch displays the expected band of approximately 700 bp</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>13.09</td><br />
<td>Purification</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>LssmOrange1 c = 51,4 ng/µL 260/280 = 1,69 230/260 = 1,59</li><br />
<li>LssmOrange1 c = 51,6 ng/µL 260/280 = 1,66 230/260 = 1,55</li><br />
<li>mKate1 c = 38,4 ng/µL 260/280 = 1,64 230/260 = 1,43</li><br />
<li>mKate2 c = 37,8 ng/µL 260/280 = 1,59 230/260 = 1,42</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>13.09</td><br />
<td>Restriction of pPR-IBA2, LssmOrange and mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>double digest with NheI and PstI at 37°C for 4 hours</li> <br><br />
<li>preparative 1% agarose gel <ul><br />
<li>LssmOrange and mKate show a band of the expected size </li><br />
<li>pPR-IBA2 shows intense band at 3000bp, weak band at 2250bp and weak band below 100bp<ul><br />
<li>we were unsure which band the correct one is, and cut out the 3000band as well as 2250band</li><br />
</ul></li><br />
<br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>14.09</td><br />
<td>Purification of pPR-IBA2, LssmOrange and mKate</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pPR-IBA2 (3000band) c = 31,2 ng/µL 260/280 = 1,88 230/260 = 1,00</li><br />
<li>pPR-IBA2 (2250band) c = 9,5 ng/µL 260/280 = 1,39 230/260 = 0,38 (neglected due to low purity)</li><br />
<li>LssmOrange c = 57,3 ng/µL 260/280 = 1,74 230/260 = 1,53</li><br />
<li>mKate c = 22,0 ng/µL 260/280 = 2,04 230/260 = 1,16</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>14.09</td><br />
<td>Ligation</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>ligation batch of pPR-IBA2[LssmOrange]<ul><br />
<li>3 µL pPR-IBA2 3000 (93 ng) </li><br />
<li>1,5 µL LssmOrange (72 ng)</li><br />
<li>2 µL 10x T4 Ligase Buffer (NEB)</li><br />
<li>1 µL T4 Ligase (NEB) </li><br />
<li>12,5 µL nucleasefree water</li><br />
</ul></li> <br><br />
<li>ligation batch of pPR-IBA2[mKate]<ul><br />
<li>3 µL pPR-IBA2 3000 (93 ng)</li><br />
<li>3,5 µL mKate (72 ng)</li><br />
<li> 2 µL 10x T4 Ligase Buffer (NEB)</li><br />
<li> 1 µL T4 Ligase (NEB)</li><br />
<li>10,5 µL nucleasefree water</li><br />
</ul></li> <br><br />
<li>incubation at room temperature for 30 minutes</li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>15.09</td><br />
<td>Transformation of pPR-IBA2[LssmOrange] and pPR-IBA2[mKate]</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>2 µL of ligation batch were transformed into E.coli DH5? according to heat shock protocol <ul><br />
<li>a lot of colonies grew on LB-Amp plates</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>16.09</td><br />
<td>Colony PCR of pPR-IBA2[LssmOrange] and pPR-IBA2[mKate]</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL forward primer</li><br />
<li>1 µL reverse primer</li><br />
<li>colony</li><br />
<li>2 µl MgCl2</li><br />
<li>1 µl dNTP Mix</li><br />
<li>1 µl DMSO</li><br />
<li>5 µl 10x Taq buffer</li><br />
<li>1 µl Taq-Polymerase </li><br />
<li>38 µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pSB1C3[LssmOrange]: lssmorange pprf + lssmorange ppr </li><br />
<li>pSB1C3[mKate]: mkate pprf + mkate ppr</li><br />
</ul></li> <br><br />
<li>PCR Program<ul><br />
<li>initial Denaturation 300s 95°C</li><br />
<li>Denaturation 30s 95°C</li><br />
<li>Annealing 30s 65°C</li><br />
<li>Elongation 60s 72°C </li><br />
<li>Final Elongation 300s 72°C </li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>2 positive clones for pPR-IBA2-LssmOrange</li><br />
<li>7 positive clones for pPR-IBA2-mKate</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/e/ef/Darmstadt13_biobricks_colonyPCR.png" alt="xxx"></td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>19.09</td><br />
<td>Plasmid prep</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>using PureYield Plasmid Miniprep System (Promega)<ul><br />
<li>pPR-IBA2[LssmOrange] clone 5 c = 51,2 ng/µL</li><br />
<li>pPR-IBA2[LssmOrange] clone 10 c = 55,5 ng/µL</li><br />
<li>pPR-IBA2[mKate] clone 2 c = 32,3 ng/µL</li><br />
<li>pPR-IBA2[mKate] clone 3 c = 35,2 ng/µL</li><br />
<li>pPR-IBA2[mKate] clone 4 c = 29,1 ng/µL</li><br />
<li>pPR-IBA2[mKate] clone 8 c = 37,6 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>19.09</td><br />
<td>Transformation in BL21DE3</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>1 µL of plasmid was transformed into E.coli DH5? according to heat shock protocol<ul><br />
<li>biofilm grew on LB-Amp plates </li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>21.09</td><br />
<td>Induction with IPTG</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>pPR-IBA2[LssmOrange] and pPR-IBA2[mKate] clones were cultivated in SOC-Amp medium at 37°C and 150 rpm to an OD600 = 0,6</li><br />
<li>subsequent induction with IPTG (2 mM end concentration) and cultivation overnight at 30°C and 150 rpm<ul><br />
<li>induction worked for pPR-IBA2[LssmOrange] clone 10 and pPR-IBA2[mKate] clone 3 and clone 4</li><br />
</ul></li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>23.09</td><br />
<td>Spectral analysis of induced clones</td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>cells and cell lysate was analyzed using an fluorospectrometer</li><br />
</ul></td><br />
<td>&nbsp;</td><br />
</tr><br />
<br />
<br />
<tr><br />
<td>27.09</td><br />
<td>SDS-PAGE</td><br />
<td>&nbsp;</td><br />
</tr><br />
<tr><br />
<td>&nbsp;</td><br />
<td><ul><br />
<li>Gel of Biobrick producing cells<br><br />
M.Marker NEB Color Prestaind Broadrange<br><br />
2.LssmOrange supernatant<br><br />
3.mKate cells<br><br />
4.Control (no visible band)<br><br />
</li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/c/cf/Darmstadt13_biobrick_Sdspage_29_09_13.png" width="50%" alt="xxx"></td><br />
</tr><br />
<br />
</table><br />
<br />
<br />
<br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/labbook/DetectionConstruct
Team:TU Darmstadt/labbook/DetectionConstruct
2013-10-05T02:16:28Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
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<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Construction of the Detection Construct</font></h2><br />
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<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
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The detection construct pSB1C3-pBAD-CheB-mKate-terminator-pBAD-tar-LssmOrange<br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">Traditional cloning approach</font></h2><br />
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<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
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Was abandoned due to major difficulties during restriction, ligation and transformation<br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">gBLOCK approach</font></h2><br />
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<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
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Was abandoned due to major difficulties during restriction, ligation and transformation<br />
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<h2><font size="6" color="#F0F8FF" face="Arial regular">InFusion approach</font></h2><br />
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<font size="3" color="#F0F8FF" face="Arial regular"><br />
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<table border="1" rules="rows" style="margin-left:50px; margin-right:50px"><br />
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<br />
<tr><br />
<td >13.08</td><br />
<td>gBLOCK assembly of CMK and TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>PCR mixture<br />
<ul><li>1 µL of each gBLOCK<br />
<ul><br />
<li>CMK: B_01 - B_04</li><br />
<li>TLO: A_01 - A_06</li><br />
</ul></li><br />
<li>10 µL 5x Q5 Reaction Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>1 µL Q5 Hot Start Polymerase</li><br />
<li>10 µL 5x Q5 High GC Enhancer</li><br />
<li>1 µL primer suffix-R (10 mM)</li><br />
<li>1 µL primer prefix_R (10 mM)</li><br />
</ul></ul></li><br><br />
<ul><li>PCR program (40 cycles)<br />
<ul><li>initial denaturation 94°C, 100s</li><br />
<li>denaturation 94°C, 55s</li><br />
<li>annealing 64°C, 55s</li><br />
<li>elongation 72°C, 120s</li><br />
<li>final elongation 72°C, 300s</li><br />
</li></ul></ul><br><br />
<ul><li>preparative 1% agarose gel<br />
<ul><li>gel displays bands of expected size, therefore assembly was successful</li><br />
</ul></li></ul><br />
</td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/6c/Darmstadt13_InFusion_130813.png" alt=""></td><br />
</tr><br />
<tr><br />
<td>13.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><li>using Wizard SV Gel and PCR Clean-Up System (Promega)<br />
<ul><br />
<li>TLO c = 34,4 ng/µL</li><br />
<li>CMK c = 35,6 ng/µL</li><br />
</ul><br />
</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 19.8. --><br />
<tr><br />
<td>19.08</td><br />
<td>Overlap extension PCR</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<br />
<td><ul><br />
<li>reaction mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pSB1C3[BBa_K206000] + frag1_for + frag4_rev</li><br />
<li>CMK: CMK gBLOCK assembly + frag2_for + frag2_rev</li><br />
<li>terminator: pSB1AK3[BBa_B0014] + frag3_for + frag3_rev</li><br />
<li>pBAD4: pSB1C3[BBa_K206000] + frag4_for + frag4_rev</li><br />
<li>TLO: TLO gBLOCK assembly + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3[fsC] + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 60°C, 40s</li><br />
<li>elongation: 72°C, 90s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays byproducts<ul><br />
<li>subsequent purification and amplification of desired bands (framed)</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/56/Darmstadt13_infusion_130819_InFusion_overlap_extension_PCR.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Purification</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 10 ng/µL</li><br />
<li>CMK c = 30 ng/µL </li><br />
<li>terminator c = 7 ng/µL</li><br />
<li>pBAD4 c = 11 ng/µL</li><br />
<li>TLO c = 22 ng/µL</li><br />
<li> pSB1C3 c = 16 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>19.08 </td><br />
<td>Amplification PCR of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer (10 mM)</li><br />
<li>1 µL reverse primer (10 mM)</li><br />
<li>9,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li> pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev </li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation: 98°C, 60s</li><br />
<li>denaturation: 98°C, 45s</li><br />
<li>annealing: 70°C, 30s</li><br />
<li>elongation: 72°C, 30s</li><br />
<li>final elongation: 72°C, 300s</li><br />
</ul> </li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pBAD1 shows single band of expected size, 70°C is the optimal annealing temperature </li><br />
<li>terminator and pBAD4 show no bands</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>19.08</td><br />
<td>Gradient PCR of the amplification of pBAD1, terminator and pBAD4</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (25 µL total volume)<ul><br />
<li>12,5 µL Q5 High Fidelity 2x Master Mix (NEB)</li><br />
<li>4 µL template</li><br />
<li> 1 µL forward primer (10 mM) </li><br />
<li> 1 µL reverse primer (10 mM)</li><br />
<li>6,5 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>pBAD1: pBAD1 (c = 10 ng/µL, 19.08) + frag1_for + frag4_rev</li><br />
<li>terminator: terminator (c = 7 ng/µL, 19.08) + frag3_for + frag3_rev</li><br />
<li>pBAD4: pBAD4 (c = 11 ng/µL, 19.08) + frag4_for + frag4_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s</li><br />
<li>annealing 55°C/57°C/61°C/65°C, 30s</li><br />
<li>elongation 72°C, 30s</li><br />
<li> final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>optimal annealing temperature for terminator is 55°C</li><br />
<li>optimal annealing temperature for pBAD4 is 55°C</li><br />
</ul></li><br />
<br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_infusion_130820_InFusion_Gradient_PCR.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 20.8. --><br />
<br />
<tr><br />
<td>20.08</td><br />
<td>Purification of PCR batches pBAD1 (Amplification PCR, 19.08), terminator 55°C (Gradient PCR, 19.08) and pBAD4 55°C (Gradient PCR, 19.08)</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>pBAD1 c = 45 ng/µL</li><br />
<li>terminator c = 44 ng/µL 260/280 = 1,67 260/230 = 0,73</li><br />
<li>pBAD4 c = 57,7 ng/µL 260/280 = 1,81 260/230 = 0,67</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 21.8. --><br />
<tr><br />
<td>21.08 </td><br />
<td>Amplification PCR of CMK, TLO and pSB1C3</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>25 µL Q5 High Fidelity 2x Master Mix</li><br />
<li>1 µL template</li><br />
<li>1 µL forward primer</li><br />
<li>1 µL reverse primer</li><br />
<li>22 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>CMK: CMK (c = 30 ng/µL, 19.08) + frag2_for + frag2_rev</li><br />
<li>TLO: TLO (c = 22 ng/µL, 19.08) + frag5_for + frag5_rev</li><br />
<li>pSB1C3: pSB1C3 (c = 16 ng/µL, 19.08) + vector_for + vector_rev</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 45s </li><br />
<li>annealing 55°C, 40s</li><br />
<li>elongation 72°C, 90s </li><br />
<li>final elongation 72°C, 300s</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>gel displays critical amount of byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<!-- 21.8 --><br />
<tr><br />
<td>22.08</td><br />
<td>Purification of CMK, TLO and pSB1C3 from preparative gel</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>CMK c = 154,2 ng/µL 260/280 = 1,92 260/230 = 1,88</li><br />
<li>TLO c = 37,9 ng/µL 260/280 = 1,90 260/230 = 0,60</li><br />
<li> pSB1C3 c = 120,8 ng/µL 260/280 = 1,89 260/230 = 1,64</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<!-- 22.8. --><br />
<tr><br />
<td>23.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<br />
<!--23.8.--><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs </li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/6/66/Darmstadt13_infusion_130823_InFusion_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<br />
<!-- 24.8.--><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 37,9 ng/µL, 22.08)</li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O </li><br />
<br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li> annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands (just like on 23.08)<ul><br />
<li>probably the template is poorly </li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>24.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL) </li><br />
<li> 10 µL 5x Phusion-Buffer </li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO </li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li> 2 µL MgCl2 (50 mM) </li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>27 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li> initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s</li><br />
<li>annealing 56°C, 30s</li><br />
<li>elongation 72°C, 720s</li><br />
<li>final elongation 72°C, 600s</li><br />
<br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/39/Darmstadt13_infusion_130824_2_PCR_TLO.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>2 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li>10 µL 5x Phusion-Buffer</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 4 µL Pfu-Polymerase</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>26 µL nuclease-free H2O</li><br />
</ul></li> <br><br />
<li> PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s</li><br />
<li> elongation 72°C, 300s </li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>25.08</td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (71 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li> 1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li>1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>42µl TLO (2440 bp, c = 19 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li>3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>18µl 5x InFusion Pfu MasterMix</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li><br />
<br><br><br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>no colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li> 27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li> 2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/3/3b/Darmstadt13_infusion_130826_PCR_TLOamplification_AS.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>26.08 </td><br />
<td>Purification of TLO</td><br />
<td></td><br />
<br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega)<ul><br />
<li>TLO c = 19,7 ng/µL</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
<br />
</tr><br />
<br />
<tr><br />
<td>26.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO (c = 19,7 ng/µL)</li><br />
<li> 1 µL frag5_rev (10 mM)</li><br />
<li> 1 µL frag5_for (10 mM)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>27 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs</li><br />
<li>2 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles) <ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 55°C, 30s</li><br />
<li>elongation 72°C, 300s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li> preparative 1% agarose gel<ul><br />
<li>no distinctive bands</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_infusion_130828_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>27.08</td><br />
<td> Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li> PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:4 dilution, final amount = 5 ng)</li><br />
<li>1 µL frag5_rev (10 mM)</li><br />
<li>1 µL frag5_for (1:10)</li><br />
<li>10 µL 5x Phusion Buffer</li><br />
<li>25,5 µL nucleasefree H2O</li><br />
<li>2 µL dNTPs (10 mM)</li><br />
<li>2,5 µL DMSO</li><br />
<li>2 µL MgCl2 (50 mM)</li><br />
<li>4 µL Pfu-Polymerase</li><br />
<li>2 µL BSA (10 mg/mL, final concentration = 0,4 µg/µL)</li><br />
<br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li> denaturation 98°C, 30s </li><br />
<li>annealing 55°C, 30s </li><br />
<li>elongation 72°C, 480s</li><br />
<li>final elongation 72°C, 600s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel <ul><br />
<li>no distinctive bands (just like on 26.08)</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1 µL TLO (c = 19,7 ng/µL, 1:20 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O </li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s </li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul> </li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li>no distinctive bands (just like on 26.08)<ul><br />
<li>probably the template is poorly</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>28.08</td><br />
<td>Amplification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume) <ul><br />
<li>1 µL TLO gBLOCK assembly (c = 34,4 ng/µL, 1:30 dilution, final amount = 1 ng)</li><br />
<li>2,5 µL primer frag5_rev (10 mM)</li><br />
<li>2,5 µL primer frag5_for (10 mM)</li><br />
<li>19 µL nucleasefree H2O</li><br />
<li>25 µL 2x Q5 Mastermix (NEB)</li><br />
</ul></li> <br><br />
<li>PCR program (35 cycles)<ul><br />
<li>initial denaturation 98°C, 60s</li><br />
<li>denaturation 98°C, 30s</li><br />
<li>annealing 66°C, 30s</li><br />
<li>elongation 72°C, 90s</li><br />
<li>final elongation 72°C, 120s</li><br />
</ul></li> <br><br />
<li>preparative 1% agarose gel<ul><br />
<li> gel displays the desired band of 2500 bp as well as byproducts</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/f/ff/Darmstadt_infusion_130829_PCR_TLOamplification.png" alt="x"></td><br />
</tr><br />
<br />
<tr><br />
<td>29.08</td><br />
<td>Purification of TLO</td><br />
<td></td><br />
</tr><br />
<tr><br />
<td></td><br />
<td><ul><br />
<li>using Wizard SV Gel and PCR Clean-Up System (Promega) <ul><br />
<li> TLO c = 241,7 ng/µL 260/280 = 1,93 260/230 = 2,02</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<td>31.08 </td><br />
<td>InFusion</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<br />
<td></td><br />
<td><ul><br />
<li>reaction mixture (20 µL total volume)<ul><br />
<li>1µl pBAD1 (130 bp, c = 45 ng/µL, 45 ng)</li><br />
<li>1µl pBAD4 (130 bp, c = 57,7 ng/µL, 58 ng)</li><br />
<li> 1µl terminator (100 bp, c = 44 ng/µL, 44 ng)</li><br />
<li>3,5µl TLO (2440 bp, c = 241,7 ng/µL, 780 ng)</li><br />
<li>4,5µl CMK (1850 bp, c = 154,2 ng/µL, 680 ng)</li><br />
<li> 3,5µl pSB1C3 (2100 bp, c = 120,8 ng/µL, 360 ng)</li><br />
<li>4µl 5x InFusion Pfu MasterMix</li><br />
<li>1,5µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>treatment of the reaction mixture according to InFusion protocol</li> <br> <br><br />
<br />
<li>transformation into E.coli Top10 according to heat shock protocol<ul><br />
<li>2 colonies grew on LB-Cam plates, most likely the reaction volume was to high</li><br />
</ul></li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>02.09 </td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using colony PCR and test restriction</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>Colony PCR<ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl VR primer (10 mM)</li><br />
<li>1µl VF2 primer (10 mM)</li><br />
<li>5µl Colony LB Medium (Colony 1/Colony 2)</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO</li><br />
<li>5µl 10x Taq Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>33µl nucleasefree H2O</li><br />
</ul></li> <br><br />
<li>PCR Programm <ul><br />
<li> initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li> final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<br />
</ul><br />
</li><br />
<li>Purification using Pure Yield Plasmid Miniprep System (Promega)<ul><br />
<li>Colonie 1 = pSB1C3-TLO-CMK 1<ul><br />
<li>c = 245,5 ng/ul </li><br />
<li>260/280 = 1,87</li><br />
<li>260/230 = 2,23</li><br />
</ul></li> <br><br />
<li>Colonie 2 = pSB1C3-TLO-CMK 2<ul><br />
<li>c = 107,7 ng/ul</li><br />
<li>260/280 = 1,95</li><br />
<li>260/230 = 2,28 </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>digestion with EcoRI and double digest with EcoRI and PstI</li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>pattern is as expected</li><br />
<li>colony PCR product is off by 2000 bp</li><br />
<li>linearized plasmid is off by 1000 bp</li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/b/ba/Darmstadt13_infusion_130902_1%282%29.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Control of pSB1C3-CMK-TLO clone 1 and 2 using PCR</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>PCR mixture (50 µL total volume)<ul><br />
<li>1µl forward primer</li><br />
<li>1µl reverse primer</li><br />
<li>1µl template</li><br />
<li>2µl MgCl2</li><br />
<li>1µl dNTP Mix</li><br />
<li>1µl DMSO </li><br />
<li>5µl 10x Taq-Buffer</li><br />
<li>1µl Taq-Polymerase</li><br />
<li>37µl H2O</li><br />
</ul></li> <br><br />
<li>template and primer specifications<ul><br />
<li>template = pSB1C3-TLO-CMK 1, c = 245,5 ng/µl, 1:10 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li>PCR tube 4: frag4_for + frag4_rev</li><br />
<li>PCR tube 5: frag5_for + frag5_rev</li><br />
</ul></li> <br><br />
<li>template = pSB1C3-TLO-CMK 2, c = 107,7 ng/µl, 1:4 dilution<ul><br />
<li>PCR tube 1: frag1_for + frag4_rev</li><br />
<li>PCR tube 2: frag2_for + frag2_rev</li><br />
<li>PCR tube 3: frag3_for + frag3_rev </li><br />
<li> PCR tube 4: frag4_for + frag4_rev </li><br />
<li>PCR tube 5: frag5_for + frag5_rev </li><br />
</ul></li> <br><br />
<br />
</ul></li><br />
<li>PCR program (30 cycles)<ul><br />
<li>initial denaturation 300s 95°C</li><br />
<li>denaturation 30s 95°C</li><br />
<li>annealing 30s 55°C</li><br />
<li>elongation 150s 72°C</li><br />
<li>final elongation 300s 72°C</li><br />
</ul></li> <br><br />
<li>analytical 1% agarose gel<ul><br />
<li>besides many byproducts all fragments could be amplified from templates (framed bands)<ul><br />
<li>clones are most likely correct</li><br />
</ul></li><br />
</ul></li><br />
</ul></td><br />
<td><img src="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_infusion_130904_pSB1C3-TLO-CMK.png" alt="x"></td><br />
</tr><br />
<tr><br />
<td>03.09</td><br />
<td>Induction of pSB1C3-TLO-CMK clones with L-Arabinose</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>induction with L-arabinose (0,02% w/v) at OD600 = 0,6<ul><br />
<li>induced clones showed no difference to native E.coli Top10 cells when analyzed with an fluorospcetrometer</li><br />
</ul> </li><br />
</ul></td><br />
<td></td><br />
</tr><br />
<tr><br />
<td>03.09 </td><br />
<td>Sequencing of pSB1C3-TLO-CMK clones</td><br />
<td></td><br />
</tr><br />
<br />
<tr><br />
<br />
<td></td><br />
<td><ul><br />
<li>were not able to sequence the whole insert, as pimers did not anneal sufficiently</li><br />
<li>results indicate that all five fragments were successfully ligated in the correct order</li><br />
<li>results show that gBLOCK assembly of TLO and CMK did not work correctly</li><br />
</ul></td><br />
<td></td><br />
</tr><br />
</table><br />
</front><br />
</p><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/Plasmids
Team:TU Darmstadt/materials/Plasmids
2013-10-05T02:05:15Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
</center><br />
<br />
<br />
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<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Labbook |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
<br />
<br />
<br><br />
<br><br />
<br><br />
<br />
<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Plasmids</font></h2><br />
</center><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<ul><br />
<li><a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3"><font size="3" color="#F0F8FF" face="Arial regular"><b>pSB1C3</b></font></a></li><br />
<li><a href=http://lucerna-chem.ch/shop/558271><font size="3" color="#F0F8FF" face="Arial regular"><b> pPR-IBA2 </b></font></a></li><br />
</ul><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/Plasmids
Team:TU Darmstadt/materials/Plasmids
2013-10-05T02:01:35Z
<p>Schiefie: </p>
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<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Labbook |</font><br />
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<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
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<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
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<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Plasmids</font></h2><br />
<br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
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<ul><br />
<li><a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3"><font size="3" color="#F0F8FF" face="Arial regular"><b>pSB1C3</b></font></a></li><br />
<li><a href=http://lucerna-chem.ch/shop/558271><font size="3" color="#F0F8FF" face="Arial regular"><b> pPR-IBA2 </b></font></a></li><br />
</ul><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/Plasmids
Team:TU Darmstadt/materials/Plasmids
2013-10-05T02:01:00Z
<p>Schiefie: </p>
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<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Labbook |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<br />
<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Plasmids</font></h2><br />
<br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<ul><br />
<li><a href="http://parts.igem.org/Part:pSB1C3?title=Part:pSB1C3"><font size="3" color="#F0F8FF" face="Arial regular"><b>pSB1C3</b></font></a></li><br />
<li><a href=http://lucerna-chem.ch/shop/558271><font size="3" color="#F0F8FF" face="Arial regular"><b> pPR-IBA2 </b></font></a></li><br />
</ul><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/protocols/Protein_Expression
Team:TU Darmstadt/protocols/Protein Expression
2013-10-05T01:59:52Z
<p>Schiefie: </p>
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<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
<br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/2/2c/Darmstadt_green_Result.jpg" width="80" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
</div><br />
<br />
<br><br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Protein Expression</font></h2><br />
<body><br />
<br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular"><br />
<B> Materials<br></B></font></p><br />
<br />
<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<br><br />
<B>Equipment<br></B><br />
<div align="left" style="margin-left:60px; margin-right:50px"><br />
<ul><br />
<li class=list1>- Incubation shaker</li><br />
<li class=list1>- Photometer</li><br />
</ul><br />
</div><br />
</font><br />
</p><br />
<br />
<p text-aligne:left style="margin-left:60px; margin-right:50px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<br><br />
<B>Chemicals & consumables<br></B><br />
<div align="left" style="margin-left:60px; margin-right:50px"><br />
<ul><br />
<li class=list1>- E. coli BL21 DE3</li><br />
<li class=list1>- <a href="https://2013.igem.org/Team:TU_Darmstadt/materials/Media"><font size="3" color="#F0F8FF" face="Arial regular"><b>DYT Medium</b></font></a></li><br />
<li class=list1>- IPTG</li><br />
<li class=list1>- 100 ml and 3 l flasks</li><br />
<li class=list1>- ice</li><br />
</ul><br />
</div><br />
</font><br />
</p><br />
<br><br />
<br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><font size="4.5" color="#F0F8FF" face="Arial regular"><br />
<B> Procedure<br></B></font></p><br><br />
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<div align="left" style="margin-left:30px; margin-right:50px"><br />
<ol><br />
<li>Inoculation of 50 mL DYT medium in the 100 mL flask with <i>E. coli</i> BL21 DE3 containing <a href=http://lucerna-chem.ch/shop/558271><font size="3" color="#F0F8FF" face="Arial regular"><b> pPR-IBA2 </b></font></a><br />
plasmid with the sequence for the respective part to be expressed.</li><br />
<li>Incubation at 180 repulsion per minute (rpm) at 30°C to an OD600= 4</li><br />
<li>Transferation of the starter culture into 1 L DYT medium in a 3 L flask resulting in an OD600= 0.2</li><br />
<li>Incubation to an OD600= 0.6 at 180 rpm and 30°C.</li><br />
<li>Incubation for 15 minutes on ice.</li><br />
<li>Induction of the proteinexpression with 20 mL of IPTG (stock conentration 1M).</li><br />
<li>Incubation of the cell suspension over night at 180 rpm at 30°C.</li><br />
</ol><br />
</div><br />
</font></p></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/Primers
Team:TU Darmstadt/materials/Primers
2013-10-05T01:43:02Z
<p>Schiefie: </p>
<hr />
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<div id="taskbar"><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
<br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/2/2c/Darmstadt_green_Result.jpg" width="80" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<br />
<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Primers</font></h2><br />
<br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<br />
VR: ATTACCGCCTTTGAGTGAGC<br><br />
VF2: TGCCACCTGACGTCTAAGAA<br><br />
<br><br />
<br />
Suffix-R: TACTAGTAGCGGCCGCTGCAGGGTAA<br><br />
Prefix-R: GTAGTAGAATTCGCGGCCGCTTCTAG<br><br />
<br><br />
<br />
LO-suf-R: GTAGTACTGCAGCGGCCGCTACTAGTATTATGATTTGTACAGCTCGTCCATGCC<br><br />
LO-pre-F: GTTGTTGAATTCGCGGCCGCTTCTAGATGGTGAGCAAAGGCGAAGAAAATAACATGG<br><br />
<br><br />
<br />
mKate-suf-R: GTAGTACTGCAGCGGCCGCTACTAGTATTATTAATTCAGTTTATGACCCAGGTTGCTCG<br><br />
mKate-pre-ATG-F: GTTGTTGAATTCGCGGCCGCTTCTAGATGGTAAGCAAAGGTGAAGAGTTAATCAAGGAGA<br><br />
<br><br />
<br />
lssmorange ppr: CCCCCTGCAGGTTTATTATTTGTACAGCTCGTCC<br><br />
lssmorange pprf: ATATGGCTAGCATGGTGAGCAAAGGCG<br><br />
<br><br />
<br />
mkate ppr: GTCCCCCTGCAGGTTTATTAATTCAGTTTATG<br><br />
mkate pprf: TACATATGGCTAGCATGGTAAGCAAAGGTG<br><br />
<br><br />
<br />
frag4_rev: CTCTTTCCGCTAGCCCAAAAAAACGGTATGGAG<br><br />
frag1_for: CGGCCGCTTCTAGAGACATTGATTATTTGCACGGCGT<br><br />
<br><br />
<br />
frag2_rev: CAGTGTGATTATTAATTCAGTTTATGACCCAGGTTGCTCG<br><br />
frag2_for: GGCTAGCGGAAAGAGGGGACAAATGTCAAAAATCC<br><br />
<br><br />
<br />
frag3_rev: ATCAATGTAAATAATAAAAAAGCCGGATTAATAATCTGGCTTTTTATATTCT<br><br />
frag3_for: TTAATAATCACACTGGCTCACCTTCGG<br><br />
<br><br />
<br />
frag4_rev: CTCTTTCCGCTAGCCCAAAAAAACGGTATGGAG<br><br />
frag4_for: ATTATTTACATTGATTATTTGCACGGCGT<br><br />
<br><br />
<br />
frag5_rev: CGGCCGCTACTAGTATTATCATTTGTACAGCTCGTCCATGC<br><br />
frag5_for: TTTTTTTGGGCTAGCGGAAAGAGGGGACAAATGATTAACCG<br><br />
<br><br />
<br />
vector_rev: CTCTAGAAGCGGCCGCGA<br><br />
vector_for: TACTAGTAGCGGCCGCTGC<br><br />
<br><br />
<br />
Pre-PezT: GGATCCTGCAGCGGCCGCTACTAGTACTCGAGCGCTGCTGCCA<br><br />
For-PezT: ATACCGAATTCGCGGCCGCTTCTAGATGGAAATCCAAGATTATACTG<br><br />
<br><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/result/electrical_engineering
Team:TU Darmstadt/result/electrical engineering
2013-10-05T01:25:05Z
<p>Schiefie: </p>
<hr />
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<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
<br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/c/ca/04._Result_(angewählt).jpg" width="80" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/f/f3/Darmstadt_green_Labbook.jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
</div><br />
<br />
<br />
<br><br><br> <br> <br><br />
<h1 align="center"><font size="6" color="#F0F8FF" face="Arial regular">Electrical engineering</font></h1><br />
<body><br />
<br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<br />
<b>Handheld development:</b><br><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
blabblabalalbal <sup><span style="color:blue">[1]</span></sup> showed that LSSmOrange has an excitation maximum by<br />
blablabla<br />
</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2013/9/9b/Darmstadt13_elEng_handheld_Steckplatine.png" alt="" width="50%"><br />
<b>LED Sensing:</b><br><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYy<br />
</p><br />
<br><br><br />
<br />
<b>App development:</b><br><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<div align="left"><br />
<img alt="Choose Mykotoxine" src="/wiki/images/8/89/Choose_Mykotoxin.png" width="200" height="400"> <br><br />
Graph 1: "Screenshot from an smart phone display"<br><br />
<br><br><br><br><br />
<br><br />
</div><br />
<br />
<div align="right"><br />
<img alt="Plug" src="/wiki/images/e/ee/Screenshot_plug.png" width="200" height="400"><br><br />
Graph 1: "Screenshot from an smart phone display"<br><br />
<br><br><br><br><br />
</div><br />
<br />
</p><br />
<br><br><br />
<br />
<b>Bluetooth communication:</b><br><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYy<br />
</p><br />
<br><br><br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<center><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">References</font></h2></center><br />
<body><br />
<br />
<ol><br />
<br />
<li style="margin-left:15px; margin-right:50px; text-align:justify">Daria M. Shcherbakova et al. (2012) <i>An Orange Fluorescent Protein with a Large Stokes Shift for Single-Excitation Multicolor FCCS and FRET Imaging.</i> J. Am. Chem. Soc. 134 (18), 7913–7923</li><br />
<br />
<li style="margin-left:15px; margin-right:50px; text-align:justify">http://www.evrogen.com/products/basicFPs.shtml</li><br />
<br />
</ol><br />
<br />
<!-- <font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
G.W.Mitchell and J.W.Hastings <br />
</body><br />
<br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/result/electrical_engineering
Team:TU Darmstadt/result/electrical engineering
2013-10-05T01:24:21Z
<p>Schiefie: </p>
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<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
<br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/c/ca/04._Result_(angewählt).jpg" width="80" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/f/f3/Darmstadt_green_Labbook.jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
</div><br />
<br />
<br />
<br><br><br> <br> <br><br />
<h1 align="center"><font size="6" color="#F0F8FF" face="Arial regular">Electrical engineering</font></h1><br />
<body><br />
<br><br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<br />
<b>Handheld development:</b><br><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
blabblabalalbal <sup><span style="color:blue">[1]</span></sup> showed that LSSmOrange has an excitation maximum by<br />
blablabla<br />
</p><br />
<br><br><br />
<img src="https://static.igem.org/mediawiki/2013/9/9b/Darmstadt13_elEng_handheld_Steckplatine.png" alt=""><br />
<b>LED Sensing:</b><br><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYYy<br />
</p><br />
<br><br><br />
<br />
<b>App development:</b><br><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<div align="left"><br />
<img alt="Choose Mykotoxine" src="/wiki/images/8/89/Choose_Mykotoxin.png" width="200" height="400"> <br><br />
Graph 1: "Screenshot from an smart phone display"<br><br />
<br><br><br><br><br />
<br><br />
</div><br />
<br />
<div align="right"><br />
<img alt="Plug" src="/wiki/images/e/ee/Screenshot_plug.png" width="200" height="400"><br><br />
Graph 1: "Screenshot from an smart phone display"<br><br />
<br><br><br><br><br />
</div><br />
<br />
</p><br />
<br><br><br />
<br />
<b>Bluetooth communication:</b><br><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
YYYYYYYYYYYYYYYYYYYYYYYYYYYYYYy<br />
</p><br />
<br><br><br />
<br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
<center><br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">References</font></h2></center><br />
<body><br />
<br />
<ol><br />
<br />
<li style="margin-left:15px; margin-right:50px; text-align:justify">Daria M. Shcherbakova et al. (2012) <i>An Orange Fluorescent Protein with a Large Stokes Shift for Single-Excitation Multicolor FCCS and FRET Imaging.</i> J. Am. Chem. Soc. 134 (18), 7913–7923</li><br />
<br />
<li style="margin-left:15px; margin-right:50px; text-align:justify">http://www.evrogen.com/products/basicFPs.shtml</li><br />
<br />
</ol><br />
<br />
<!-- <font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
G.W.Mitchell and J.W.Hastings <br />
</body><br />
<br />
</html></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_elEng_handheld_Steckplatine.png
File:Darmstadt13 elEng handheld Steckplatine.png
2013-10-05T01:22:19Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/Primers
Team:TU Darmstadt/materials/Primers
2013-10-05T01:17:19Z
<p>Schiefie: </p>
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<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
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<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
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<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Lab book |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<br />
<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">Primers</font></h2><br />
<br />
<br />
<font size="3" color="#F0F8FF" face="Arial regular"><br />
<p text-aligne:left style="margin-left:50px; margin-right:50px"><br />
<br />
<br />
VR: ATTACCGCCTTTGAGTGAGC<br><br />
VF2: TGCCACCTGACGTCTAAGAA<br><br />
<br><br />
<br />
Suffix-R:<br><br />
Prefix-R:<br><br />
<br><br />
<br />
LO-suf-R:<br><br />
LO-pre-F:<br><br />
<br><br />
<br />
mKate-suf-R:<br><br />
mKate-pre-ATG-F:<br><br />
<br><br />
<br />
lssmorange ppr:<br><br />
lssmorange pprf:<br><br />
<br><br />
<br />
mkate ppr:<br><br />
mkate pprf:<br />
<br><br />
<br />
frag4_rev:<br />
frag1_for:<br />
<br><br />
<br />
frag2_rev:<br />
frag2_for:<br />
<br><br />
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frag3_rev:<br />
frag3_for:<br />
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frag4_rev:<br />
frag4_for:<br />
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frag5_rev:<br />
frag5_for:<br />
<br><br />
<br />
vector_rev:<br />
vector_for:<br />
<br><br />
<br />
Pre-PezT: GGA TCC TGC AGC GGC CGC TAC TAG TAC TCG AGC GCT GCT GCC A<br />
For-PezT: ATA CCG AAT TCG CGG CCG CTT CTA GAT GGA AAT CCA AGA TTA TAC TG<br />
<br><br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/Team:TU_Darmstadt/materials/gBlocks
Team:TU Darmstadt/materials/gBlocks
2013-10-05T01:11:43Z
<p>Schiefie: </p>
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<!-- central main menu --><br />
<br />
<br><br />
<br><br />
<br />
<br />
<!-- Taskbar --><br />
<div id="taskbar"><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt"><br />
<img alt="Home_ausgewählt" src="/wiki/images/e/ef/Darmstadt_green_Home.jpg" width="70" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/problem"><br />
<img alt="Problem" src="/wiki/images/6/66/Darmstadt_green_Problem.jpg" width="100" height="30"></a><br />
<br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/result"><br />
<img alt="result" src="/wiki/images/2/2c/Darmstadt_green_Result.jpg" width="80" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/safety"><br />
<img alt="safety" src="/wiki/images/7/7a/Darmstadt_green_Safety.jpg" width="90" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/team"><br />
<img alt="team" src="/wiki/images/a/a4/Darmstadt_green_Team.jpg" width="70" height="30"></a><br />
<br><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/strategy"><br />
<img alt="team" src="/wiki/images/a/a0/Darmstadt_green_Strategy.jpg" width="100" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/humanpractice"><br />
<img alt="team" src="/wiki/images/4/4f/Darmstadt_green_Human_Practice.jpg" width="150" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/modelling"><br />
<img alt="team" src="/wiki/images/0/06/Darmstadt_green_Modelling.jpg" width="110" height="30"></a><br />
<br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<img alt="team" src="/wiki/images/4/4c/10._Labbook_(angewählt).jpg" width="90" height="30"></a><br />
<br />
<br />
<br />
<br />
</div><br />
<br><br><br><br><br />
<center><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/labbook"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Labbook |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/materials"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Materials |</font><br />
</a><br />
<a href="https://2013.igem.org/Team:TU_Darmstadt/protocols"><br />
<font size="8" color="#F0F8FF" face="Arial regular">Protocols</font><br />
</a><br />
</center><br />
<br><br />
<br />
<br />
<br />
<body><br />
<br />
<h2><font size="6" color="#F0F8FF" face="Arial regular">gBlocks</font></h2><br />
<br />
<font size="5" color="#F0F8FF" face="Arial regular"><p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
CMK<br />
</p></font><br />
<br />
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<div align="left" style="margin-left:30px; margin-right:50px"><br />
<ol><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/6/68/Darmstadt13_mat_gbl_B_01_166474000Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">B_01:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GTTGTTGAATTCGCGGCCGCTTCTAGAGGAAAGAGGGGACAAATGTCAAAAATCCGTGTCCTTAGCGTGGATGATTCTGCTTTGATGCGCCAGATCATGACTGAAATTATCAACTCACACAGTGATATGGAAATGGTGGCAACCGCACCGGATCCCCTGGTTGCGCGCGACCTTATTAAGAAATTTAACCCTGATGTCTTAACCCTTGACGTAGAAATGCCGCGTATGGATGGCCTGGACTTCCTGGAAAAACTGATGCGCCTGCGTCCCATGCCGGTGGTGATGGTCTCCTCACTGACGGGAAAGGGCTCCGAGGTGACGCTGCGTGCTCTGGAACTGGGGGCCATCGATTTCGTTACTAAACCGCAACTTGGTATCCGCGAGGGTATGCTGGCGTACAACGAAATGATTGCAGAAAAAGTACGTACGGCAGCGAAAGCGTCGTTAGCGGCGCACAAACCACTCAGCGCCCCAACAACGCTGAAAGCTGGCCCATTATT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/b/b1/Darmstadt13_mat_gbl_B_02_166486021Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">B_02:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GCTGAAAGCTGGCCCATTATTAAGTAGCGAAAAACTCATTGCAATTGGCGCGTGCACCGGGGGCACGGAAGCGATCCGTCATGTATTACAACCGCTGCCGCTCAGCTCACCGGCCCTTCTGATCACCCAACACATGCCACCGGGCTTTACTCGTAGTTTTGCGGATCGCCTGAATAAGTTATGCCAAATTGGGGTAAAAGAAGCGGAAGATGGCGAGCGTGTCCTGCCGGGCCATGCCTATATTGCGCCGGGCGATCGTCATATGGAGCTTTCACGGAGTGGCGCAAATTATCAGATTAAAATCCATGACGGTCCGGCTGTGAATCGGCATCGGCCGTCCGTGGACGTCCTGTTCCATTCCGTGGCGAAACAGGCCGGCCGCAATGCTGTCGGCGTCATTCTGACAGGCATGGGGAATGATGGCGCGGCCGGTATGCTGGCAATGCGCCAAGCTGGCGCGTGGACCCTGGCACAAAATGAGGCATCCTGCGTAGTGTTT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/1/1f/Darmstadt13_mat_gbl_B_03_166489532Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">B_03:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GGCATCCTGCGTAGTGTTTGGGATGCCTCGTGAAGCGATTAATATGGGAGGTGTATGTGAGGTGGTGGACCTTTCGCAGGTCAGTCAGCAGATGTTAGCCAAAATTAGCGCGGGACAGGCTATCCGTATTGGGGTAAGCAAAGGTGAAGAGTTAATCAAGGAGAACATGCACATGAAATTGTATATGGAAGGTACGGTGAATAATCACCATTTCAAATGCACAAGTGAAGGCGAAGGGAAGCCGTACGAAGGCACGCAGACGATGCGCATTAAGGTAGTGGAAGGCGGTCCTTTACCGTTCGCTTTTGATATCCTGGCCACCTCCTTCATGTATGGGTCCAAGACCTTTATCAACCATACCCAGGGCATTCCGGACTTTTTTAAACAGTCTTTTCCAGAAGGTTTCACTTGGGAGCGTGTAACGACCTACGAGGATGGTGGCGTGCTGACGGCAACCCAGGACACGAGCCTTCAGGATGGATGTCTTATTTACAACGT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/1/15/Darmstadt13_mat_gbl_B_04_166470013Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">B_04</a>:</li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GGATGGATGTCTTATTTACAACGTGAAAATTCGCGGAGTGAACTTTCCGAGCAACGGTCCAGTTATGCAGAAAAAAACCCTGGGGTGGGAGGCCTCAACCGAAATGCTGTACCCAGCTGATGGTGGTCTGGAAGGTCGCTCGGACATGGCGCTGAAGCTGGTCGGTGGCGGCCATCTTATTTGTAACCTTAAAACGACCTACCGTAGCAAAAAGCCTGCGAAAAACCTGAAAATGCCCGGTGTCTACTACGTTGACCGGCGTCTGGAACGTATTAAGGAAGCCGACAAAGAGACATATGTGGAGCAGCACGAAGTTGCCGTTGCACGCTATTGCGATCTCCCGAGCAACCTGGGTCATAAACTGAATTAATAATACTAGTAGCGGCCGCTGCAGTACTAC</li><br />
</ol></div></font></p><br />
<br><br />
<br><br />
<font size="5" color="#F0F8FF" face="Arial regular"><p text-aligne:left style="margin-left:15px; margin-right:50px"><br />
Safety<br />
</p></font><br />
<br />
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<div align="left" style="margin-left:30px; margin-right:50px"><br />
<ol><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f3/Darmstadt13_mat_gbl_C1_166475119Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C1:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> TTTCTGGAATTCGCGGCCGCTTCTAGAATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGGGTCTCTCTAGTTATTACTCGAGCGCTGCTGCCACCTGCAACATCTCCTTCTCTACCTGACTCCACTCCCCAAAGAATAACTCTTGAAGAACATCTGCTGCTGAAGTTGTATTTTCTTTTGAATCATATACACAACTTCTATCTCGTTGGTAAATTTGGATTCTTTCAAAGATAGCTAGTTCTTCCAATTTTCGTGTGTTATCAACTAGATGATTTACAATGAAATCATGATGTTCTTTTGGAGTTGCGCGTGCTTGATTTGGATTGATAATGTACAGTTCTTCATAACGGATAAGAGTACTTAGATACGACAATTCAGGCTTTGTCGCAATTAAGGCCAATT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/e/e4/Darmstadt13_mat_gbl_C2_166473991Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C2:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"> GGCTTTGTCGCAATTAAGGCCAATTGTACTTCATATCCCTTATTTTTCAAGAGTTGTGCCGTTTTCTTTGGAACATCAATTGTTCGTAAAGTTCCCTCGATCAAAAGATTGTATCCCAAACTACTCAATTTTGTTACTAAAGACTCTACCATTTTTCCTGCAAAATCTTTGGTATATTCTACACTGTCTTTGCCATATTCTTGCTGTAGTTCTAAATAGTGTGGATGCTGAGAACGAAAACTATCACCATCTATGATAACAATATTTCCTTGAAATTCTTTCTGTTTAATACGATGAATTGTAGTCTTACCGGCACCACTTTGACCTCCAAGCAAAATCGCTATAGGTTGCTTACTGGACTTTTTTCCTCTTGTCAGTGAACGAAGATTCCTTGCTAAAGCATGTTTGAACTCACTATCAGTATAATCTTGGATTTCCATAATTTGAGACCCTCCTTCTTAAAGTTAAACAAAATTATTTCTTGAGCAACCATTATCACC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/5/5f/Darmstadt13_mat_gbl_C3_166482414Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C3:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
TATTTCTTGAGCAACCATTATCACCGCCAGAGGTAAAATAGTCAACACGCACGGTGTTACTCTAGTATATAAACGCAGAAAGGCCCACCCGAAGGTGAGCCAGTGTGACTCTAGTAGAGAGCGTTCACCGACAAACAACAGATAGCACGAAAGGCCCAGTCTTTCGACTGAGCCTTTCGTTTTATTTGATGCCTGGCTCTAGTAGCGATCTACACTAGCACTATCAGCGTTATTAAGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCGCCAAACGTCTCTTCAGGCCACTGACTAGCGATAACTTTCCCCACAACGGAACAACTCTCATTGCATGGGATCATTGGGTACTGTGGGTTTAGTGGTTGTAAAAACACCTGACCGCTATCCCTGATCAGTTTCTTGAAGGTAAACTCATCACCCCCAAGTCTGGCTATGCAGAAATCACCTGGCTCAACAGCCTGCTCAGGGTCAACGAGAATTAACATTCCGTCAGGAAAG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/6/63/Darmstadt13_mat_gbl_C4_166486022Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C4:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
AGAATTAACATTCCGTCAGGAAAGCTCGGCTTGGAGCCTGTTGGTGCGGTCATGGAATTACCTTCAACCTCAAGCCAGAATGCAGAATCACTGGCTTTTTTGGTTGTGCTTACCCATCTCTCCGCATCACCTTTGGTAAAGGTTCTAAGCTCAGGTGAGAACATCCCTGCCTGAACATGAGAAAAAACAGGGTACTCATACTCACTTCTAAGTGACGGCTGCATACTAACCGCTTCATACATCTCGTAGATTTCTCTGGCGATTGAAGGGCTAAATTCTTCAACGCTAACTTTGAGAATTTTTGCAAGCAATGCGGCGTTATAAGCATTTAATGCATTGATGCCATTAAATAAAGCACCAACGCCTGACTGCCCCATCCCCATCTTGTCTGCGACAGATTCCTGGGATAAGCCAAGTTCATTTTTCTTTTTTTCATAAATTGCTTTAAGGCGACGTGCGTCCTCAAGCTGCTCTTGTGTTAATGGTTTCTTTTTTGTGC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/2/23/Darmstadt13_mat_gbl_C5_166474049Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C5:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GTGTTAATGGTTTCTTTTTTGTGCTCATCTAGTATTTCTCCTCTTTTCTTGACTCCGTTGTGATGACGCATTGGTACGCGGTATCGGGAGGTTCGAAAATTTCGAGCGATATCTTAAGAGGGGTGCCTTACGTAGAACCCCGTAGGTCATGCCCGAGGCCGGTCCTGGATGGCGCGGCGGATACGCTTGAGCAGGTTTTCGTCGAGAAGCGGCTTCAAAACCACGTCTTTTACGCCGGCCTCGGCGGCCCGGGTCGAGATGTTTTCGTCCGGATAGCCGGTGATCAGGATCACGGGCGTAGATCTCGATCCTCTACGCCGGACGCATCGTGGCCGGCATCACCGGCTCCACAGGTGCGGTTGCTGGCACCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCACCATCTCCTTGCA</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/b/b3/Darmstadt13_mat_gbl_C6_166475120Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C6:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GACTGTTGGGCGCCATCTCCTTGCATGCACCATTCCTTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGATCCCGGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTGAGGGTGGTGAATGTGGCTAGTTTTCAATCATTTGGGATACCAGGACAGCTGGAAGTCATCAAAAAAGCACTTGATCACGTGCGAGTCGGTGTGGTAATTACAGATCCCGCACTTGAAGATAATCCTATTGTCTACGTAAATCAAGGCTTTGTTCAAATGACCGGCTACGAGACGGAGGAAATTTTAGGAAAGAACTGTCGCTTCTTACAGGGGAAACACACAGATCCTGCTGAAGTGGACAACATCAGAACCGCTTTACAAAATAAAGAACCGGTCACCGTTCAGATCCAAAACTACAAAAAAGACGGAA</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f1/Darmstadt13_mat_gbl_C7_166473992Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C7:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
TCCAAAACTACAAAAAAGACGGAACGATGTTCTGGAATGAATTAAATATTGATCCAATGGAAATAGAGGATAAAACGTATTTTGTCGGTATTCAGAATGATATCACCGAGCACCAGCAGACCCAGGCACGCCTCCAGGAACTGCAATCCGAGCTCGTCCACGTCTCCAGGCTGAGCGCCATGGGCGAAATGGCGTCCGCTCTCGCGCACGAGCTCAACCAGCCGCTGGCGGCGATCAGCAACTACATGAAGGGCTCGCGGCGGCTGCTTGCCGGCAGCAGTGATCCGAACACACCGAAGGTCGAAAGCGCCCTGGACCGCGCCGCCGAGCAGGCACTACGCGCAGGACAGATCATCCGACGACTGCGAGACTTCGTTGCCCGCGGCGAATCGGAGAAGCGGGTCGAGAGTCTCTCCAAGCTGATCGAGGAGGCAGGAGCACTCGGGCTTGCAGGAGCTCGCGAGCAGAACGTGCAGCTCCGTTTCAGTCTCGATCCAGGC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/3/38/Darmstadt13_mat_gbl_C8_166473991Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C8:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GCTCCGCTTCAGTCTCGATCCGGGCGCCGATCTCGTTCTCGCCGACCGGGTGCAGATCCAGCAGGTCCTGGTCAACCTGTTCCGCAACGCGCTGGAAGCGATGGCTCAGTCGCAGCGACGCGAGCTCGTCGTCACCAACACCCCCGCCGCCGACGACATGATCGAGGTCGAAGTGTCCGACACCGGCAGCGGTTTCCAGGACGACGTCATTCCGAACCTGTTTCAGACTTTCTTCACCACCAAGGACACCGGCATGGGCGTGGGACTGTCCATCAGCCGCTCGATCATCGAAGCTCACGGCGGGCGCATGTGGGCCGAGAGCAACGCATCGGGCGGGGCGACCTTCCGCTTCACCCTCCCGGCAGCCGACGAGATGATAGGAGGTCTAGCATGACGACCAAGGGACATATCTACGTCATCGACGACGACGCGGCGATGCGGGATTCGCTGAATTTCCTGCTGGATTCTGCCGGCTTCGGCGTCACGCTGTTTGACGACG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f3/Darmstadt13_mat_gbl_C9_166482415Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C9:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
TCGGCGTCACGCTGTTTGACGACGCGCAAGCCTTTCTTGACGCCCTGCCGGGACTCTCCTTCGGCTGTGTCGTCTCCGACGTGCGCATGCCGGGCCTTGACGGCATCGAGCTGTTGAAGCGGATGAAGGCGCAGCAAAGCCCCTTTCCGATCCTCATCATGACCGGTCACGGCGACGTGCCGCTCGCGGTAGAGGCGATGAAGTTAGGGGCGGTGGACTTTCTGGAAAAGCCTTTCGAGGACGACCGCCTCACCGCCATGATCGAATCGGCGATCCGCCAGGCCGAGCCGGCCGCCAAGAGCGAGGCCGTCGCGCAGGATATCGCCGCCCGCGTCGCCTCTTTGAGCCCCAGGGAGCGCCAGGTCATGGAAGGGCTGATCGCCGGCCTTTCCAACAAGCTGATCGCCCGCGAGTACGACATCAGCCCGCGCACCATCGAGGTGTATCGGGCCAACGTCATGACCAAGATGCAGGCCAACAGCCTTTCGGAGCTGGTTCGC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/d/df/Darmstadt13_mat_gbl_C10_166470014Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">C10:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
CAACAGCCTTTCGGAGCTGGTTCGCCTCGCGATGCGCGCCGGCATGCTCAACGATTGACAATTGATGTAAGTTAGCTCACTCATTAGGCACCGGGATCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATGACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAGCGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGTACTAGTAGCGGCCGCTGCAGTCCGGC</li><br />
</ol></div></font></p><br />
<br><br />
<br><br />
<font size="5" color="#F0F8FF" face="Arial regular"><p text-aligne:left style="margin-left:0px; margin-right:50px"><br />
TLO<br />
</p></font><br />
<p text-aligne:left style="margin-left:60px; margin-right:60px"><font size="3" color="#F0F8FF" face="Arial regular"><br />
<div align="left" style="margin-left:30px; margin-right:50px"><br />
<ol><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/f/f2/Darmstadt13_mat_gbl_A_01_166474041Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_01:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GTTGTTGAATTCGCGGCCGCTTCTAGAGGAAAGAGGGGACAAATGATTAACCGTATTCGTGTCGTGACGTTGCTGGTCATGGTTCTGGGTGTTTTCGCGCTCCTCCAGCTGATCTCCGGCTCGCTTTTTTTTTCGTCGCTCCATCATTCCCAGAAATCTTTTGTGGTGAGTAATCAACTGCGGGAACAGCAGGGTGAGCTCACCAGCACCTGGGATTTAATGCTCCAGACGCGCATCAATCTGAGCCGCTCGGCCGTCCGTATGATGATGGATAGTAGCAACCAGCAGTCCAACGCCAAAGTTGAGTTGCTGGATTCAGCCCGTAAAACCCTTGCCCAGGCAGCCACCCATTACAAAAAATTCAAGTCTATGGCGCCGCTGCCCGAGATGGTAGCGACGTCGCGGAATATTGACGAGAAGTATAAAAATTATTACACCGCTCTGACTGAACTGATTGATTATTTGGACTATGGCAACACC</li><br />
<br><br />
</li><li class=list1><a href="https://static.igem.org/mediawiki/2013/c/cf/Darmstadt13_mat_gbl_A_02_166474053Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_02:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
GTTGTTGAATTCGCGGCCGCTTCTAGAGGAAAGAGGGGACAAATGATTAACCGTATTCGTGTCGTGACGTTGCTGGTCATGGTTCTGGGTGTTTTCGCGCTCCTCCAGCTGATCTCCGGCTCGCTTTTTTTTTCGTCGCTCCATCATTCCCAGAAATCTTTTGTGGTGAGTAATCAACTGCGGGAACAGCAGGGTGAGCTCACCAGCACCTGGGATTTAATGCTCCAGACGCGCATCAATCTGAGCCGCTCGGCCGTCCGTATGATGATGGATAGTAGCAACCAGCAGTCCAACGCCAAAGTTGAGTTGCTGGATTCAGCCCGTAAAACCCTTGCCCAGGCAGCCACCCATTACAAAAAATTCAAGTCTATGGCGCCGCTGCCCGAGATGGTAGCGACGTCGCGGAATATTGACGAGAAGTATAAAAATTATTACACCGCTCTGACTGAACTGATTGATTATTTGGACTATGGCAACACC</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/5/59/Darmstadt13_mat_gbl_A_03_166475118Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_03:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;">AGCCCTGGAAGAAACCGCAGCGTCTATGGAACAGTTAACGGCAACTGTTAAGCAGAACGCGGATAATGCGCGTCAGGCCTCTCAGCTTGCACAGAGTGCGTCTGATACGGCCCAGCACGGTGGAAAAGTCGTCGACGGAGTGGTTAAAACGATGCATGAAATTGCCGATTCGTCCAAGAAAATCGCTGATATTATTAGCGTCATCGACGGTATTGCATTTCAGACCAATATTCTGGCTCTGAACGCGGCGGTAGAAGCAGCGCGTGCGGGGGAACAGGGCCGTGGCTTTGCGGTAGTTGCCGGTGAAGTGCGTAACCTGGCGTCTCGTAGCGCCCAGGCGGCAAAAGAGATTAAGGCACTGATCGAAGACAGTGTGTCGCGGGTCGATACGGGAAGCGTGTTAGTGGAATCAGCAGGCGAAACAATGAATAATATCGTGAATGCTGTGACTCGCGTGACCGACATTATGGGCGAGATCGCTAGCGCCTCTGATGAACAG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/4/4e/Darmstadt13_mat_gbl_A_04_166474048Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_04:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;">CTAGCGCCTCTGATGAACAGTCCCGTGGTATTGATCAAGTGGCTCTTGCGGTTAGCGAGATGGATCGTGTTACGCAGCAGAACGCCAGCTTGGTCCAGGAATCTGCGGCAGCGGCTGCGGCCCTGGAAGAACAAGCTTCTCGTCTGACGCAAGCAGTCAGCGCCTTTCGCCTGGCAGCGAGCCCACTTACAAACAAACCGCAGACGCCGAGTCGTCCAGCATCAGAACAGCCGCCGGCCCAACCTCGCCTGCGGATCGCTGAACAGGACCCCAACTGGGAAACCTTTGGGGTGAGCAAAGGCGAAGAAAATAACATGGCTATTATCAAGGAATTTATGCGCTTTAAAGTGCGTATGGAAGGGAGTGTGAACGGTCATGAATTTGAAATTGAAGGCGAGGGTGAAGGCCGTCCTTATGAAGGGTTTCAGACCGTGAAACTGAAGGTGACCAAAGGTGGTCCCCTTCCCTTCGCCTGGGATATTCTGTCGCCGCAGTT</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/8/8f/Darmstadt13_mat_gbl_A_05_166469986Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_05:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;">GATATTCTGTCGCCGCAGTTCACCTATGGCAGCAAGGCATATGTGAAGCATCCCGCCGATATCCCCGATTATTTGAAACTGTCGTTTCCTGAGGGCTTTAAGTGGGAACGTGTGATGAATTTCGAAGATGGCGGGGTCGTGACGGTAACTCAAGACAGTTCGCTTCAGGATGGCGAGTTTATTTATAAGGTGAAGTTGCGTGGTACCAATTTCCCAAGCGACGGTCCGGTGATGCAAAAAAAAACAATGG</li><br />
<br><br />
<li class=list1><a href="https://static.igem.org/mediawiki/2013/b/b8/Darmstadt13_mat_gbl_A_06_166469994Spec.pdf"><font size="3" color="#F0F8FF" face="Arial regular">A_06:</a></li><br />
<li class=list1 style="max-width:1000px;word-wrap:break-word;"><br />
CGGTGATGCAAAAAAAAACAATGGGTATGGAAGCGTCGTCGGAACGTATGTATCCAGAAGACGGTGCACTGAAAGGCGAAGACAAATTACGTCTGAAACTGAAGGACGGTGGCCATTACACGAGCGAAGTAAAAACCACCTATAAGGCGAAAAAACCGGTGCAACTTCCAGGTGCATACATCGTTGACATTAAGTTGGATATTACGAGCCATAACGAGGATTACACCATTGTTGAGCAGTATGAGCGTGCCGAGGGGCGTCATAGCACCGGCGGCATGGACGAGCTGTACAAATGATAATACTAGTAGCGGCCGCTGCAGTACTAC</li><br />
</ol></div></font></p><br />
<br />
</body><br />
</html></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_mat_gbl_A_06_166469994Spec.pdf
File:Darmstadt13 mat gbl A 06 166469994Spec.pdf
2013-10-05T01:07:27Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_mat_gbl_A_05_166469986Spec.pdf
File:Darmstadt13 mat gbl A 05 166469986Spec.pdf
2013-10-05T01:07:05Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_mat_gbl_A_03_166475118Spec.pdf
File:Darmstadt13 mat gbl A 03 166475118Spec.pdf
2013-10-05T01:06:46Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_mat_gbl_A_02_166474053Spec.pdf
File:Darmstadt13 mat gbl A 02 166474053Spec.pdf
2013-10-05T01:06:20Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_mat_gbl_A_01_166474041Spec.pdf
File:Darmstadt13 mat gbl A 01 166474041Spec.pdf
2013-10-05T01:05:57Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_mat_gbl_C10_166470014Spec.pdf
File:Darmstadt13 mat gbl C10 166470014Spec.pdf
2013-10-05T01:05:36Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_mat_gbl_C9_166482415Spec.pdf
File:Darmstadt13 mat gbl C9 166482415Spec.pdf
2013-10-05T01:05:12Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_mat_gbl_C8_166473991Spec.pdf
File:Darmstadt13 mat gbl C8 166473991Spec.pdf
2013-10-05T01:04:51Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_mat_gbl_C7_166473992Spec.pdf
File:Darmstadt13 mat gbl C7 166473992Spec.pdf
2013-10-05T01:04:33Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie
http://2013.igem.org/File:Darmstadt13_mat_gbl_C6_166475120Spec.pdf
File:Darmstadt13 mat gbl C6 166475120Spec.pdf
2013-10-05T01:04:12Z
<p>Schiefie: </p>
<hr />
<div></div>
Schiefie