Team:USTC CHINA/Notebook/Protocols/Colony PCR

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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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<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> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/Colony PCR">protocols Colony PCR</a></div></div>
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<h1>Gel Extraction</h1>
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<h1>Colony PCR</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 Thermo Scientific Taq DNA Polymerase (recombinant), 5U/μl</br>
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Protocol
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<table width="580" border="2">
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1. Excise gel slice containing DNA fragment of interest.
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  <tr>
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2. Add 3×sample volume of Buffer DE-A.
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    <td></td>
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Incubate at 75° C for 15-20 min or until gel melts completely.
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    <td>Volume (μl)</td>
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Add 0.5 × Buffer DE-A volume of Buffer DE-B.
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  </tr>
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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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    <td>10×Taq Buffer</td>
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    <td>5</td>
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4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
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  </tr>
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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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    <td>dNTP Mix, 2 mM each</td>
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    <td>5 (0.2 mM of each)</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>Forward primer</td>
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    <td>0.1-1.0 μM</td>
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  </tr>
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    <td>Reverse primer</td>
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    <td>0.1-1.0 μM</td>
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  </tr>
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    <td>25 mM MgCl<sub>2</sub>*</td>
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    <td>1-4 mM</td>
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  </tr>
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<tr>
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    <td>Template DNA</td>
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    <td>Picked from the colony after transformation</td>
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  </tr>
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<tr>
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    <td>Taq DNA Polymerase</td>
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    <td>0.25 U</td>
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  </tr><tr>
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    <td>ddH<sub>2O</sub></td>
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    <td>To 10</td>
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  </tr>
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    <td>Total</td>
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    <td>10</td>
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  </tr>
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Revision as of 16:44, 25 September 2013

Colony PCR

Performed with Thermo Scientific Taq DNA Polymerase (recombinant), 5U/μl

Volume (μl)
10×Taq Buffer 5
dNTP Mix, 2 mM each 5 (0.2 mM of each)
Forward primer 0.1-1.0 μM
Reverse primer 0.1-1.0 μM
25 mM MgCl2* 1-4 mM
Template DNA Picked from the colony after transformation
Taq DNA Polymerase 0.25 U
ddH2O To 10
Total 10

notebook

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