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Team:Sumbawagen/Notebook/protocol4
From 2014.igem.org
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<a class="brand" style="padding:0px 15px;width:70px;height:50px;" href="https://igem.org"><img width="70px" height="50px" src="https://static.igem.org/mediawiki/2013/1/17/IGEM_basic_Logo_white_stylized.png"> </a> | <a class="brand" style="padding:0px 15px;width:70px;height:50px;" href="https://igem.org"><img width="70px" height="50px" src="https://static.igem.org/mediawiki/2013/1/17/IGEM_basic_Logo_white_stylized.png"> </a> | ||
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<li><a href="https://2014.igem.org/Team:Sumbawagen/parts">Parts</a></li> | <li><a href="https://2014.igem.org/Team:Sumbawagen/parts">Parts</a></li> | ||
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<li><a href="https://2014.igem.org/Team:Sumbawagen/Notebook/Protocol">Protocol</a></li> | <li><a href="https://2014.igem.org/Team:Sumbawagen/Notebook/Protocol">Protocol</a></li> | ||
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<li><a href="https://2014.igem.org/Team:Sumbawagen/Team">Meet the Team</a></li> | <li><a href="https://2014.igem.org/Team:Sumbawagen/Team">Meet the Team</a></li> | ||
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<li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/moyo">Moyo Festival</a></li> | <li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/moyo">Moyo Festival</a></li> | ||
<li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/virtual">Virtual Outreach</a></li> | <li><a href="https://2014.igem.org/Team:Sumbawagen/Human_practice/virtual">Virtual Outreach</a></li> | ||
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Latest revision as of 09:17, 19 February 2015
Team:Sumbawagen/Team
From 2014.igem.org
Notebook – Protocol – 4. DNA Purification
Spin column based purification was used. The kit is Gel/PCR extraction kit (Nippon Genetics). Protocols from the kit were followed by adaptation because we have no high speed microcentrifuge, but only a 6,000 rpm small microcentrifuge. All steps were done at room temperature.
DNA Purification from Liquid Samples
1. Into a clean, sterilized 0.5 ul polypropylene tube, add PCR products : buffer GP1 = 1 : 5 (e.g. 40 ul : 200 ul). Vortexing.
2. Load the samples onto the spin column equipped with collection tube. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.
3. Add 600 ul of GP2. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.
4. Centrifuge for 6,000 rpm; 5 minutes to dry the column.
5. Replace collecting tube with clean, sterilized 1.5 ml size polypropylene tube.
6. Add 30 ul of GP3. Incubate 2 minutes at room temperature. Centrifuge for 6,000 rpm; 5 minutes.
7. Obtain the DNA as the flowthrough.
DNA Purification from Agarose Gel
1. Into a clean, sterlized 1.5 ul polypropylene tube, add cutted gel containing DNA of interest : buffer GP1 = < 300 mg : 500 ul. Vortexing. Incubate at 55 oC; 10-15 minutes. Invert the tube.
2. Load the samples onto the spin column equipped with collection tube. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.
3. Add 600 ul of GP2. Centrifuge for 6,000 rpm; 1 minute. Discard the flowthrough.
4. Centrifuge for 6,000 rpm; 5 minutes to dry the column.
5. Replace collecting tube with clean, sterilized 1.5 ml size polypropylene tube
6. Add 30 ul of GP3. Incubate 2 minutes at room temperature. Centrifuge for 6,000 rpm; 5 minutes.
7. Obtain the DNA as the flowthrough.
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