Difference between revisions of "Team:Freiburg/Labjournals/Cloning/September"

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<html>
 
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<!-- Labjournal content goes in here -->
 
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<div class="content_box">
 
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<h1 class="sectionedit1">Labjournal Surchem September</h1>
 
−
<div class="level1">
 
−
</div>
 
−
<!-- EDIT1 SECTION "Labjournal Surchem September" [1-43] -->
 
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<h2 class="sectionedit2">Experiment 65b:  PDITC slides  for iRIf</h2>
 
−
<div class="level2">
 
−
<ul>
 
−
<li class="level1"><div class="li"> protocols used :<a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/Plasmaactivation" title="protocols:plasma_activation">Plasma activation</a>, <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/SlidePrep" title="irif_slide_preparation">iRIf slide preparation</a></div>
 
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</li>
 
−
</ul>
 
−
</div>
 
−
<!-- EDIT2 SECTION "Experiment 65b:  PDITC slides  for iRIf" [44-218] -->
 
−
<h3 class="sectionedit3">2015.09.04</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<ul>
 
−
<li class="level1"><div class="li"> 4 PDITC slides were prepared</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Plasma activation: 40 L/h, 5 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> APTES incubation: 30 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> PDITC incubation: 2 h</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> desiccator: 30 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> stored at 4 °C</div>
 
−
</li>
 
−
</ul>
 
−
</div>
 
−
<!-- EDIT3 SECTION "2015.09.04" [219-436] -->
 
−
<h2 class="sectionedit4">Experiment 65b:  Tetanus antigen PDITC slides  for iRIf V</h2>
 
−
<div class="level2">
 
−
<ul>
 
−
<li class="level1"><div class="li"> protocols used :<a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/Plasmaactivation" title="protocols:plasma_activation">Plasma activation</a>, <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/SlidePrep" title="irif_slide_preparation">iRIf slide preparation</a></div>
 
−
</li>
 
−
</ul>
 
−
</div>
 
−
<!-- EDIT4 SECTION "Experiment 65b:  Tetanus antigen PDITC slides  for iRIf V" [437-627] -->
 
−
<h3 class="sectionedit5">2015.09.04</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<ul>
 
−
<li class="level1"><div class="li"> 4 PDITC slides were prepared</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Plasma activation: 40 L/h, 5 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> APTES incubation: 30 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> PDITC incubation: 2 h</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> desiccator: 30 min</div>
 
−
</li>
 
−
</ul>
 
−
<p>
 
−
Spotting pattern: <img alt="" class="media" src="https://static.igem.org/mediawiki/2015/d/dd/Freiburg_labjournal-surchem-spotting_pattern6.png" width="300"/><br/>
 
−
</p>
 
−
<div class="table sectionedit6"><table class="inline">
 
−
<tr class="row0">
 
−
<th class="col0"> # </th><th class="col1 leftalign"> spot  </th><th class="col2"> Concentration </th>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0"> 1 </td><td class="col1"> pos. control (GFP) </td><td class="col2"> 0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0"> 2 </td><td class="col1"> BSA </td><td class="col2"> 5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0"> 3 </td><td class="col1 leftalign"> Tetanus Antigen  </td><td class="col2"> ~0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0"> 4 </td><td class="col1"> - </td><td class="col2"> - </td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0"> 5 </td><td class="col1">-</td><td class="col2"> - </td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0"> 6 </td><td class="col1">bBSA </td><td class="col2"> 200 µg/ml</td>
 
−
</tr>
 
−
</table></div>
 
−
<!-- EDIT6 TABLE [905-1088] -->
 
−
</div>
 
−
<!-- EDIT5 SECTION "2015.09.04" [628-1089] -->
 
−
<h3 class="sectionedit7">2015.09.05</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<p>
 
−
Flush protocol:
 
−
</p>
 
−
<p>
 
−
Tet serum (-): <br/>
 
−
 
−
Tet serum (+):
 
−
</p>
 
−
<div class="table sectionedit8"><table class="inline">
 
−
<tr class="row0">
 
−
<th class="col0"> Reagent </th><th class="col1"> Step </th><th class="col2"> Flow rate (ul/min) </th><th class="col3"> Priming time </th><th class="col4"> Concentration </th>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">Buffer</td><td class="col1">1</td><td class="col2">60</td><td class="col3">600</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BSA</td><td class="col1">2</td><td class="col2">60</td><td class="col3">600</td><td class="col4">10 mg/ml</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Buffer</td><td class="col1">3</td><td class="col2">60</td><td class="col3">600</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">anti-GFP</td><td class="col1">4</td><td class="col2">30</td><td class="col3">600</td><td class="col4">1.5 ug/ml</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">Buffer</td><td class="col1">5</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">Serum SIG002 (-)/(+)</td><td class="col1">6</td><td class="col2">30</td><td class="col3">600</td><td class="col4">1:10/1:30</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">Buffer</td><td class="col1">7</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0">StrepCy5</td><td class="col1">8</td><td class="col2">30</td><td class="col3">600</td><td class="col4">5 ug/ml</td>
 
−
</tr>
 
−
<tr class="row9">
 
−
<td class="col0">Buffer</td><td class="col1">9</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
</table></div>
 
−
<!-- EDIT8 TABLE [1191-1490] -->
 
−
</div>
 
−
<!-- EDIT7 SECTION "2015.09.05" [1090-1492] -->
 
−
<h2 class="sectionedit9">Experiment 65a: cell free GFP on NiNTA and PDITC slides  for iRIf</h2>
 
−
<div class="level2">
 
−
<ul>
 
−
<li class="level1"><div class="li"> protocols used :<a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/Plasmaactivation" title="protocols:plasma_activation">Plasma activation</a>, <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/SlidePrep" title="irif_slide_preparation">iRIf slide preparation</a></div>
 
−
</li>
 
−
</ul>
 
−
</div>
 
−
<!-- EDIT9 SECTION "Experiment 65a: cell free GFP on NiNTA and PDITC slides  for iRIf" [1493-1692] -->
 
−
<h3 class="sectionedit10">2015.09.04</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<ul>
 
−
<li class="level1"><div class="li"> 2 PDITC slides were prepared (216,254)</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Plasma activation: 40 L/h, 5 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> APTES incubation: 30 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> PDITC incubation: 2 h</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> desiccator: 30 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> 2 Ni NTA slides from experiment 63b were also spotted (102,502)</div>
 
−
</li>
 
−
</ul>
 
−
<p>
 
−
Spotting pattern:<img alt="" class="media" src="https://static.igem.org/mediawiki/2015/0/09/Freiburg_labjournal-surchem-spotting_pattern_8.png" width="300"/><br/>
 
−
</p>
 
−
<div class="table sectionedit11"><table class="inline">
 
−
<tr class="row0">
 
−
<th class="col0"> # </th><th class="col1 leftalign"> spot  </th><th class="col2"> Concentration </th>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0"> 1 </td><td class="col1"> pos. control (GFP) </td><td class="col2"> 0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0"> 2 </td><td class="col1"> cell free expression mix without DNA (KK)</td><td class="col2"> </td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0"> 3 </td><td class="col1 leftalign"> cell free expressed HA-GFP-His6-His6 (KK)  </td><td class="col2 leftalign"> </td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0"> 4 </td><td class="col1">pos. control (GFP) </td><td class="col2"> 0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0"> 5 </td><td class="col1">cell free expression mix without DNA (KK)</td><td class="col2"> </td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0"> 6 </td><td class="col1">cell free expressed His10-GFP-Spy (KK)</td><td class="col2"> </td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0"> 7 </td><td class="col1">BSA</td><td class="col2"> 5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0"> 8 </td><td class="col1">bBSA</td><td class="col2"> 200 µg/ml </td>
 
−
</tr>
 
−
</table></div>
 
−
<!-- EDIT11 TABLE [2048-2405] -->
 
−
</div>
 
−
<!-- EDIT10 SECTION "2015.09.04" [1693-2406] -->
 
−
<h3 class="sectionedit12">2015.09.05</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<p>
 
−
Flush protocol:
 
−
</p>
 
−
<div class="table sectionedit13"><table class="inline">
 
−
<tr class="row0">
 
−
<th class="col0"> Reagent </th><th class="col1"> Step </th><th class="col2"> Flow rate (ul/min) </th><th class="col3"> Priming time </th><th class="col4"> Concentration </th>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">Buffer</td><td class="col1">1</td><td class="col2">60</td><td class="col3">600</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BSA</td><td class="col1">2</td><td class="col2">60</td><td class="col3">600</td><td class="col4">10 mg/ml</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Buffer</td><td class="col1">3</td><td class="col2">60</td><td class="col3">600</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">anti-GFP</td><td class="col1">4</td><td class="col2">30</td><td class="col3">600</td><td class="col4">1.5 ug/ml</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">Buffer</td><td class="col1">5</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">StrepCy5</td><td class="col1">8</td><td class="col2">30</td><td class="col3">600</td><td class="col4">5 ug/ml</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">Buffer</td><td class="col1">9</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
</table></div>
 
−
<!-- EDIT13 TABLE [2473-2709] -->
 
−
</div>
 
−
<!-- EDIT12 SECTION "2015.09.05" [2407-2710] -->
 
−
<h2 class="sectionedit14">Experiment 64c:  Tetanus antigen PDITC slides  for iRIf IV</h2>
 
−
<div class="level2">
 
−
<ul>
 
−
<li class="level1"><div class="li"> protocols used :<a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/Plasmaactivation" title="protocols:plasma_activation">Plasma activation</a>, <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/SlidePrep" title="irif_slide_preparation">iRIf slide preparation</a></div>
 
−
</li>
 
−
</ul>
 
−
</div>
 
−
<!-- EDIT14 SECTION "Experiment 64c:  Tetanus antigen PDITC slides  for iRIf IV" [2711-2902] -->
 
−
<h3 class="sectionedit15">2015.08.31</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<ul>
 
−
<li class="level1"><div class="li"> 4 PDITC slides were prepared</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Plasma activation: 40 L/h, 5 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> APTES incubation: 30 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> PDITC incubation: 2 h</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> desiccator: 15 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> stored at 4 °C</div>
 
−
</li>
 
−
</ul>
 
−
</div>
 
−
<!-- EDIT15 SECTION "2015.08.31" [2903-3120] -->
 
−
<h3 class="sectionedit16">2015.09.03</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<p>
 
−
Spotting pattern: <img alt="" class="media" src="https://static.igem.org/mediawiki/2015/d/dd/Freiburg_labjournal-surchem-spotting_pattern6.png" width="300"/><br/>
 
−
</p>
 
−
<div class="table sectionedit17"><table class="inline">
 
−
<tr class="row0">
 
−
<th class="col0"> # </th><th class="col1 leftalign"> spot  </th><th class="col2"> Concentration </th>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0"> 1 </td><td class="col1"> pos. control (GFP) </td><td class="col2"> 0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0"> 2 </td><td class="col1"> BSA </td><td class="col2"> 5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0"> 3 </td><td class="col1 leftalign"> Tetanus Antigen  </td><td class="col2"> ~0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0"> 4 </td><td class="col1"> - </td><td class="col2"> - </td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0"> 5 </td><td class="col1"> mCherry</td><td class="col2"> 0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0"> 6 </td><td class="col1">bBSA </td><td class="col2"> 200 µg/ml</td>
 
−
</tr>
 
−
</table></div>
 
−
<!-- EDIT17 TABLE [3249-3447] -->
 
−
</div>
 
−
<!-- EDIT16 SECTION "2015.09.03" [3121-3449] -->
 
−
<h3 class="sectionedit18">2015.09.04</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<p>
 
−
Flush protocol:
 
−
</p>
 
−
<p>
 
−
Tet serum (-): 500(1:10 diluted serum)<br/>
 
−
 
−
Tet serum (+): 024 (1:10 diluted serum), 504 (1:30 diluted serum)
 
−
</p>
 
−
<div class="table sectionedit19"><table class="inline">
 
−
<tr class="row0">
 
−
<th class="col0"> Reagent </th><th class="col1"> Step </th><th class="col2"> Flow rate (ul/min) </th><th class="col3"> Priming time </th><th class="col4"> Concentration </th>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">Buffer</td><td class="col1">1</td><td class="col2">60</td><td class="col3">600</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BSA</td><td class="col1">2</td><td class="col2">60</td><td class="col3">600</td><td class="col4">10 mg/ml</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Buffer</td><td class="col1">3</td><td class="col2">60</td><td class="col3">600</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">anti-GFP</td><td class="col1">4</td><td class="col2">30</td><td class="col3">600</td><td class="col4">1.5 ug/ml</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">Buffer</td><td class="col1">5</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">Serum SIG002 (-)/(+)</td><td class="col1">6</td><td class="col2">30</td><td class="col3">600</td><td class="col4">1:10/1:30</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">Buffer</td><td class="col1">7</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0">StrepCy5</td><td class="col1">8</td><td class="col2">30</td><td class="col3">600</td><td class="col4">5 ug/ml</td>
 
−
</tr>
 
−
<tr class="row9">
 
−
<td class="col0">Buffer</td><td class="col1">9</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
</table></div>
 
−
<!-- EDIT19 TABLE [3625-3924] -->
 
−
</div>
 
−
<h4>Results</h4>
 
−
<div class="level4">
 
−
<p>
 
−
Serum(-):
 
−
</p>
 
−
<div class="thumb2 tleft" style="width:310px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/8/84/Freiburg_labjournal-surchem-exp_64c_slide500.png" width="300"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64c_slide500.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 500: Quotion picture, Tet serum (-), 1:10 diluted</div></div></div><div class="thumb2 trien" style="width:510px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/2/28/Freiburg_labjournal-surchem-exp_64c_slide500_binding.png" width="500"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64c_slide500_binding.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 500: binding curves</div></div></div><hr/>
 
−
<p>
 
−
Serum(+):
 
−
</p>
 
−
<div class="thumb2 tleft" style="width:310px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/d/d3/Freiburg_labjournal-surchem-exp_64c_slide024.png" width="300"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64c_slide024.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 024: Quotion picture, Tet serum (+), 1:10 diluted</div></div></div>
 
−
<p>
 
−
→ bad measurement, no binding curves were obtained
 
−
</p>
 
−
<div class="thumb2 tleft" style="width:310px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/8/88/Freiburg_labjournal-surchem-exp_64c_slide504.png" width="300"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64c_slide504.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 504: Quotion picture, Tet serum (+), 1:30 diluted</div></div></div><div class="thumb2 trien" style="width:510px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/5/55/Freiburg_labjournal-surchem-exp_64c_slide504_binding.png" width="500"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64c_slide504_binding.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 504: binding curves</div></div></div>
 
−
<p>
 
−
→ signal for 1:10 dilution of the blood serum much better → continue with 1:10 dilution
 
−
</p>
 
−
</div>
 
−
<!-- EDIT18 SECTION "2015.09.04" [3450-4640] -->
 
−
<h2 class="sectionedit20">Experiment 64b: Tetanus antigen PDITC slides  for iRIf III</h2>
 
−
<div class="level2">
 
−
<ul>
 
−
<li class="level1"><div class="li"> protocols used :<a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/Plasmaactivation" title="protocols:plasma_activation">Plasma activation</a>, <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/SlidePrep" title="irif_slide_preparation">iRIf slide preparation</a></div>
 
−
</li>
 
−
</ul>
 
−
</div>
 
−
<!-- EDIT20 SECTION "Experiment 64b: Tetanus antigen PDITC slides  for iRIf III" [4641-4832] -->
 
−
<h3 class="sectionedit21">2015.08.31</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<ul>
 
−
<li class="level1"><div class="li"> 4 PDITC slides were prepared</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Plasma activation: 40 L/h, 5 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> APTES incubation: 30 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> PDITC incubation: 2 h</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> desiccator: 15 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> stored at 4 °C</div>
 
−
</li>
 
−
</ul>
 
−
</div>
 
−
<!-- EDIT21 SECTION "2015.08.31" [4833-5051] -->
 
−
<h3 class="sectionedit22">2015.09.02</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<p>
 
−
Spotting pattern: <img alt="" class="media" src="https://static.igem.org/mediawiki/2015/d/dd/Freiburg_labjournal-surchem-spotting_pattern6.png" width="300"/><br/>
 
−
</p>
 
−
<div class="table sectionedit23"><table class="inline">
 
−
<tr class="row0">
 
−
<th class="col0"> # </th><th class="col1 leftalign"> spot  </th><th class="col2"> Concentration </th>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0"> 1 </td><td class="col1"> pos. control (GFP) </td><td class="col2"> 0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0"> 2 </td><td class="col1"> mCherry </td><td class="col2"> 0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0"> 3 </td><td class="col1 leftalign"> Tetanus Antigen  </td><td class="col2"> ~0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0"> 4 </td><td class="col1"> BSA </td><td class="col2"> 10 mg/ml </td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0"> 5 </td><td class="col1"> BSA +bBSA</td><td class="col2"> 10 mg/ml </td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0"> 6 </td><td class="col1">bBSA </td><td class="col2"> 200 µg/ml</td>
 
−
</tr>
 
−
</table></div>
 
−
<!-- EDIT23 TABLE [5180-5394] -->
 
−
<p>
 
−
note: bBSA on spot 5 was incubated for only a minute and then replaced with BSA 
 
−
</p>
 
−
</div>
 
−
<!-- EDIT22 SECTION "2015.09.02" [5052-5478] -->
 
−
<h3 class="sectionedit24">2015.09.03</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<p>
 
−
Flush protocol:
 
−
</p>
 
−
<p>
 
−
Tet serum (-): 287 (1:10 diluted serum),429 (1:30 diluted serum)<br/>
 
−
 
−
Tet serum (+): 443 (1:10 diluted serum), 423 (1:30 diluted serum)
 
−
</p>
 
−
<div class="table sectionedit25"><table class="inline">
 
−
<tr class="row0">
 
−
<th class="col0"> Reagent </th><th class="col1"> Step </th><th class="col2"> Flow rate (ul/min) </th><th class="col3"> Priming time </th><th class="col4"> Concentration </th>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">Buffer</td><td class="col1">1</td><td class="col2">60</td><td class="col3">600</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BSA</td><td class="col1">2</td><td class="col2">60</td><td class="col3">600</td><td class="col4">10 mg/ml</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Buffer</td><td class="col1">3</td><td class="col2">60</td><td class="col3">600</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">anti-GFP</td><td class="col1">4</td><td class="col2">30</td><td class="col3">600</td><td class="col4">1.5 ug/ml</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">Buffer</td><td class="col1">5</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">Serum SIG002 (-)/(+)</td><td class="col1">6</td><td class="col2">20</td><td class="col3">900</td><td class="col4">1:10</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">Buffer</td><td class="col1">7</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0">StrepCy5</td><td class="col1">8</td><td class="col2">30</td><td class="col3">600</td><td class="col4">5 ug/ml</td>
 
−
</tr>
 
−
<tr class="row9">
 
−
<td class="col0">Buffer</td><td class="col1">9</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
</table></div>
 
−
<!-- EDIT25 TABLE [5680-5974] -->
 
−
</div>
 
−
<h4>Results</h4>
 
−
<div class="level4">
 
−
<p>
 
−
Serum(-):
 
−
</p>
 
−
<div class="thumb2 tleft" style="width:310px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/a/a4/Freiburg_labjournal-surchem-exp_64b_slide287.png" width="300"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64b_slide287.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 287: Quotion picture, Tet serum (-), 1:10 diluted</div></div></div><div class="thumb2 trien" style="width:510px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/d/da/Freiburg_labjournal-surchem-exp_64b_slide287_binding.png" width="500"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64b_slide287_binding.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 287: binding curves</div></div></div><div class="thumb2 tleft" style="width:310px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/b/b4/Freiburg_labjournal-surchem-exp_64b_slide429.png" width="300"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64b_slide429.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 429: Quotion picture, Tet serum (-), 1:30 diluted</div></div></div><div class="thumb2 trien" style="width:510px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/7/70/Freiburg_labjournal-surchem-exp_64b_slide429_binding.png" width="500"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64b_slide429_binding.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 429: binding curves</div></div></div>
 
−
<p>
 
−
→ higher dilution of the blood serum leads to weaker iRif signal for mCherry, BSA,BSA+bBSA and bBSA but also for the tetanus antigen
 
−
</p>
 
−
<hr/>
 
−
<p>
 
−
Serum(+):
 
−
</p>
 
−
<div class="thumb2 tleft" style="width:310px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/5/50/Freiburg_labjournal-surchem-exp_64b_slide443.png" width="300"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64b_slide443.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 443: Quotion picture, Tet serum (+), 1:10 diluted</div></div></div><div class="thumb2 trien" style="width:510px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/7/7e/Freiburg_labjournal-surchem-exp_64b_slide443_binding.png" width="500"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64b_slide443_binding.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 443: binding curves</div></div></div><div class="thumb2 tleft" style="width:310px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/3/33/Freiburg_labjournal-surchem-exp_64b_slide423.png" width="300"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64b_slide423.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 423: Quotion picture, Tet serum (+), 1:30 diluted</div></div></div><div class="thumb2 trien" style="width:510px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/5/5e/Freiburg_labjournal-surchem-exp_64b_slide423_binding.png" width="500"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:surchem:exp_64b_slide423_binding.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 423: binding curves</div></div></div>
 
−
<p>
 
−
→ higher dilution of the blood serum leads to weaker iRif signal for mCherry, BSA,BSA+bBSA and bBSA but also for the tetanus antigen
 
−
</p>
 
−
<p>
 
−
→ signal of the tetanus antigen spot very weak compared to experiment 59a → use fresh stock of tetanus antigen next time
 
−
</p>
 
−
<p>
 
−
→ unspecific binding to the BSA spot is most of the time weaker than to mCherry → from now on BSA will be used as negative contol for blood serum
 
−
</p>
 
−
</div>
 
−
<!-- EDIT24 SECTION "2015.09.03" [5479-7388] -->
 
−
<h2 class="sectionedit26">Experiment 64a: Salmonella antigen on PDITC slides  for iRIf II</h2>
 
−
<div class="level2">
 
−
<ul>
 
−
<li class="level1"><div class="li"> protocols used :<a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/Plasmaactivation" title="protocols:plasma_activation">Plasma activation</a>, <a class="wikilink1" href="https://2015.igem.org/Team:Freiburg/Protocols/SlidePrep" title="irif_slide_preparation">iRIf slide preparation</a></div>
 
−
</li>
 
−
</ul>
 
−
</div>
 
−
<!-- EDIT26 SECTION "Experiment 64a: Salmonella antigen on PDITC slides  for iRIf II" [7389-7586] -->
 
−
<h3 class="sectionedit27">2015.08.31</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<ul>
 
−
<li class="level1"><div class="li"> 3 PDITC slides were prepared</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> Plasma activation: 40 L/h, 5 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> APTES incubation: 30 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> PDITC incubation: 2 h</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> desiccator: 15 min</div>
 
−
</li>
 
−
<li class="level1"><div class="li"> stored at 4 °C</div>
 
−
</li>
 
−
</ul>
 
−
</div>
 
−
<!-- EDIT27 SECTION "2015.08.31" [7587-7804] -->
 
−
<h3 class="sectionedit28">2015.09.01</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<p>
 
−
Spotting pattern: <img alt="" class="media" src="https://static.igem.org/mediawiki/2015/8/8a/Freiburg_labjournal-surchem-spotting_pattern_3.png" width="300"/><br/>
 
−
</p>
 
−
<div class="table sectionedit29"><table class="inline">
 
−
<tr class="row0">
 
−
<th class="col0"> # </th><th class="col1 leftalign"> spot  </th><th class="col2"> Concentration </th>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0"> 1 </td><td class="col1"> pos. control (GFP) </td><td class="col2"> 0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0"> 2 </td><td class="col1"> bBSA </td><td class="col2"> 0.5 mg/ml </td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0"> 3 </td><td class="col1"> Salmonella Antigen (15) </td><td class="col2"> undiluted </td>
 
−
</tr>
 
−
</table></div>
 
−
<!-- EDIT29 TABLE [7934-8074] -->
 
−
<p>
 
−
Slides (3): 208,215,216
 
−
</p>
 
−
</div>
 
−
<!-- EDIT28 SECTION "2015.09.01" [7805-8100] -->
 
−
<h3 class="sectionedit30">2015.09.02</h3>
 
−
<div class="level3">
 
−
</div>
 
−
<h4>Experiment/Protocol</h4>
 
−
<div class="level4">
 
−
<p>
 
−
Flush protocol:
 
−
</p>
 
−
<div class="table sectionedit31"><table class="inline">
 
−
<tr class="row0">
 
−
<th class="col0"> Reagent </th><th class="col1"> Step </th><th class="col2"> Flow rate (ul/min) </th><th class="col3"> Priming time </th><th class="col4"> Concentration </th>
 
−
</tr>
 
−
<tr class="row1">
 
−
<td class="col0">Buffer (TBS)</td><td class="col1">1</td><td class="col2">60</td><td class="col3">600</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row2">
 
−
<td class="col0">BSA</td><td class="col1">2</td><td class="col2">60</td><td class="col3">600</td><td class="col4">10 mg/ml</td>
 
−
</tr>
 
−
<tr class="row3">
 
−
<td class="col0">Buffer (TBS)</td><td class="col1">3</td><td class="col2">60</td><td class="col3">600</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row4">
 
−
<td class="col0">anti-GFP</td><td class="col1">4</td><td class="col2">30</td><td class="col3">600</td><td class="col4">1,5 ug/ml</td>
 
−
</tr>
 
−
<tr class="row5">
 
−
<td class="col0">Buffer (TBS)</td><td class="col1">5</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row6">
 
−
<td class="col0">anti-Salmonella (13, desalt)</td><td class="col1">6</td><td class="col2">30</td><td class="col3">900</td><td class="col4">undiluted</td>
 
−
</tr>
 
−
<tr class="row7">
 
−
<td class="col0">Buffer (TBS)</td><td class="col1">7</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
<tr class="row8">
 
−
<td class="col0">StrepCy5</td><td class="col1">8</td><td class="col2">30</td><td class="col3">600</td><td class="col4">5 ug/ml</td>
 
−
</tr>
 
−
<tr class="row9">
 
−
<td class="col0">Buffer (TBS)</td><td class="col1">9</td><td class="col2">60</td><td class="col3">300</td><td class="col4">1x</td>
 
−
</tr>
 
−
</table></div>
 
−
<!-- EDIT31 TABLE [8168-8505] -->
 
−
<p>
 
−
last slide (208) changed protocol: only 1 ug/ml anti-GFP used; anti-Salmonella desalt was mixed 1:1 with anti-Salmonella lysate
 
−
</p>
 
−
</div>
 
−
<h4>Results</h4>
 
−
<div class="level4">
 
−
<p>
 
−
Note: Slide 208 had the best signal.
 
−
</p>
 
−
<div class="thumb2 trien" style="width:210px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/8/89/Freiburg_labjournal-roi_selection_20150902_208.png" width="200"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:roi_selection_20150902_208.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 208: ROI selection</div></div></div><div class="thumb2 trien" style="width:310px"><div class="thumbinner"><img alt="" class="mediabox2" src="https://static.igem.org/mediawiki/2015/b/bc/Freiburg_labjournal-binding_curve_20150902_208.png" width="300"/><div class="thumbcaption"><div class="magnify"><a class="internal" href="/igem2015/lib/exe/detail.php?id=labjournal%3Asurchem%3Aseptember&amp;media=labjournal:binding_curve_20150902_208.png" title="vergrößern"><img alt="" height="11" src="/igem2015/lib/plugins/imagebox/magnify-clip.png" width="15"/></a></div>Slide 208: Binding curves</div></div></div>
 
−
</div>
 
−
<!-- EDIT30 SECTION "2015.09.02" [8101-] -->
 
−
</div>
 
−
</html>
 
−
<!-- Labjournal content ends here -->
 
−
{{Freiburg/wiki_content_end}}
 

Revision as of 09:56, 6 September 2015