View source for Team:Freiburg/Protocols/Gel Extraction


{{Freiburg/CSS}}
{{Freiburg/Menubar}}
{{Freiburg/LabjournalCSS}}
{{Freiburg/wiki_content_start}}
<html>
<style>
/*========= BEGIN: style for navigation bar ==========*/
#notebook {
    background: url(https://static.igem.org/mediawiki/2015/c/cd/Freiburg_icon_notebook_active_yellow.png) no-repeat;
}

#notebook a {
    color: #ecdc18;
}

#runningchip {
    left: 85.5%;


/*========= END: style for navigation bar ==========*/
</style>


<!--========= BEGIN: GoBack button ==========-->
<div class="button_back">
      <a style="color:#FFF" href="https://2015.igem.org/Team:Freiburg/Protocols"> << Back</a>
</div>
<!--========= END: GoBack button ==========-->

<div class="content_box">

<h1 class="sectionedit1">Gel Extraction</h1>
<div class="level1">

<p>
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.
</p>

<p>
<strong>Protocol by QIAGEN, modified</strong>
</p>
<p>
<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/>
<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/>
</p>
<hr/>
<ol>
<li>Cut out the desired DNA band (<strong>use UV protection</strong>) and transfer the gel slice into an Eppendorff tube.
</li>
<li>Weigh out the gel slice.
</li>
<li>Add QG-Buffer (three times the volume of the gel slice; assume 1 g ~ 1 mL)
</li>
<li>Incubate at 50°C until the agarose is completely solved (~10 min, vortex several times)
</li>
<li>Add isopropanole (one volume) and mix by inverting the tube several times.
</li>
<li>Load the sample onto a spin column (max. 750 µL at once).
</li>
<li> Centrifuge for 1 min at full speed (RT) and discard the flow through.
</li>
<li>Load 750 µL PE washing buffer onto the column.
</li>
<li>Centrifuge for 1 min for 1 min at full speed (RT) and discard the flow through.
</li>
<li>Transfer column into a new Eppendorff tube.
</li>
<li>Apply 25 µL dH2O onto the membrane (may be warmed up before).
</li>
<li>Incubate the column for 10 min at 45°C (may be shaking at 300 rpm)
</li>
<li>Centrifuge for 1 min at full speed (RT) to elute the DNA from the membrane.
</li>
<li>The final concentration may be determined by NanoDrop.
</li>
</ol>
<p>
<br/>
<strong>Note:</stong> to increase the yield two bands at once may be loaded onto one column.
</p>
<div class="tags"><span>
<a class="wikilink1" href="/igem2015/doku.php?id=tag:protocols_old&amp;do=showtag&amp;tag=protocols_old" rel="tag" title="tag:protocols_old">protocols old</a>
</span></div>
</div>

</div>
</html>
<!-- Labjournal content ends here -->
{{Freiburg/wiki_content_end}}