Team:HKUST/Protocols/PCR cleanup

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(New page: 3. PCR product clean-up Purpose: To clean up primers, dNTPs, enzymes, short-failed PCR products in the PCR reaction. Materials: FAPC Buffer，Wash Buffer, Elution Buffer, PCR Product...)
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3. PCR product clean-up
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  Purpose: To clean up primers, dNTPs, enzymes, short-failed PCR products in the PCR reaction.
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<head>
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  Materials: FAPC Buffer，Wash Buffer, Elution Buffer, PCR Product (all provided in FavorPrepTM PCR 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.Transfer 30μL of PCR product and add 5 volumes of FAPC buffer to a 1.5mL microcentrifuge tube, then mix well by vortexing.
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<title>Salt and Soap template</title>
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   b.Place a FAPC Column into a Collection Tube and transfer the sample mixture to FAPC Column.
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</head>
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   c.Centrifuge at 6,000 rpm for1 min then discard the flow-through.
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   d.Add 750 μl of Wash Buffer (ethanol added) to FAPC Column. Centrifuge at 6,000 rpm for 1 min, then discard the flow-through.
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   e.Centrifuge at 14,000 rpm for an additional 3 min to dry the column.
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   f.Place FAPC Column into an Elution Tube.
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   g.Add 40 μl of Elution Buffer or ddH2O (pH 7.0~8.5) to the membrane center of FAPC Column. Stand FAPC Column for 2 minutes.
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   h.Centrifuge at 14,000 rpm for 1 min to elute the DNA.
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  Tips:
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<div id="borderxx">
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  •In step f, for effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely.
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<li><a href="https://2009.igem.org/Team:HKUST">Home</a></li>
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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>PCR product clean-up</p>
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<p> Purpose: To clean up primers, dNTPs, enzymes, short-failed PCR products in the PCR reaction.</p>
 +
Materials: FAPC Buffer，Wash Buffer, Elution Buffer, PCR Product (all provided in FavorPrepTM PCR Clean-Up Kit)<br> <br>
 +
 
 +
<p>Procedure: </p>
 +
a.Transfer 30μL of PCR product and add 5 volumes of FAPC buffer to a 1.5mL microcentrifuge tube, then mix well by vortexing.<br>
 +
   b.Place a FAPC Column into a Collection Tube and transfer the sample mixture to FAPC Column.<br>
 +
   c.Centrifuge at 6,000 rpm for1 min then discard the flow-through.<br>
 +
   d.Add 750 μl of Wash Buffer (ethanol added) to FAPC Column. Centrifuge at 6,000 rpm for 1 min, then discard the flow-through.<br>
 +
   e.Centrifuge at 14,000 rpm for an additional 3 min to dry the column.<br>
 +
   f.Place FAPC Column into an Elution Tube.<br>
 +
   g.Add 40 μl of Elution Buffer or ddH2O (pH 7.0~8.5) to the membrane center of FAPC Column. Stand FAPC Column for 2 minutes.<br>
 +
   h.Centrifuge at 14,000 rpm for 1 min to elute the DNA.<br>
 +
<p> Tips: </p>
 +
In step f, for effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely.
 +
 
 +
 
 +
<ul>
 +
<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Agarose_gel" >Agarose gel preparation and gel
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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>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Enzyme_digestion"> Enzyme digestion (vector, insert)</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/plasmid_extraction"> Plasmid DNA extraction</a></li>
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<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>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Ligation"> Ligation</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_Ecoli"> Transformation to E.coli</a></li>
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<li><a href="https://2009.igem.org/Team:HKUST/Protocols/Transformation_to_yeast">Transformation to
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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>
 +
 
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</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 class="clear"></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 id="footerend"></div>
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</div>
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</bodyxx>
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</html>

Revision as of 15:30, 19 October 2009

Salt and Soap template

a

PCR product clean-up

Purpose: To clean up primers, dNTPs, enzymes, short-failed PCR products in the PCR reaction.

Materials: FAPC Buffer，Wash Buffer, Elution Buffer, PCR Product (all provided in FavorPrepTM PCR Clean-Up Kit)

Procedure:

a.Transfer 30μL of PCR product and add 5 volumes of FAPC buffer to a 1.5mL microcentrifuge tube, then mix well by vortexing.
b.Place a FAPC Column into a Collection Tube and transfer the sample mixture to FAPC Column.
c.Centrifuge at 6,000 rpm for1 min then discard the flow-through.
d.Add 750 μl of Wash Buffer (ethanol added) to FAPC Column. Centrifuge at 6,000 rpm for 1 min, then discard the flow-through.
e.Centrifuge at 14,000 rpm for an additional 3 min to dry the column.
f.Place FAPC Column into an Elution Tube.
g.Add 40 μl of Elution Buffer or ddH2O (pH 7.0~8.5) to the membrane center of FAPC Column. Stand FAPC Column for 2 minutes.
h.Centrifuge at 14,000 rpm for 1 min to elute the DNA.

Tips:

In step f, for effective elution, make sure that the elution solution is dispensed onto the membrane center and is absorbed completely.
HKUST

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