Difference between revisions of "Team:Freiburg/Protocols/TerrificBroth"

 
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</div>
 
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<h4>Spotting DNA</h4>
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<h4>Terrific Broth</h4>
 
<div class="level4">
 
<div class="level4">
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<ol>
 
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<li class="level1"><div class="li"> Spot DNA (c= 25 ng/µl) on flow cell (spot size can be 0.5 µl or more)</div>
 
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</li>
 
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<li class="level1"><div class="li"> Incubate over night at room temperature in humid atmosphere in petri dish sealed with Parafilm </div>
 
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</li>
 
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<li class="level1"><div class="li"> Heat whole petri dish in oven at 60°C for 30min in order to dry the DNA (leave humid tissue inside and make sure the petri dish is still sealed)</div>
 
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</li>
 
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<li class="level1"><div class="li"> put the flow cell in a petri dish filled with H<sub>2</sub>O and wash it </div>
 
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</li>
 
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</ol>
 
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<p>
 
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<p><div class="noteimportant">DNA has bound to surface if water drops appear on it during washing step
 
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</div></p>
 
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</p>
 
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<ol>
 
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<li class="level1"><div class="li"> dry flow cell with wafergun</div>
 
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</li>
 
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</ol>
 
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</div>
 
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<h4>Storage of spotted flow cells</h4>
 
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<div class="level4">
 
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<ol>
 
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<li class="level1"><div class="li"> store in fridge sealed with parafilm without humid tissue</div>
 
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</li>
 
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</ol>
 
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</div>
 
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<h4>Cellfree Expression "on air"</h4>
 
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<div class="level4">
 
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<ol>
 
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<li class="level1"><div class="li"> Put flow cell with its frontside (with spots) on a clean glass slide </div>
 
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</li>
 
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<li class="level1"><div class="li"> Make sure the flow cell does not collapse!</div>
 
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</li>
 
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<li class="level1"><div class="li"> Add a second glass slide orthogonally to the first one on the backside of the flow cell to give some stability (should look like a cross now)</div>
 
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</li>
 
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<li class="level1"><div class="li"> Prepare 15 µl cellfree Expression mix per flow cell</div>
 
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</li>
 
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</ol>
 
 
<div class="table sectionedit2"><table class="inline">
 
<div class="table sectionedit2"><table class="inline">
 
<tr class="row0">
 
<tr class="row0">
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<th class="col0"> AG Roth Mix </th><td class="col1"></td><td class="col2"></td>
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<th class="col0">Component </th><th class="col1"> for 1 litre medium </th><td class="col2"> <b>remarks</b> </td>
 
</tr>
 
</tr>
 
<tr class="row1">
 
<tr class="row1">
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<th class="col0"> component </th><th class="col1"> concentration </th><th class="col2"> volume </th>
+
<td class="col0"> Monopotassium phosphate </td><td class="col1"> 5.6 g </td><td class="col2"> </td>
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</tr>
+
 
<tr class="row2">
 
<tr class="row2">
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<td class="col0">H<sub>2</sub>O</td><td class="col1"> </td><td class="col2">3,75 µl</td>
+
<td class="col0"> Potassium phosphate dibasic  </td><td class="col1"> 28.9 g </td><td class="col2"> </td>
−
</tr>
+
 
<tr class="row3">
 
<tr class="row3">
−
<td class="col0">Buffer</td><td class="col1"> </td><td class="col2">6 µl</td>
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<td class="col0"> yeast extract </td><td class="col1"> 10  </td>
 
</tr>
 
</tr>
 
<tr class="row4">
 
<tr class="row4">
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<td class="col0">Lysate</td><td class="col1"> </td><td class="col2">5,25 µl</td>
+
<td class="col0"> Thiamine </td><td class="col1"> 10 mg </td><td class="col2"> added after sterilisation (filter sterilized) </td>
 +
</tr>
 +
<tr class="row5">
 +
<td class="col0"> Glucose (20%) </td><td class="col1"> 40ml </td><td class="col2"> added after sterilisation (filter sterilized) </td>
 
</tr>
 
</tr>
 
</table></div>
 
</table></div>
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<!-- EDIT2 TABLE [1300-1419] --><ol>
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<!-- EDIT2 TABLE [412-920] -->
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<li class="level1"><div class="li"> Put flow cells in petri dish with humid tissue and sealed with parafilm</div>
+
</div>
 +
<li class="level1"><div class="li"> autoclave medium than add filter sterilized Thiamine and Glucose</div>
 
</li>
 
</li>
−
<li class="level1"><div class="li"> Incubate at 37°C</div>
+
<li class="level1"><div class="li"> Store at 4°C</div>
 
<ol>
 
<ol>
−
<li class="level2"><div class="li"> for 25 min (for a quick check)</div>
+
 
−
</li>
+
−
<li class="level2"><div class="li"> for 2 h (for full Expression)</div>
+
−
</li>
+
−
</ol>
+
−
</li>
+
−
<li class="level1"><div class="li"> Measure fluorescence in microscope of AG Eimer (only possible with Tobi)</div>
+
−
</li>
+
−
</ol>
+
 
<div class="tags"><span>
 
<div class="tags"><span>
 
<a class="wikilink1" href="/igem2015/doku.php?id=tag:protocols&amp;do=showtag&amp;tag=protocols" rel="tag" title="tag:protocols">protocols</a>,
 
<a class="wikilink1" href="/igem2015/doku.php?id=tag:protocols&amp;do=showtag&amp;tag=protocols" rel="tag" title="tag:protocols">protocols</a>,

Latest revision as of 22:07, 14 September 2015

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Preparation of Teriffic Broth

material: chemicals, used kits, …
duration: 90 min

Terrific Broth

Component for 1 litre medium remarks
Monopotassium phosphate 5.6 g
Potassium phosphate dibasic 28.9 g
yeast extract 10
Thiamine 10 mg added after sterilisation (filter sterilized)
Glucose (20%) 40ml added after sterilisation (filter sterilized)
  • autoclave medium than add filter sterilized Thiamine and Glucose
  • Store at 4°C
      ,