Team:UESTC-China/Protocol

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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>
       <div id="SectionTitles1" style="width:1100px;">Fragments Ligation
       <div id="SectionTitles1" style="width:1100px;">Fragments Ligation
         </h1>
         </h1>
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           <tr>
           <tr>
             <th class="top">Component</th>
             <th class="top">Component</th>
-
             <th class="top">20µl Reaction</th>
+
             <th class="top">20µl Reaction </th>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">1.33X Master Mix</td>
+
             <td class="mid">Vector DNA</td>
-
             <td class="mid"> 15µl</td>
+
             <td class="mid">50ng</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">Insert DNA </td>
+
             <td class="mid">Insert DNA</td>
-
             <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">Vector DNA</td>
+
             <td class="mid">10X T4 DNA ligase buffer</td>
-
             <td class="mid"> 50ng</td>
+
             <td class="mid">2µl</td>
 +
          </tr>
 +
          <tr>
 +
            <td class="mid">T4 DNA ligase</td>
 +
            <td class="mid">1µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">ddH2O</td>
             <td class="mid">ddH2O</td>
-
             <td class="mid"> To 20µl</td>
+
             <td class="mid">to 20µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">Incubate at 50℃ for 1 hour</td>
+
             <td class="mid">Incubate at 25℃ for 12 hours</td>
             <td class="mid"></td>
             <td class="mid"></td>
           </tr>
           </tr>
         </table>
         </table>
 +
 +
       </div>
       </div>
-
       <div id="SectionTitles2" style="width:1100px;">DNA Ligation with Gibson Assembly
+
       <div id="SectionTitles2" style="width:1100px;">Gibson Assembly
         </h1>
         </h1>
 +
 +
 +
 +
 +
         <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;">
         <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">20µl Reaction </th>
+
             <th class="top">20µl Reaction</th>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">Vector DNA</td>
+
             <td class="mid">1.33X Master Mix</td>
-
             <td class="mid">50ng</td>
+
             <td class="mid"> 15µl</td>
           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">Insert DNA</td>
+
             <td class="mid">Insert DNA </td>
-
             <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>
+
             <td class="mid">Vector DNA</td>
-
             <td class="mid">2µl</td>
+
             <td class="mid"> 50ng</td>
-
          </tr>
+
-
          <tr>
+
-
            <td class="mid">T4 DNA ligase</td>
+
-
            <td class="mid">1µl</td>
+
           </tr>
           </tr>
           <tr>
           <tr>
             <td class="mid">ddH2O</td>
             <td class="mid">ddH2O</td>
-
             <td class="mid">to 20µl</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:1100px;">Fragment Digestion
       <div id="SectionTitles3" style="width:1100px;">Fragment Digestion
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           <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>
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           <tr>
           <tr>
             <td class="mid">10mM dNTPs</td>
             <td class="mid">10mM dNTPs</td>
-
             <td class="mid">5ug</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">1µl</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">1µl</td>
             <td class="mid">1µl</td>
           </tr>
           </tr>
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           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">10uM forward primer</td>
+
             <td class="mid">10µM forward primer</td>
             <td class="mid">1µl</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">1µl</td>
             <td class="mid">1µl</td>
           </tr>
           </tr>
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           </tr>
           </tr>
           <tr>
           <tr>
-
             <td class="mid">10uM forward primer</td>
+
             <td class="mid">10µM forward primer</td>
             <td class="mid">0.5µl</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">0.5µl</td>
             <td class="mid">0.5µl</td>
           </tr>
           </tr>
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     </div>
     </div>
     <div class="textEditingArea" >
     <div class="textEditingArea" >
 +
       <h1 class="textEditingTitle" style="width: 1100px">E. coli Transformation</br>
       <h1 class="textEditingTitle" style="width: 1100px">E. coli Transformation</br>
-
         </h1><p class="textEditingstyle">1)Streak E.coli cells (DH5α) on an LB plate, (BL21 (DE3) LysS cells on LB plate + 25 mg/ml chloramphenicol);<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>
           2) Allow cells to grow at 37℃ overnight;<br>
           2) Allow cells to grow at 37℃ overnight;<br>
           3)Place one colony in 10 ml LB media (+ antibiotic selection if necessary), grow overnight at 37℃;<br>
           3)Place one colony in 10 ml LB media (+ antibiotic selection if necessary), grow overnight at 37℃;<br>
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           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>
           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>
           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>
           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>
-
           9) Centrifuge cells at 4O℃ for 10 min at 3,000 g;<br>
+
           9) Centrifuge cells at 40℃ for 10 min at 3,000 g;<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>
           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>
           11) Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells in step;<br>
           11) Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells in step;<br>
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           15) Incubate on ice for 2 minutes;<br>
           15) Incubate on ice for 2 minutes;<br>
           16) Spread the appropriate quantity of cells (50 to 100 ml) on selective media. Store the remaining cells at 4℃.<br>
           16) Spread the appropriate quantity of cells (50 to 100 ml) on selective media. Store the remaining cells at 4℃.<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>
+
           (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>
-
           (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>
+
           (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>
           17) Incubate all plates overnight at 37℃.<br>
           18) Count the number of colonies.<br>
           18) Count the number of colonies.<br>
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           <tr>
           <tr>
             <td class="lastmid">Qualitative detection</td>
             <td class="lastmid">Qualitative detection</td>
-
             <td class="lastmid">Have 6 positive seedlings 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 a week. </td>
+
             <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>
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    <tr>
    <tr>
             <td class="lastmid"><br>Quantitative detection</td>
             <td class="lastmid"><br>Quantitative detection</td>
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             <td class="lastmid"><br> Have 4 positive seedlings from every transgenic line (about 8 leaves age) in a 650ml culture bottle. Treat with 10µl 37% formaldehyde for 2 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="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="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>
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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>
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           </tr>
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