Team:HKUST/Protocols/gel extraction

From 2009.igem.org

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7. Gel extraction
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  Purpose: To clean up the gel after band cutting from the gel and leave only the desired DNA fragment.
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<head>
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  Materials: GEX Buffer, WN Buffer, WS Buffer, Elution Buffer (all provided in FavorPrepTM Gel/PCR DNA Clean-Up Kit)
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<meta http-equiv="Content-Type" content="text/html; charset=iso-8859-1" />
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  Procedure:
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<link href="http://igem2009hkust.fileave.com/wiki/template/12092009/style.css " rel="stylesheet" type="text/css" />
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   a.Add 0.5ml GEX buffer into the tube with gel fragment in it.  
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<title>Salt and Soap template</title>
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   b.Incubate at 60°C for 5-10mins until the gel is completely dissolved. Invert the tube every 2 mins during the incubation. Stop incubation after the gel is completely dissolved. Let the gel mixture to cool down to room temperature.
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</head>
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   c.Place a GP column onto a collection tube. Load no more than 0.7ml gel mixture into each column. Centrifuge for 30s-60s (9000rpm). Discard the flow through.
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   d.Repeat step c for the excessive gel mixture.
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<bodyxx>
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   e.Wash the column once with 0.5ml WN buffer by centrifuging for 30s-60s (9000rpm). Discard the flow through.
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<div id="containerxx">
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   f.Wash the column once with 0.5ml WS buffer by centrifuging for 30s-60s (9000rpm).
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<div id="headerxx">
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   g.Centrifuge for another 3 mins (13200rpm) to remove all the ethanol
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   h.Place the column onto a new 1.5ml centrifuge tube. Add 15μL -30μL elution buffer onto the center of the membrane. (Preheated the Elution buffer to 60°C).
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</div>
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   i.Stand the column for another 3mins and then centrifuge at full speed for 1-2 mins to elute DNA.
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<div id="borderxx">
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  Tips:
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<div id="mainxx">
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  •The gel extraction has a low recovery rate of about 10%. Stand the column longer in step i may give relatively higher recovery rate.
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<div class="contentlist"> <h3>a</h3>
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</div>
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<div class="contentxx">
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<p>Gel extraction</p>
 +
 
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<p> Purpose: To clean up the gel after band cutting from the gel and leave only the desired DNA fragment.</p>
 +
Materials: GEX Buffer, WN Buffer, WS Buffer, Elution Buffer (all provided in FavorPrepTM Gel/PCR DNA Clean-Up Kit) <br><br>
 +
 
 +
<p>Procedure: </p>
 +
   a.Add 0.5ml GEX buffer into the tube with gel fragment in it. <br>
 +
   b.Incubate at 60°C for 5-10mins until the gel is completely dissolved. Invert the tube every 2 mins during the incubation. Stop incubation after the gel is completely dissolved. Let the gel mixture to cool down to room temperature.<br>
 +
   c.Place a GP column onto a collection tube. Load no more than 0.7ml gel mixture into each column. Centrifuge for 30s-60s (9000rpm). Discard the flow through.<br>
 +
   d.Repeat step c for the excessive gel mixture.<br>
 +
   e.Wash the column once with 0.5ml WN buffer by centrifuging for 30s-60s (9000rpm). Discard the flow through.<br>
 +
   f.Wash the column once with 0.5ml WS buffer by centrifuging for 30s-60s (9000rpm).<br>
 +
   g.Centrifuge for another 3 mins (13200rpm) to remove all the ethanol<br>
 +
   h.Place the column onto a new 1.5ml centrifuge tube. Add 15μL -30μL elution buffer onto the center of the membrane. (Preheated the Elution buffer to 60°C).<br>
 +
   i.Stand the column for another 3mins and then centrifuge at full speed for 1-2 mins to elute DNA.<br><br>
 +
<p> Tips: </p>
 +
The gel extraction has a low recovery rate of about 10%. Stand the column longer in step i may give relatively higher recovery rate.
 +
 
 +
<ul>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel
 +
electrophoresis</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR">PCR (Taq, Vent)</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/PCR_cleanup">PCR product clean-up</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_cut"> Gel cutting</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/gel_extraction"> Gel extraction</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ethanol_precipitation"> Ethanol precipitation</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to
 +
yeast</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Cell_lysis"> Cell lysis</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Yeast_genomic_DNA_extraction"> Yeast genomic DNA
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extraction</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Western_blotting"> Western blotting</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/GFP_testing"> GFP testing</a></li>
 +
 
 +
</ul>
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</div>
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<div class="productxx">
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<div class="clear"></div>
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</div>
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</div>
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<div class="clear"></div>
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</div>
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</div>
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<div id="footerxx">
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<div id="copyright">
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<span> iGEM 2009 <br /> </span>
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</div>
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<div id="payment"><img src="http://igem2009hkust.fileave.com/wiki/template/12092009/images/HKUSTLogo.jpg" alt="HKUST" /></div>
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</div>
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<div id="footerend"></div>
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</div>
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</bodyxx>
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</html>

Revision as of 15:45, 19 October 2009

Salt and Soap template

a

Gel extraction

Purpose: To clean up the gel after band cutting from the gel and leave only the desired DNA fragment.

Materials: GEX Buffer, WN Buffer, WS Buffer, Elution Buffer (all provided in FavorPrepTM Gel/PCR DNA Clean-Up Kit)

Procedure:

a.Add 0.5ml GEX buffer into the tube with gel fragment in it.
b.Incubate at 60°C for 5-10mins until the gel is completely dissolved. Invert the tube every 2 mins during the incubation. Stop incubation after the gel is completely dissolved. Let the gel mixture to cool down to room temperature.
c.Place a GP column onto a collection tube. Load no more than 0.7ml gel mixture into each column. Centrifuge for 30s-60s (9000rpm). Discard the flow through.
d.Repeat step c for the excessive gel mixture.
e.Wash the column once with 0.5ml WN buffer by centrifuging for 30s-60s (9000rpm). Discard the flow through.
f.Wash the column once with 0.5ml WS buffer by centrifuging for 30s-60s (9000rpm).
g.Centrifuge for another 3 mins (13200rpm) to remove all the ethanol
h.Place the column onto a new 1.5ml centrifuge tube. Add 15μL -30μL elution buffer onto the center of the membrane. (Preheated the Elution buffer to 60°C).
i.Stand the column for another 3mins and then centrifuge at full speed for 1-2 mins to elute DNA.

Tips:

The gel extraction has a low recovery rate of about 10%. Stand the column longer in step i may give relatively higher recovery rate.
HKUST

Retrieved from "http://2009.igem.org/Team:HKUST/Protocols/gel_extraction"