Team:NCTU Formosa/protocol

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</head>
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<body>
<body>
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<br>
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<ul id = "pmenu">
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<li><a>Team &#187;</a>
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<div id="banner"><a href = "https://2011.igem.org/Team:NCTU_Formosa"><img src = "https://static.igem.org/mediawiki/2011/8/8c/Banner010.jpg" height="236" width="944"/></a></div>
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<ul>
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/members">Members</a></li>
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<li><a href="https://2011.igem.org/Team:NCTU_Formosa/school">School</a></li>
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<ul id="cm-nav">
-
<li><a href="https://2011.igem.org/Team:NCTU_Formosa/gallery">Gallery</a></li>
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  <li><a href="https://2011.igem.org/Team:NCTU_Formosa">Home</a></li>
-
</ul>
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  <li><a onClick="out('cm-nav')" class="arrow">Team </a>
 +
      <ul>
 +
        <li><a href="https://2011.igem.org/Team:NCTU_Formosa/members">Members</a></li>
 +
        <li><a href="http://www.nctu.edu.tw/english/index.php">School</a></li>
 +
        <li><a href="https://2011.igem.org/Team:NCTU_Formosa/gallery">Gallery</a></li>
 +
      </ul>
 +
  </li>
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  <li><a onClick="out('cm-nav')" class="arrow">Project</a>
 +
      <ul class="arrow-pad">
 +
        <li><a href="https://2011.igem.org/Team:NCTU_Formosa/introduction">Introduction</a></li>
 +
        <li><a onClick="out('cm-nav')" class="arrow">RNA Thermometer</a>
 +
            <ul>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_design">Design</a></li>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_data">Data</a></li>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_discussion">Modeling</a></li>
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            </ul>
 +
        </li>
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        <li><a onClick="out('cm-nav')" class="arrow">CI promoter </a>
 +
            <ul>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_design">Design</a></li>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_data">Data</a></li>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_discussion">Modeling</a></li>
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            </ul>
 +
        </li>
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        <li><a onClick="out('cm-nav')" class="arrow">Carotenoid synthesis pathway</a>
 +
            <ul>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_design">Design</a></li>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_data">Data</a></li>
 +
            </ul>
 +
        </li>
 +
        <li><a onClick="out('cm-nav')" class="arrow">Butanol pathway</a>
 +
            <ul>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_design">Design</a></li>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_data">Data</a></li>
 +
            </ul>
 +
        </li>
 +
        <li><a onClick="out('cm-nav')" class="arrow">Violacein pathway</a>
 +
            <ul>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/VP_design">Design</a></li>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/VP_data">Data</a></li>
 +
            </ul>
 +
        </li>       
 +
      </ul>
 +
  </li>
 +
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/modeling">Measurements</a></li>
 +
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/parts">Parts</a></li>
 +
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/safty">Safety</a></li>
 +
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/humanpractice">Human Practice</a></li>
 +
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/contributions">Attribution</a></li>
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  <li><a onClick="out('cm-nav')" class="arrow">Notebook </a>
 +
      <ul>
 +
          <li><a onClick="out('cm-nav')" class="arrow">Protocols</a>
 +
            <ul>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol">Mutation</a></li>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol_F">Flow Cytometry</a></li>
 +
              <li><a href="https://2011.igem.org/Team:NCTU_Formosa/protocol_G">GC</a></li>
 +
            </ul>
 +
          <li><a href="https://2011.igem.org/Team:NCTU_Formosa/calendar">Calendar</a></li>
 +
      </ul>
 +
  </li>
 +
</ul>
 +
 
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<br><br>
 +
<div id="blueBox"><p>Protocols</p></div>
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<div id="Box"><h2>Point Mutation</h2>
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<p>The procedure is as follows : </p>
 +
<ol type="decimal">
 +
<li> Design primers </li>
 +
<li> Find the best PCR condition by gradient PCR<br>
 +
<li> KOD PCR condition<br>
 +
<table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all">
 +
<tr><td> Plasmid 0.5 μl<br>pF 0.5<br>pR 0.5<br>MgCl2      1<br>dNTP 2.5<br>buffer 2.5<br>
 +
KOD enzyme    0.5<br>H2O 17<br>Total 25<br></td>
 +
<td>94℃  5    min<br>94℃  30  sec<br>55℃  30  sec<br>72℃  5  min<br>72℃  7~10  min<br>Cycles : 25<br><br><br><br></td>
 +
</tr></table>
</li>
</li>
-
<li><a>Project &#187;</a>
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<li> Confirm the PCR product with electrophoresis</li>
-
<ul>
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<li> DPN1 37℃ for 3hr~overnight <br>
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  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/introduction">Introduction</a></li>
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<table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all">
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<li><a>RNA Thermometer &#187;</a>
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<tr><td> DPN1 0.5<br>Buffer2 2<br>PCR product 17.5<br>Total 20<br>
-
  <ul>
+
</td></tr></table>
-
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_design">Design</a></li>
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</li>
-
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_data">Data</a></li>
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<li> 80℃ 20mins to denature the DPN1</li>
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  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/RNA_discussion">Discussion</a></li>  
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<li> Confirm with electrophoresis </li>
-
  </ul>
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<li> Self ligation room temperature for 2~3 hr <br>
-
</li>
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<table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all">
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<li><a>CI promoter &#187;</a>
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<tr><td> Enzyme 1<br> Buffer 2<br> ATP 2<br> H2O 5<br> PCR product    10<br>Total 20<br>
-
  <ul>
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</td></tr></table>
-
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_design">Design</a></li>
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</li>
-
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_data">Data</a></li>
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<li> Transform DH5alpha with the self-ligation product <br>
-
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CI_discussion">Discussion</a></li>  
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<table style="border: 1px dotted rgb(0, 0, 0);width: 300px; align="left" cellpadding="5" cellspacing="5" frame="border" rules="all">
-
  </ul>
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<tr><td> Self-ligation product  20<br>DH5alfa 50<br>
-
</li>
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</td></tr></table>
-
<li><a>Carotenoid synthesis pathway &#187;</a>
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</li>
-
  <ul>
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<li> Incubate in Ap25 plate then transfer to Ap50 plate</li>
-
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_design">Design</a></li>
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</ol>
-
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_data">Data</a></li>
+
</div>
-
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/CSP_discussion">Discussion</a></li>
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-
  </ul>
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</li>
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-
<li><a>Butanol pathway &#187;</a>
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-
  <ul>
+
-
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_design">Design</a></li>
+
-
  <li><a href="https://2011.igem.org/Team:NCTU_Formosa/BP_data">Data</a></li>
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<li><a>Violacein pathway &#187;</a>
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Latest revision as of 13:41, 5 October 2011



Protocols

Point Mutation

The procedure is as follows :

  1. Design primers
  2. Find the best PCR condition by gradient PCR
  3. KOD PCR condition
    Plasmid 0.5 μl
    pF 0.5
    pR 0.5
    MgCl2 1
    dNTP 2.5
    buffer 2.5
    KOD enzyme 0.5
    H2O 17
    Total 25
    		 94℃ 5 min
    94℃ 30 sec
    55℃ 30 sec
    72℃ 5 min
    72℃ 7~10 min
    Cycles : 25
  4. Confirm the PCR product with electrophoresis
  5. DPN1 37℃ for 3hr~overnight
    DPN1 0.5
    Buffer2 2
    PCR product 17.5
    Total 20
  6. 80℃ 20mins to denature the DPN1
  7. Confirm with electrophoresis
  8. Self ligation room temperature for 2~3 hr
    Enzyme 1
    Buffer 2
    ATP 2
    H2O 5
    PCR product 10
    Total 20
  9. Transform DH5alpha with the self-ligation product
    Self-ligation product 20
    DH5alfa 50
  10. Incubate in Ap25 plate then transfer to Ap50 plate

Retrieved from "http://2011.igem.org/Team:NCTU_Formosa/protocol"