Team:USTC CHINA/Notebook/Protocols/Ligation

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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>Ligation</h1>
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<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
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<p><span>Performed with TaKaRa Ligation Kit (Solution I)</span></br>
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Protocol
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1. System</br>
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1. Excise gel slice containing DNA fragment of interest.
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2. Add 3×sample volume of Buffer DE-A.
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<table width="580" border="2">
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Incubate at 75° C for 15-20 min or until gel melts completely.
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  <tr>
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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    <td></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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    <td>Volume (μl)</td>
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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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  <tr>
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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    <td>Solution I</td>
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Repeat wash with Buffer W2
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    <td>5</td>
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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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  <tr>
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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>DNA (fragment + carrier)</td>
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    <td>5</td>
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  </tr>
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</table>
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</br>
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fragment : carrier (radio in mol)= 3:1~10:1</br>
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carrier≥0.03 pmol</br>
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2. Gently vortex the samples and spin down.</br>
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3. Incubate at 16°C in a low temperature water thermostat for at least 3h.</br>
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Latest revision as of 08:58, 27 September 2013

Ligation

Performed with TaKaRa Ligation Kit (Solution I)
1. System

Volume (μl)
Solution I 5
DNA (fragment + carrier) 5

fragment : carrier (radio in mol)= 3:1~10:1
carrier≥0.03 pmol
2. Gently vortex the samples and spin down.
3. Incubate at 16°C in a low temperature water thermostat for at least 3h.

Notebook

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