Team:USTC CHINA/Notebook/Protocols/Concentrating proteins

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<div id="breadcrumb"><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">notebook</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">protocols</a></div></div>
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<h1>Gel Extraction</h1>
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<h1>Concentrating proteins</h1>
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<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
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<p>Performed with Sangon Biotech(Shanghai)</br>
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Protocol
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<span>Protocol</span>：</br>
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1. Excise gel slice containing DNA fragment of interest.
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1.Prepare a 1.5ml clean centrifuge tube and add 200ul sample protein solution to the tube.</br>
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2. Add 3×sample volume of Buffer DE-A.
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2.Add 50ul precipitaton solution A and reverse the tube vertically for 10 seconds to make it mixed up evenly. </br>
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Incubate at 75° C for 15-20 min or until gel melts completely.
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3.Put the tube on ice or the substratum of fridge and incubate it for an hour. </br>
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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4.Centrifuge the tube for 15 minutes with 15000rpm, temperature 4℃. </br>
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<img src="https://static.igem.org/mediawiki/2013/4/4d/2013igemustc-china_Protocol1.png" width="580" height="200" />  
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5.Remove the supernatant and the liquid on the tube wall and tube bottom, and remain the sediment. </br>
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3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
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6.Add 600ul washing liquor and reverse the tube vertically for 10 seconds. </br>
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7.Put the tube on the substratum of fridge for 10 minutes and centrifuge the tube for 15 minutes with 12000rpm, temperature 4℃ to get the sediment.</br>
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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8.Pour away the supernatant and dry out the sediment in the draught cupboard for 30 minutes or unwater the sediment with freeze drier. </br>
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Repeat wash with Buffer W2
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9.Add 20ul solution C and reverse the tube vertically for 10 seconds; if the solution turns yellow, add 1-5ul buffer solution to make it blue </br>
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Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
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10.Boiling the solution for 5 minutes and centrifuge it for 5 minutes with 12000rpm at room temperature.</br>
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12.Sample the supernatant and carry out SDS-PAGE electrophoresis and analyze the molecule weight.</br>
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5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
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Latest revision as of 09:11, 27 September 2013

Concentrating proteins

Performed with Sangon Biotech(Shanghai)
Protocol：
1.Prepare a 1.5ml clean centrifuge tube and add 200ul sample protein solution to the tube.
2.Add 50ul precipitaton solution A and reverse the tube vertically for 10 seconds to make it mixed up evenly.
3.Put the tube on ice or the substratum of fridge and incubate it for an hour.
4.Centrifuge the tube for 15 minutes with 15000rpm, temperature 4℃.
5.Remove the supernatant and the liquid on the tube wall and tube bottom, and remain the sediment.
6.Add 600ul washing liquor and reverse the tube vertically for 10 seconds.
7.Put the tube on the substratum of fridge for 10 minutes and centrifuge the tube for 15 minutes with 12000rpm, temperature 4℃ to get the sediment.
8.Pour away the supernatant and dry out the sediment in the draught cupboard for 30 minutes or unwater the sediment with freeze drier.
9.Add 20ul solution C and reverse the tube vertically for 10 seconds; if the solution turns yellow, add 1-5ul buffer solution to make it blue
10.Boiling the solution for 5 minutes and centrifuge it for 5 minutes with 12000rpm at room temperature.
12.Sample the supernatant and carry out SDS-PAGE electrophoresis and analyze the molecule weight.

Notebook

Retrieved from "http://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/Concentrating_proteins"