Team:USTC CHINA/Notebook/Protocols/Single Digestion

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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>Single Digestion</h1>
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<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
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<p>1.Performed with Thermo Scientific FastDigest. There are four restriction endonucleases we used in our experiments: EcoRI, XbaI, SpeI, PstI.</br>
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Protocol
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<span>Single Digestion</span></br>
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1. Excise gel slice containing DNA fragment of interest.
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<table width="580" border="2">
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2. Add 3×sample volume of Buffer DE-A.
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  <tr>
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Incubate at 75° C for 15-20 min or until gel melts completely.
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    <td></td>
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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    <td>Volume (μl)</td>
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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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  </tr>
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3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
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  <tr>
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    <td>Plasmid DNA</td>
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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    <td>Up to 1μg</td>
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Repeat wash with Buffer W2
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  </tr>
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Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
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  <tr>
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    <td>10×FastDigest Buffer</td>
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5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
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    <td>2</td>
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  </tr>
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  <tr>
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    <td>Enzyme</td>
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    <td>1</td>
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  </tr>
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  <tr>
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    <td>ddH<sub>2</sub>O</td>
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    <td>To 20</td>
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  </tr>
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  <tr>
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    <td>Total</td>
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    <td>20</td>
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  </tr>
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</table>
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2. Mix gently and spin down.</br>
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3. Incubate at 37°C in a heat block or water thermostat for 30 min.</br>
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4. Inactivate the enzyme by adding glycerol DNA loading buffer, or by heating for 5 min at 80°C(only for EcoRI) or 20min at 65°C(only for XbaI)</br>
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Latest revision as of 08:55, 27 September 2013

Single Digestion

1.Performed with Thermo Scientific FastDigest. There are four restriction endonucleases we used in our experiments: EcoRI, XbaI, SpeI, PstI.
Single Digestion

Volume (μl)
Plasmid DNA Up to 1μg
10×FastDigest Buffer 2
Enzyme 1
ddH2O To 20
Total 20
2. Mix gently and spin down.
3. Incubate at 37°C in a heat block or water thermostat for 30 min.
4. Inactivate the enzyme by adding glycerol DNA loading buffer, or by heating for 5 min at 80°C(only for EcoRI) or 20min at 65°C(only for XbaI)

Notebook

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