Team:UESTC-China/Protocol

From 2014.igem.org

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<a href="https://2014.igem.org/Team:UESTC-China/Lecture.html"><li>Lecture</li></a>
<a href="https://2014.igem.org/Team:UESTC-China/Lecture.html"><li>Lecture</li></a>
<a href="https://2014.igem.org/Team:UESTC-China/Communication.html"><li>Communication</li></a>
<a href="https://2014.igem.org/Team:UESTC-China/Communication.html"><li>Communication</li></a>
 +
<a href="https://2014.igem.org/Team:UESTC-China/Art"><li>Art</li></a>
</ul>
</ul>
</div>
</div>
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       <div id="SectionTitles1" style="width:500px;">DNA ligation with Gibson Assemble
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<h1 style="color:#1b1b1b; position:relative; left:25px; padding:15 5px; font-size:40px; font-family: calibri, arial, helvetica, sans-serif; font-weight: bold;font-style: Italic; text-align:center; width:1140px;">Protocol</h1>
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       <div id="SectionTitles1" style="width:1100px;">Fragments Ligation
         </h1>
         </h1>
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         <table border="1" id="BiosensorsTable" cellspacing="0";>
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         <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;">
           <tr>
           <tr>
             <th class="top">Component</th>
             <th class="top">Component</th>
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             <th class="top">20ul Reaction</th>
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             <th class="top">20µl Reaction </th>
           </tr>
           </tr>
           <tr>
           <tr>
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             <td class="mid">1.33X Master Mix</td>
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             <td class="mid">Vector DNA</td>
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             <td class="mid"> 15ul</td>
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             <td class="mid">50ng</td>
           </tr>
           </tr>
           <tr>
           <tr>
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             <td class="mid">Insert DNA </td>
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             <td class="mid">Insert DNA</td>
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             <td class="mid"> the moles of insert DNA to vector DNA is 5:1</td>
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             <td class="mid"> the moles rate of insert DNA to vector DNA is 5:1</td>
           </tr>
           </tr>
           <tr>
           <tr>
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             <td class="mid">>Vector DNA</td>
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             <td class="mid">10X T4 DNA ligase buffer</td>
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             <td class="mid"> 50ng</td>
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            <td class="mid">2µl</td>
 +
          </tr>
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          <tr>
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            <td class="mid">T4 DNA ligase</td>
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             <td class="mid">1µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">ddH2O</td>
             <td class="mid">ddH2O</td>
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             <td class="mid"> To 20ul</td>
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             <td class="mid">to 20µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
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             <td class="mid">Incubate at 50℃ for 1 hour</td>
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             <td class="mid">Incubate at 25℃ for 12 hours</td>
             <td class="mid"></td>
             <td class="mid"></td>
           </tr>
           </tr>
         </table>
         </table>
 +
 +
       </div>
       </div>
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       <div id="SectionTitles2" style="width:500px;">DNA ligation with T4 DNA Ligase
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       <div id="SectionTitles2" style="width:1100px;">Gibson Assembly
         </h1>
         </h1>
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           <tr>
           <tr>
             <th class="top">Component</th>
             <th class="top">Component</th>
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             <th class="top">20ul Reaction </th>
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             <th class="top">20µl Reaction</th>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">Vector DNA</td>
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             <td class="mid">1.33X Master Mix</td>
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             <td class="mid">50ng</td>
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             <td class="mid"> 15µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
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             <td class="mid">Insert DNA</td>
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             <td class="mid">Insert DNA </td>
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             <td class="mid"> the moles of insert DNA to vector DNA is 5:1</td>
+
             <td class="mid"> the moles rate of insert DNA to vector DNA is 5:1</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">10X T4 DNA ligase buffer</td>
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             <td class="mid">Vector DNA</td>
-
             <td class="mid">2ul</td>
+
             <td class="mid"> 50ng</td>
-
          </tr>
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-
          <tr>
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-
            <td class="mid">T4 DNA ligase</td>
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-
            <td class="mid">1ul</td>
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           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">ddH2O</td>
             <td class="mid">ddH2O</td>
-
             <td class="mid">to 20ul</td>
+
             <td class="mid"> To 20µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">Incubate at 25℃ for 12 hours</td>
+
             <td class="mid">Incubate at 50℃ for 1 hour</td>
             <td class="mid"></td>
             <td class="mid"></td>
           </tr>
           </tr>
         </table>
         </table>
 +
 +
 +
 +
       </div>
       </div>
-
       <div id="SectionTitles3" style="width:500px;">Double Digestion of DNA
+
       <div id="SectionTitles3" style="width:1100px;">Fragment Digestion
         </h1>
         </h1>
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           <tr>
             <th class="top">Component</th>
             <th class="top">Component</th>
-
             <th class="top">50ul Reaction </th>
+
             <th class="top">50µl Reaction </th>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">10x FastDigest buffer</td>
             <td class="mid">10x FastDigest buffer</td>
-
             <td class="mid">5ul</td>
+
             <td class="mid">5µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">Restriction enzyme 1</td>
             <td class="mid">Restriction enzyme 1</td>
-
             <td class="mid"> 1ul</td>
+
             <td class="mid"> 1µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">Restriction enzyme 2</td>
             <td class="mid">Restriction enzyme 2</td>
-
             <td class="mid">1ul</td>
+
             <td class="mid">1µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">DNA</td>
             <td class="mid">DNA</td>
-
             <td class="mid">1-2ug</td>
+
             <td class="mid">1-2µg</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">ddH2O</td>
             <td class="mid">ddH2O</td>
-
             <td class="mid">to 50ul</td>
+
             <td class="mid">to 50µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
Line 677: Line 769:
         </table>
         </table>
       </div>
       </div>
-
       <div id="SectionTitles4" style="width:500px;">PCR protocol for KOD-Plus-Neo
+
       <div id="SectionTitles4" style="width:1100px;">PCR Amplication Protocol for KOD-Plus-Neo
         </h1>
         </h1>
-
         <table border="1" id="BiosensorsTable" cellspacing="0";>
+
         <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;">
           <tr>
           <tr>
             <th class="top">Component</th>
             <th class="top">Component</th>
-
             <th class="top">50ul Reaction </th>
+
             <th class="top">50µl Reaction </th>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">ddH2O</td>
             <td class="mid">ddH2O</td>
-
             <td class="mid">33ul</td>
+
             <td class="mid">33µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">10X buffer for KOD-Plus-Neo</td>
             <td class="mid">10X buffer for KOD-Plus-Neo</td>
-
             <td class="mid"> 5ul</td>
+
             <td class="mid"> 5µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">MgSO4</td>
             <td class="mid">MgSO4</td>
-
             <td class="mid">1ul</td>
+
             <td class="mid">3µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">10mM dNTPs</td>
             <td class="mid">10mM dNTPs</td>
-
             <td class="mid">1ug</td>
+
             <td class="mid">5µg</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">10uM forward primer</td>
+
             <td class="mid">10µM forward primer</td>
-
             <td class="mid">1ul</td>
+
             <td class="mid">1µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">10uM reverse primer</td>
+
             <td class="mid">10µM reverse primer</td>
-
             <td class="mid"> 1ul</td>
+
             <td class="mid">1µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">Template</td>
             <td class="mid">Template</td>
-
             <td class="mid">1ul </td>
+
             <td class="mid">1µl </td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">KOD enzyme</td>
             <td class="mid">KOD enzyme</td>
-
             <td class="mid"> 1ul</td>
+
             <td class="mid">1µl</td>
           </tr>
           </tr>
         </table>
         </table>
       </div>
       </div>
-
       <div id="SectionTitles5" style="width:500px;">
+
       <div id="SectionTitles5" style="width:1100px;">
-
         <table border="1" id="BiosensorsTable" cellspacing="0";>
+
         <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;">
           <tr>
           <tr>
             <th class="top"></th>
             <th class="top"></th>
Line 728: Line 820:
           <tr>
           <tr>
             <td class="mid">Step1</td>
             <td class="mid">Step1</td>
 +
            <td class="mid">94℃</td>
 +
            <td class="mid">5min</td>
 +
            <td class="mid">X1 cycle</td>
 +
          </tr>       
 +
          <tr>
 +
            <td class="mid">Step2</td>
             <td class="mid">94℃</td>
             <td class="mid">94℃</td>
             <td class="mid">30s</td>
             <td class="mid">30s</td>
Line 756: Line 854:
             <td class="mid"></td>
             <td class="mid"></td>
           </tr>
           </tr>
-
          <tr>
+
 
-
            <td class="mid">10uM reverse primer</td>
+
-
            <td class="mid"> 1ul</td>
+
-
            <td class="mid">ddH2O</td>
+
-
            <td class="mid">33ul</td>
+
-
          </tr>
+
         </table>
         </table>
       </div>
       </div>
-
       <div id="SectionTitles6" style="width:500px;">PCR protocol for KOD-Plus-Neo
+
       <div id="SectionTitles6" style="width:1100px;">Fusion PCR Amplication
         </h1>
         </h1>
-
        <table border="1" id="BiosensorsTable" cellspacing="0";>
+
      <p id="underTitle">Each template will be diluted by the number of moles into 2-10ng/µl.</p>
 +
      <div id="fusionArea" ><table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;">
           <tr>
           <tr>
             <th class="top">Component</th>
             <th class="top">Component</th>
-
             <th class="top">50ul Reaction </th>
+
             <th class="top">50µl Reaction </th>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">ddH2O</td>
             <td class="mid">ddH2O</td>
-
             <td class="mid">32ul</td>
+
             <td class="mid">32µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">10X buffer for KOD-Plus-Neo</td>
             <td class="mid">10X buffer for KOD-Plus-Neo</td>
-
             <td class="mid"> 5ul</td>
+
             <td class="mid"> 5µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">MgSO4</td>
             <td class="mid">MgSO4</td>
-
             <td class="mid">3ul</td>
+
             <td class="mid">3µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">10mM dNTPs</td>
             <td class="mid">10mM dNTPs</td>
-
             <td class="mid">5ul</td>
+
             <td class="mid">5µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">10uM forward primer</td>
+
             <td class="mid">10µM forward primer</td>
-
             <td class="mid">1ul</td>
+
             <td class="mid">1µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">10uM reverse primer</td>
+
             <td class="mid">10µM reverse primer</td>
-
             <td class="mid"> 1ul</td>
+
             <td class="mid">1µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">Template</td>
+
             <td class="mid">Template one</td>
-
             <td class="mid">1ul </td>
+
             <td class="mid">1µl </td>
 +
          </tr>
 +
          <tr>
 +
            <td class="mid">Template two</td>
 +
            <td class="mid">1µl </td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">KOD enzyme</td>
             <td class="mid">KOD enzyme</td>
-
             <td class="mid"> 1ul</td>
+
             <td class="mid">1µl</td>
-
          </tr>
+
-
          <tr>
+
-
            <td class="mid">Each template will be diluted by the number of moles into 2-10ng/ul.</td>
+
-
            <td class="mid"></td>
+
           </tr>
           </tr>
         </table>
         </table>
       </div>
       </div>
-
       <div id="SectionTitles7" style="width:500px;">
+
      </div>
-
         <table border="1" id="BiosensorsTable" cellspacing="0";>
+
       <div id="SectionTitles7" style="width:1100px;">
 +
         <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;">
           <tr>
           <tr>
             <th class="top"></th>
             <th class="top"></th>
Line 854: Line 949:
           </tr>
           </tr>
         </table>
         </table>
-
      </div>
+
        </div>
-
       <div id="SectionTitles8" style="width:500px;">Colony PCR with Taq DNA polymerase
+
       <div id="SectionTitles8" style="width:1100px;">Colony PCR
         </h1>
         </h1>
-
         <table border="1" id="BiosensorsTable" cellspacing="0";>
+
         <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;">
           <tr>
           <tr>
             <th class="top">Component</th>
             <th class="top">Component</th>
-
             <th class="top">25ul Reaction </th>
+
             <th class="top">25µl Reaction </th>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">ddH2O</td>
             <td class="mid">ddH2O</td>
-
             <td class="mid">15.8ul</td>
+
             <td class="mid">15.8µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">10X Taq buffer</td>
             <td class="mid">10X Taq buffer</td>
-
             <td class="mid"> 2.5ul</td>
+
             <td class="mid"> 2.5µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">2.mM dNTPs</td>
             <td class="mid">2.mM dNTPs</td>
-
             <td class="mid">0.5ul</td>
+
             <td class="mid">0.5µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">10uM forward primer</td>
+
             <td class="mid">10µM forward primer</td>
-
             <td class="mid">0.5ul</td>
+
             <td class="mid">0.5µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">10uM reverse primer</td>
+
             <td class="mid">10µM reverse primer</td>
-
             <td class="mid">0.5ul</td>
+
             <td class="mid">0.5µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">Taq enzyme</td>
             <td class="mid">Taq enzyme</td>
-
             <td class="mid"> 0.2ul</td>
+
             <td class="mid"> 0.2µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">Water with colony </td>
             <td class="mid">Water with colony </td>
-
             <td class="mid">5ul </td>
+
             <td class="mid">5µl </td>
           </tr>
           </tr>
         </table>
         </table>
       </div>
       </div>
-
       <div id="SectionTitles9" style="width:500px;">
+
       <div id="SectionTitles9" style="width:1100px;">
-
         <table border="1" id="BiosensorsTable" cellspacing="0";>
+
         <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;">
           <tr>
           <tr>
             <th class="top"></th>
             <th class="top"></th>
Line 940: Line 1,035:
     </div>
     </div>
     <div class="textEditingArea" >
     <div class="textEditingArea" >
-
       <h1 class="textEditingTitle" style="width: 600px">E. coli Calcium Chloride competent cell protocol</br>
+
 
-
         </h1><p class="textEditingstyle">1)Streak E.coli cells (DH5a) on an LB plate; (BL21(DE3)LysS cells on LB plate+34 mg/ml chloramphenicol)<br>
+
       <h1 class="textEditingTitle" style="width: 1100px">E. coli Transformation</br>
-
           2) Allow cells to grow at 37℃ overnight<br>
+
         </h1><p class="textEditingstyle">1)Streak <i>E.coli</i> cells (DH5α) on an LB plate, (BL21 (DE3) LysS cells on LB plate + 25 mg/ml chloramphenicol);<br>
-
           3)Place one colony in 10 ml LB media (+antibiotic selection if necessary), grow overnight at 37℃<br>
+
           2) Allow cells to grow at 37℃ overnight;<br>
-
           4) Take 2 ml LB media and save for blank. Transfer 5 ml overnight DH5a culture into 500 ml LB media in 3 L flask<br>
+
           3)Place one colony in 10 ml LB media (+ antibiotic selection if necessary), grow overnight at 37℃;<br>
-
           5) Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (~2-3 hours)<br>
+
           4) Take 2 ml LB media and save for blank, transfer 5 ml overnight DH5α culture into 500 ml LB media in 3 L flask;<br>
-
           6) Transfer cells to 2 centrifuge bottles (250 ml), and place cells on ice for 20 min<br>
+
           5) Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (~2-3 hours);<br>
-
           7) Centrifuge cells in at 4oC for 10 min at 3,000 g and subsequent resuspension may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room<br>
+
           6) Transfer cells to 2 centrifuge bottles (250 ml), and place cells on ice for 20 min;<br>
-
           8) Pour off media and resuspend cells in 30 ml of cold 0.1 M CaCl2. Transfer the suspended cells into 50 ml polypropylene tubes, and incubate on ice for 30 min<br>
+
           7) Centrifuge cells in at 4℃ for 10 min at 3,000 g and subsequent resuspension may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room;<br>
-
           9) Centrifuge cells at 4O℃ for 10 min at 3,000 g<br>
+
           8) Pour off media and resuspend cells in 30 ml of cold 0.1 M CaCl2. Transfer the suspended cells into 50 ml polypropylene tubes, and incubate on ice for 30 min;<br>
-
           10) Pour supernatant and resuspend cells (by pipetting) in 8 ml cold 0.1M CaCl2 containing 15% glycerol. Transfer 140 ml into (1.5 ml) Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80oC can be used for transformation for up to ~6 months<br>
+
           9) Centrifuge cells at 40℃ for 10 min at 3,000 g;<br>
-
           11) Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells in step<br>
+
           10) Pour supernatant and resuspend cells (by pipetting) in 8 ml cold 0.1M CaCl2 containing 15% glycerol Transfer 140 ml into (1.5 ml).Eppendorf tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80℃ can be used for transformation for up to ~6 months;<br>
-
           12) Incubate the mixture on ice for 30 minutes.<br>
+
           11) Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells in step;<br>
-
           13) Transfer the reaction to a 42℃ water for 1min.<br>
+
           12) Incubate the mixture on ice for 30 minutes;<br>
-
           14) Add 0.9 ml of LB culture to each tube and incubate at 37℃ for 1 hour in a roller drum (250 rpm) to allow cells to recover and express the antibiotic resistance marker. <br>
+
           13) Transfer the reaction to a 42℃ water for 1min;<br>
-
           15) Incubate on ice for 2 minutes. <br>
+
           14) Add 0.9 ml of LB culture to each tube and incubate at 37℃ for 1 hour in a roller drum (250 rpm) to allow cells to recover and express the antibiotic resistance marker; <br>
-
           16) Spread the appropriate quantity of cells (50 to 100 ml) on selective media. Store the remaining cells at 4℃. <br>
+
           15) Incubate on ice for 2 minutes;<br>
-
           (A) E. coli cells from the control tube without DNA in step 12 above are plated on selective medium and nonselective medium. The first plating ensures that the selective medium is working properly since no growth should be observed. The second plating provides the number of viable cells in the absence of selective medium. <br>
+
           16) Spread the appropriate quantity of cells (50 to 100 ml) on selective media. Store the remaining cells at 4℃.<br>
-
           (B) E. coli cells being tested for competency are plated on LB agar containing ampicillin (50 mg/ml final concentration) to ensure that the transformation efficiency has not decreased over time due to storage.<br>
+
           (A) <i>E. coli</i> cells from the control tube without DNA in step 12 above are plated on selective medium and nonselective medium. The first plating ensures that the selective medium is working properly since no growth should be observed. The second plating provides the number of viable cells in the absence of selective medium. <br>
-
           17) Incubate all plates overnight at 37℃ (agar side up). <br>
+
           (B) <i>E. coli</i> cells being tested for competency are plated on LB agar containing ampicillin (50 mg/ml final concentration) to ensure that the transformation efficiency has not decreased over time due to storage.<br>
 +
           17) Incubate all plates overnight at 37℃.<br>
           18) Count the number of colonies.<br>
           18) Count the number of colonies.<br>
         </p>
         </p>
-
<h1 class="textEditingTitle" style="width: 600px">Agrobacterium tumefacien EHA105 Mediated Transformation with freeze-thawing steps</br></h1>
+
<h1 class="textEditingTitle" style="width: 1100px">A.tume Transformation</br></h1>
         <p class="textEditingstyle">
         <p class="textEditingstyle">
-
(1)Have 0.5~1 g plasmid DNA into 100  L competent Cells,on ice for 30 min;<br>
+
1)Have 0.5~1 g plasmid DNA into 100  L competent cells, on ice for 30 min;<br>
-
(2)Frozen in liquid nitrogen for 5 min, grow at 37 C for 5 min,on ice for 2 min;<br>
+
2)Frozen in liquid nitrogen for 5 min, grow at 37 C for 5 min, on ice for 2 min;<br>
-
(3)Pour in 1000 LYEB,200 rpm、grow at 28 C for 2~3 hr;<br>
+
3)Pour in 1000 LYEB, 200 rpm, grow at 28℃ for 2~3 h;<br>
-
(4)6000rpm,2min. Suspend collected bacteria with 100ul YEB and evenly coat that at a YEB medium. (125 mg/L Sm or 50 mg/L rif, and 50 mg/L Kan included)<br>
+
4)6000rpm,2min. Suspend collected bacteria with 100µl YEB and evenly coat that at a YEB medium. (125 mg/L Sm or 50 mg/L rif, and 50 mg/L Kan included);<br>
-
(5)Grow upside down at 28℃,for 48h。<br>
+
5)Grow upside down at 28℃, for 48h;<br>
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(6)Pick positive clones and grow them in LB medium with antibiotic at 28℃for 48h。<br>
+
6)Pick positive clones and grow them in LB medium with antibiotic at 28℃ for 48h;<br>
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(7)Inject to LB medium in flasks by the day of transformation, in the rate of 1:50. Grow to OD600 = 0.5。Ready to infect tobacco leaf discs.<br>
+
7)Inject to LB medium in flasks by the day of transformation, in the rate of 1:50. Grow to OD600 = 0.5. Ready to infect tobacco leaf discs.<br>
</p>
</p>
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<h1 class="textEditingTitle" style="width:600px">Agrobacterium-mediated genetic transformation to tobacco </br></h1>
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<h1 class="textEditingTitle" style="width:1100px">Agrobacterium-mediated Tobacco Transformation</br></h1>
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         <p class="textEditingstyle">Tobacco was transformed essentially by following the leaf disk co-cultivation protocol of Horsch et al[1]. Co-cultivation was initiated by dipping leaf disks in an Agrobacterium suspension, blotting them on sterile tissue paper, and incubating them for 2 d on MS medium (Murashige and Skoog 1962 )  containing naphthalene acetic acid (NAA 0.1mg/L), 6-Benzylaminopurine (6-BA,2.0mg/L). Cefotaxime sodium (Cef) was included in the medium (500mg/L) to inhibit Agrobacterium growth. The leaf disks were then transferred onto a medium containing antibiotics for transgenic plant selection(kanamycin, 50 mg/L), and NAA (0.1 mg/L), 6-BA (2.0 mg/L), Cef (500mg/L). And incubate them for 1 month on the medium above. At last, cut off the bud from the callus, put the buds into the mudium containing NAA (0.1mg/L), Cef (500mg/L) and kanamycin (25 mg/L).
+
         <p class="textEditingstyle">Tobacco was transformed essentially by following the leaf disk co-cultivation protocol of <i>(McCormick et al., 1986)</i>. Co-cultivation was initiated by dipping leaf disks in an Agrobacterium suspension, blotting them on sterile tissue paper, and incubating them for 2 d on MS medium <i>(Murashige and Skoog 1962)</i> containing naphthalene acetic acid (NAA 0.1mg/L), 6-Benzylaminopurine (6-BA, 2.0mg/L). Cefotaxime sodium (Cef) was included in the medium (500mg/L) to inhibit Agrobacterium growth. The leaf disks were then transferred onto a medium containing antibiotics for transgenic plant selection(kanamycin, 50 mg/L), and NAA (0.1 mg/L), 6-BA (2.0 mg/L), Cef (500mg/L). And incubate them for 1 month on the medium above. At last, cut off the bud from the callus, put the buds into the mudium containing NAA (0.1mg/L), Cef (500mg/L) and kanamycin (25 mg/L).
</p>
</p>
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<h1 class="textEditingTitle" style="width: 600px">Detection method </br></h1>
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<h1 class="textEditingTitle" style="width: 1100px">Detection Method </br></h1>
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         <p class="textEditingstyle">1)Molecular identification<br>
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         <p class="textEditingstyle">1)Molecular Identification<br>
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Extract Genomic DNA from leaves of tobacco seedlings. Using specific primers to amplify the target gene. Extract leaf mRNA from leaves of grown tobacco which DNA testing was positive. Detected by the method of RT-PCR whether target gene is expressed.<br>
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Extract genomic DNA from leaves of tobacco seedlings. Using specific primers to amplify the target gene. Extract leaf mRNA from leaves of grown tobacco which DNA testing was positive. Detected by the method of RT-PCR whether target gene is expressed.<br>
2)Phenotype testing<br>
2)Phenotype testing<br>
</p>
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        <table border="1" id="TextTable" cellspacing="0";>
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           <tr>
             <th class="top"></th>
             <th class="top"></th>
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             <td class="mid">Qualitative detection</td>
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             <td class="lastmid">Qualitative detection</td>
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             <td class="mid">Have 6 positive seedlings from every strain(about 8 leaves age) , and 6 wild-type seedings with the same growth equally distributed into 3 650ml culture bottles. Treat with 10ul 37% formaldehyde for a week. </td>
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             <td class="lastmid">Have 6 positive plants from every transgenic line (about 8 leaves age) , and 6 wild-type seedings with the same growth equally distributed into three 650ml culture bottles. Treat with 10µl 37% formaldehyde for two weeks. </td>
<td class="mid"></td>
<td class="mid"></td>
           </tr>
           </tr>
    <tr>
    <tr>
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             <td class="mid"><br>Quantitative detection</td>
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             <td class="lastmid"><br>Quantitative detection</td>
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             <td class="mid"><br> Have 4 positive seedlings from every strain(about 8 leaves age) in a 650ml culture bottle. Treat with 10ul 37% formaldehyde for 2 weeks. Using a HCHO detector (Gastec Passive Dositube, 91D, Ayase, Kanagawa, Japan) was set in the hole to detect gaseous HCHO. Three and a half hours later, the measurement was stopped and the results were photographed[2].</td>
+
             <td class="lastmid"><br> Have 4 positive plants from every transgenic line (about 8 leaves age) in a 650ml culture bottle. Treat with 10µl 37% formaldehyde for 3 weeks. Using a formaldehyde detector (Gastec Passive Dositube, 91D, Ayase, Kanagawa, Japan) was set in the hole to detect gaseous formaldehyde. Three and a half hours later, the measurement was stopped and the results were photographed <i>(Chen et al., 2010)</i>.</td>
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            <td class="mid"><br>Put 4 wild -type seedlings with the same growth of seedlings in experimental group into 650ml culture bottle , with same processing as the case of the experimental group .</td>
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            <td class="lastmid"><br>Put 4 wild -type seedlings with the same growth of seedlings in experimental group into 650ml culture bottle,with same processing as the case of the experimental group.</td>
           </tr>
           </tr>
  </table>
  </table>
  </p>
  </p>
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  <p id="references">References<br>
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[1] Horsch RB, Fry JE, Hoffmann NL, Eichholts D, Rogers SG, and Fraley RT, Science,227, 1229-1231 (1985).<br>
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  <p id="references"><b>References</b><br>
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[2] Li-mei CHEN et al, Biosci. Biotechnol. Biochem, 74(3), 627-635 (2010)<br>
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<i>McCormick, S., J. Niedermeyer, J. Fry, A. Barnason, R. Horsch and R. Fraley (1986). "Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens."Plant Cell Rep 5(2): 81-84.<br>
 +
Chen, L. M., H. Yurimoto, K. Z. Li, I. Orita, M. Akita, N. Kato, Y. Sakai and K. Izui (2010). "Assimilation of formaldehyde in transgenic plants due to the introduction of the bacterial ribulose monophosphate pathway genes." Biosci Biotechnol Biochem</i> 74(3): 627-635.</i><br>
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