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
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         </h1>
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       <h1 class="textEditingTitle" style="width: 1100px">E. coli Transformation</br>
       <h1 class="textEditingTitle" style="width: 1100px">E. coli Transformation</br>
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         </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>
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         </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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           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>
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           (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>
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           (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>
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           (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>
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           (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>
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             <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 two weeks. </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>
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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 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> 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>
           </tr>
           </tr>

Latest revision as of 03:29, 18 October 2014

UESTC-China