Difference between revisions of "Team:Freiburg/Protocols/Gel Extraction"

 
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<h1 class="sectionedit1">Gel Extraction</h1>
 
<h1 class="sectionedit1">Gel Extraction</h1>
 
<div class="level1">
 
<div class="level1">
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<p>
 
<p>
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<strong>Protokoll zur Aufreinigung von DNA nach Gel-Elektrophorese</strong><br/>
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DNA fragments that are received by PCR amplification or restriction digest are commonly analyzed by agarose gel electrophoresis. To recover the desired fragment from the gel it may be cut out and purified by using a specialized kit. Therefore, it is applied to a membrane located in a spin column. After several washing steps, it is eluted from this membrane and transferred into a new tube. The concentration may be determined by NanoDrop afterwards.
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<strong>Protocol on the purification of DNA after gel electrophoresis</strong><br/>
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<em>Protocol by QIAGEN, modified</em>
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<a class="media" href="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png" title="wiki:symbols:orga.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/></a> material: Kit (z.B. QIAGEN …)<br/>
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<strong>Protocol by QIAGEN, modified</strong>
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<a class="media" href="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png" title="wiki:symbols:alarm2.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/></a> duration: 90 min<br/>
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<a class="media" href="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png" title="wiki:symbols:orga.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/4/4b/Freiburg_wiki-symbols-orga.png"/></a> Material: Gel Extraction kit (e.g. QIAGEN)<br/>
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<a class="media" href="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png" title="wiki:symbols:alarm2.png"><img alt="" class="media" src="https://static.igem.org/mediawiki/2015/b/bb/Freiburg_wiki-symbols-alarm2.png"/></a> Duration: 45 min<br/>
 
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<ol>
 
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<li class="level1"><div class="li"> cut out DNA band (use UV protection) and transfer into Eppendorff tube (weight gel slice)</div>
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<li>Cut out the desired DNA band (<strong>use UV protection</strong>) and transfer the gel slice into an Eppendorff tube.
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</li>
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<li>Weigh out the gel slice.
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</li>
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<li>Add QG-Buffer (three times the volume of the gel slice; assume 1 g ~ 1 mL)
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</li>
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<li>Incubate at 50°C until the agarose is completely solved (~10 min, vortex several times)
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</li>
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<li>Add isopropanole (one volume) and mix by inverting the tube several times.
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</li>
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<li>Load the sample onto a spin column (max. 750 µL at once).
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</li>
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<li> Centrifuge for 1 min at full speed (RT) and discard the flow through.
 
</li>
 
</li>
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<li class="level1"><div class="li"> <strong>500 µL</strong> QG-Buffer (3/1000 of the mass of the gel slice)</div>
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<li>Load 750 µL PE washing buffer onto the column.
 
</li>
 
</li>
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<li class="level1"><div class="li"> <strong>incubate</strong> 10 min @ 50°C, vortex from time to time</div>
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<li>Centrifuge for 1 min for 1 min at full speed (RT) and discard the flow through.
 
</li>
 
</li>
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<li class="level1"><div class="li"> load onto the column</div>
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<li>Transfer column into a new Eppendorff tube.
 
</li>
 
</li>
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<li class="level1"><div class="li"> <strong>centrifuge</strong> 1 min, full speed @ RT (discard flow through)</div>
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<li>Apply 25 µL dH2O onto the membrane (may be warmed up before).
 
</li>
 
</li>
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<li class="level1"><div class="li"> <strong>500 µL QG-Buffer</strong></div>
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<li>Incubate the column for 10 min at 45°C (may be shaking at 300 rpm)
 
</li>
 
</li>
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<li class="level1"><div class="li"> <strong>centrifuge</strong> 1 min, full speed @ RT (discard flow through)</div>
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<li>Centrifuge for 1 min at full speed (RT) to elute the DNA from the membrane.
 
</li>
 
</li>
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<li class="level1"><div class="li"> <strong>750 µL PE washing buffer*
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<li>The final concentration may be determined by NanoDrop.
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  - </strong>incubate<strong> 2-5 min @ RT
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  - </strong>centrifuge<strong> 1 min, full speed @ RT
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  - turn tubes 180°
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  - </strong>centrifuge<strong> 1 min full speed @ RT
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  - transfer column into Eppendorff tube
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  - pipet 25 µL water on the membran (may be warmed up before)
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  - </strong>incubate<strong> 10 min RT
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  - </strong>incubate<strong> 3 min, 55°C
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  - </strong>centrifuge** 1min, full speed RT</div>
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</li>
 
</li>
 
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<p>
 
<p>
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⇒ DNA is transferred into the Eppendorff tube<br/>
 
 
<br/>
 
<br/>
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<em>To increase yield two bands at once may be loaded onto the column</em>
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<strong>Note:</stong> to increase the yield two bands at once may be loaded onto one column.
 
</p>
 
</p>
 
<div class="tags"><span>
 
<div class="tags"><span>

Latest revision as of 07:07, 20 November 2015

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Gel Extraction

DNA fragments that are received by PCR amplification or restriction digest are commonly analyzed by agarose gel electrophoresis. To recover the desired fragment from the gel it may be cut out and purified by using a specialized kit. Therefore, it is applied to a membrane located in a spin column. After several washing steps, it is eluted from this membrane and transferred into a new tube. The concentration may be determined by NanoDrop afterwards.

Protocol by QIAGEN, modified

Material: Gel Extraction kit (e.g. QIAGEN)
Duration: 45 min

  1. Cut out the desired DNA band (use UV protection) and transfer the gel slice into an Eppendorff tube.
  2. Weigh out the gel slice.
  3. Add QG-Buffer (three times the volume of the gel slice; assume 1 g ~ 1 mL)
  4. Incubate at 50°C until the agarose is completely solved (~10 min, vortex several times)
  5. Add isopropanole (one volume) and mix by inverting the tube several times.
  6. Load the sample onto a spin column (max. 750 µL at once).
  7. Centrifuge for 1 min at full speed (RT) and discard the flow through.
  8. Load 750 µL PE washing buffer onto the column.
  9. Centrifuge for 1 min for 1 min at full speed (RT) and discard the flow through.
  10. Transfer column into a new Eppendorff tube.
  11. Apply 25 µL dH2O onto the membrane (may be warmed up before).
  12. Incubate the column for 10 min at 45°C (may be shaking at 300 rpm)
  13. Centrifuge for 1 min at full speed (RT) to elute the DNA from the membrane.
  14. The final concentration may be determined by NanoDrop.


Note: to increase the yield two bands at once may be loaded onto one column.