Difference between revisions of "Team:Pasteur Paris/Microbiology week10"

 
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<h2><B>Microbiology Notebook</B></h2>
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  <a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><h2><B>Microbiology Notebook</B></h2><a/>  
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</div>  
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<div id="home">
 
<div id="home">
 
<center></a><a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><img src="https://static.igem.org/mediawiki/2016/5/5a/Labwork_pasteur.png" width="40%" alt=""/></img></a></center>
 
<center></a><a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><img src="https://static.igem.org/mediawiki/2016/5/5a/Labwork_pasteur.png" width="40%" alt=""/></img></a></center>
 
</div>
 
</div>
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  <div id="week10">
 
  <div id="week10">
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    <p><h5><B>Week 10</B></h5></p>
+
<p><h5><B>Week 10 </B></h3></p>
−
   
+
    <p><h3><B>August 8, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp1"><h4> 150. Culture for miniprep of C2 v2 and B1 v2 </h4></a><br/>
 +
        <a href="#exp2"><h4> 151. Digestion of B2, E1 and C2 from the 4<sup>th</sup> of August </h4></a><br/>
 +
        <a href="#exp3"><h4>  152. Electrophoresis with the results of digestion </h4></a><br/>
 +
        <a href="#exp4"><h4>  153. PCR of inserts A1/A2/D1/D2 </h4></a><br/>
 +
        <a href="#exp5"><h4>  154. Gel extraction of A1/A2/D1/D2 </h4></a><br/>
 +
        <a href="#exp6"><h4>  155. Gel extraction of B2, E1 and E2 </h4></a><br/>
 +
        <a href="#exp7"><h4>  156. Resuspension of inserts B2/E1/E2 </h4></a><br/>
 +
    </p>
 +
    <p><h3><B>August 9, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp8"><h4>  157. Ligation of inserts B2&#8260;E1&#8260;E2 with pET 43.1a(+) </h4></a><br/>
 +
        <a href="#exp9"><h4>  158. Miniprep of C2 v2 (TOP 10) and B1 v2 (TOP 10 ) </h4></a><br/>
 +
        <a href="#exp10"><h4>  159. Miniprep preculture of C1 v2 in pET 43.1a(+) </h4></a><br/>
 +
        <a href="#exp11"><h4>  160. Preparation of 10 aliquots of carbenicillin at 50 mg&#8260;m </h4></a><br/>
 +
            <a href="#exp12"><h4>  161. Electrophoresis of the PCR done on the 8<sup>th</sup> of August with A1&#8260;A2&#8260;D1&#8260;D2 </h4></a><br/>
 +
        <a href="#exp13"><h4>  162. Transformation of A1&#8260;A2&#8260;D1&#8260;D2 in TOP 10 </h4></a><br/>
 +
        <a href="#exp14"><h4>  163. Transformation of E1&#8260;E2 and B2 in TOP 10 </h4></a><br/>
 +
</p>
 +
    <p><h3><B>August 10, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp15"><h4>  164. Miniprep of C1 v2 (culture from the 9<sup></sup> of August) </h4></a><br/>
 +
            <a href="#exp16"><h4>  165. Digestion of C1 v2 before electrophoresis </h4></a><br/>
 +
            <a href="#exp17"><h4>  166. Digestion of pET 43.1 (a+) with Xba I and Hind III </h4></a><br/>
 +
            <a href="#exp18"><h4>  167. Electrophoresis and gel extraction </h4></a><br/>
 +
            <a href="#exp19"><h4>  168. Electrophoresis of C1 digested </h4></a><br/>
 +
            <a href="#exp20"><h4>  169. Transformation of B2 (4, 7 and 9), E1 (1 and 2) and E2 (2, 3, 4, 5, 6 and 7) </h4></a><br/>
 +
            <a href="#exp21"><h4>  170. Aliquot of antibodies 462 </h4></a><br/>
 +
            <a href="#exp22"><h4>  171. Culture of A1&#8260;A2&#8260;D1&#8260;D2 </h4></a><br/>
 +
            <a href="#exp23"><h4>  172. Ligation of A1&#8260;A2&#8260;D1&#8260;D2 in TOPO </h4></a><br/>
 +
            <a href="#exp24"><h4>  173. Transformation of A1&#8260;A2&#8260;D1&#8260;D2 with TOPO in TOP 10 competent cells </h4></a><br/>
 +
            <a href="#exp25"><h4>  174. Transformation of B1 v2 and C2 v2 in BL21DE3 </h4></a><br/>
 +
            <a href="#exp26"><h4>  175. Dosage of digested pET 43.1 (a+) </h4></a><br/>
 +
            <a href="#exp27"><h4>  176. Transformation of C2 v2 and B1 v2 in pET 43.1a(+) and DH3&#945; </h4></a><br/>
 +
</p>
 +
    <p><h3><B>August 11, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp28"><h4>  177. Absorbance of precultures C2 v2(1, 2, 3) and B1 v2 (1, 2, 16) </h4></a><br/>
 +
        <a href="#exp29"><h4>  178. Dephosphorylation of pET 43.1a(+) digested on the 10<sup>th</sup> of August </h4></a><br/>
 +
        <a href="#exp30"><h4>  179. Miniprep of B1 v2 &#8260;E1&#8260;E2 in TOPO </h4></a><br/>
 +
        <a href="#exp31"><h4>  180. Transformation of B1 v2 colony 8 and C2 v2 colony 16 in pET 43.1 (a+) and in DH 3&#945;  </h4></a><br/>
 +
            <a href="#exp32"><h4>  181. Precultures of B1 v2 and C2 v2 </h4></a><br/>
 +
            <a href="#exp33"><h4>  182. Digestion of pET 43.1 (a+) with Xba I and Hind III </h4></a><br/>
 +
</p>
 +
    <p><B><h3> August 12, 2016:</B></h3></p>
 +
    <p>
 +
        <a href="#exp34"><h4>  183. Miniprep from precultures of B2 v2/E1/E2 in TOPO </h4></a><br/>
 +
            <a href="#exp35"><h4>  184. Digestion of inserts B2 v2/E1/E2 with XbaI and HindIII </h4></a><br/>
 +
            <a href="#exp36"><h4>  185. Culture of C2 v2 and B1 v2 in 1 l of LB  </h4></a><br/>
 +
            <a href="#exp37"><h4>  186. Agarose gel to analyze digestion of pET 43.1(a+) done on the 11<sup>th</sup> of August </h4></a><br/>
 +
</p>
 +
   
 +
 
 +
 
 +
    <div class="lightbox" id="exp1">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
            <figcaption>
 +
              <p>
 +
              <h6><U> Aim :</U></h6> Increase the quantity of DNA before extraction. <br /><br />
 +
              <h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4b/T--Pasteur_Paris--Bacterial_culture_protocol.pdf">link</a><br /><br />
 +
              <h6><U>What we did in the lab :</U></h6><br />
 +
              <h6><U>Materials :</U></h6>
 +
                  &bull; 50 ml Falcon tube<br />
 +
                  &bull; Shaking incubator (INFORS HT)<br />
 +
                  &bull; Swing bucket centrifuge (JOUAN GR41)<br />
 +
                  &bull; Colonies of C2 v2 and B1 v2 <br />
 +
                  &bull; carbenicillin at 50 mg/ml <br />
 +
                  &bull; LB medium <br />
 +
                  &bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>)
 +
<br /><br />
 +
              <h6><U>Method :</U></h6>
 +
        1. In a 50 ml falcon, put 48 ml of LB and 48 &#181;l of carbenicillin. <br />
 +
              2. For B1 v2 : <br />
 +
&emsp; 2.a Prepare 13 Eppendorfs of 1.5 ml in which add 1 ml of the previous mix. <br />
 +
&emsp; 2.b Take with a toothpick colonies on the petri dish. <br />
 +
&emsp; 2.c Place the toothpick in a tube. <br />
 +
&emsp; 2.d Let incubate overnight at 37°C and 150 rpm. <br />
 +
                3. For C2 v2 : <br />
 +
&emsp; 3.a Prepare 20 eppendorfs of 1.5 ml with 1 ml of LB and carbenicillin mix.<br />
 +
&emsp; 3.b Take colonies and place it as previously explained.<br />
 +
&emsp; 3.c Let incubate overnight. <br />
 +
<br /><br /><br />
 +
              </p>
 +
            </figcaption>
 +
      </figure>
 
     </div>
 
     </div>
 +
 +
 +
 +
    <div class="lightbox" id="exp2">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
              <figcaption>
 +
                  <p>
 +
<h6><U> Aim :</U></h6> As the transformations did not work (B2, E1 and E2 in pET 43.1(a+) ) with TOP 10 competent cells, we take the products of the midiprep done on the 4<sup>th</sup> of August and we digest before redoing the transformation.<br /><br />
 +
                  <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a><br /><br />
 +
                  <h6><U>What we did in the lab :</U></h6><br />
 +
                  <h6><U>Materials :</U></h6>
 +
                    &bull; Restriction enzymes: Xba I, Hind III (New England Biolabs, NEB) <br />
 +
                    &bull; Restriction enzyme buffers <br />
 +
                    &bull; 37 &#176;C water bath<br />
 +
                    &bull; Shaking incubator (INFORS HT)<br />
 +
                    &bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>)<br /><br />
 +
                  <h6><U>Method :</U></h6>
 +
                      1. Realize a Mastermix and store it on ice : <br />
 +
                  <table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 113</U></p></i></caption>
 +
                    <thead>
 +
                        <tr>
 +
                            <th>Reactants</th>
 +
                            <th>Volumes (&#181;l) </th>
 +
                        </tr>
 +
                  </thead>
 +
                    <tbody>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>Xba I</p></strong></td>
 +
                            <td align="center"; valign="center"> 30 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>Hind III</p></strong></td>
 +
                            <td align="center"; valign="center"> 30 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>Buffer 2.1 </p></strong></td>
 +
                            <td align="center"; valign="center"> 90 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>Distilled H<sub>2</sub>O</p></strong></td>
 +
                            <td align="center"; valign="center"> 90 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p>Total</p></strong></td>
 +
                            <td align="center"; valign="center"> 150 </td>
 +
                          </tr>
 +
                      </tbody>
 +
                  </table><br /><br />
 +
          2. In For each insert (B2, E1, E2), prepare 10 tubes of 1.5 ml (10 eppendorfs of miniprep products). <br />
 +
          3. In each tube, put 25 &#956;l of DNA and 5 &#956;l of Master mix. <br />
 +
          4. Let digest 2 hours at 37 &#176;C, then incubate 5 minutes at 65 &#176;C. <br />
 +
<br /><br /><br />
 +
            </p>
 +
          </figcaption>
 +
      </figure>
 +
    </div>
 +
 +
 +
 +
 +
    <div class="lightbox" id="exp3">
 +
      <figure>
 +
          <a href="#" class="closemsg"></a>
 +
            <figcaption>
 +
                <p>
 +
<h6><U> Aim :</U></h6> Check if the digestion has been done, it would mean that the plasmid contains the insert.<br /><br />
 +
                    <h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br /><br />
 +
                    <h6><U>What we did in the lab :</U></h6><br />
 +
                    <h6><U>Materials :</U></h6>
 +
                          &bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <ahref="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
                          &bull; Colonies B2, E1 and E2 <br />
 +
                          &bull; Electrophoresis chamber <br />
 +
                          &bull; TAE 1X <br />
 +
                          &bull; Ethidium bromide drops (EB) <br/><br />
 +
                                    <h6><U>Method :</U></h6>
 +
        1. Make a gel with 1.4 g of agarose, 200 ml of TAE 1X and 4 droplets of EB. <br />
 +
        2. Prepare the samples with 30 &#181;l of digested mix and 6 &#181;l of Gene ruler (Thermofisher)ladder. <br />
 +
                3. Follow the deposit table : <br /><br />
 +
Ladder &#124; &#216; &#124; B2 (1) &#124; B2 (2) &#124; B2 (3) &#124; B2 (4) &#124; B2 (5) &#124; B2 (6) &#124; B2 (7) &#124; B2 (8) &#124; B2 (9) &#124; B2 (10) &#124; &#216; &#124; E1 (1) &#124; E1 (2) &#124; E1 (3) &#124; E1 (4) &#124; E1 (5) &#124; &#216; &#124; &#216; &#124; ladder  &#124; E1 (6) &#124; E1 (7) &#124; E1 (8) &#124; E1 (9) &#124; E1 (10) &#124; &#216; &#124; E2 (1)  &#124; E2 (2) &#124; E2 (3) &#124; E2 (4) &#124; E2 (5) &#124; E2 (6) &#124; E2 (7) &#124; E2 (8) &#124; E2 (9) &#124; E2 (10) &#124; &#216; &#124; &#216; <br/>
 +
        4. Launch the electrophoresis for 20 minutes at 50 V, then 45 minutes at 130 V. <br /><br />
 +
                    <h6><U>Results :</U></h6><br /><br />
 +
<center><img src = "https://static.igem.org/mediawiki/2016/c/cf/Week_10_152Electrophoresis_with_the_results_of_digestion.jpg"; alt "Gel of the results of digestion"/>
 +
<i><p> <U>Figure 18 :</U> Gel of the results of digestion </i></p></center><br />
 +
<br /><br /><br />
 +
                </p>
 +
            </figcaption>
 +
      </figure>
 +
    </div>
 +
 +
 +
 +
<div class="lightbox" id="exp4">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
        <p>
 +
            <h6><U> Aim :</U></h6> Increase the quantity of insert. <br /> <br />
 +
            <h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/b/be/T--Pasteur_Paris--PCR_protocol.pdf">link</a><br /><br />
 +
            <h6><U>What we did in the lab :</U><h6><br />
 +
            <h6><U>Materials :</U></h6>
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />               
 +
&bull; 1.5 ml eppendorfs <br />
 +
                &bull; Takara enzyme <br />
 +
                &bull; Primers S and AS <br />
 +
                &bull; dNTP mix 10 mM <br />
 +
                &bull; Tak Ex Buffer 6X <br />
 +
                &bull; Distilled H<sub>2</sub>O <br />
 +
                &bull; MgCl<sub>2</sub> <br /><br />
 +
          <h6><U>Method :</U></h6>
 +
                  1. Prepare the following tubes :<br />
 +
                  <table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 114</U></p></i></caption>
 +
                    <thead>
 +
                        <tr>
 +
                            <th> </th>
 +
                            <th> Mix with two primers </th>
 +
                            <th> Mix with primer S only </th>
 +
                            <th> Mix with primer AS only </th>
 +
                        </tr>
 +
                  </thead>
 +
                    <tbody>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> Takara enzyme (&#181;l) </p></strong></td>
 +
                            <td align="center"; valign="center"> 6 </td>
 +
                            <td align="center"; valign="center"> 6 </td>
 +
                            <td align="center"; valign="center"> 6 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> Primer S (&#181;l) </p></strong></td>
 +
                            <td align="center"; valign="center"> 6 </td>
 +
                            <td align="center"; valign="center"> 6 </td>
 +
                            <td align="center"; valign="center"> &#216; </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> Primer AS (&#181;l) </p></strong></td>
 +
                            <td align="center"; valign="center"> 6 </td>
 +
                            <td align="center"; valign="center"> &#216; </td>
 +
                            <td align="center"; valign="center"> 6 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> MgCl<sub>2</sub> (&#181;l) </p></strong></td>
 +
                            <td align="center"; valign="center"> 15 </td>
 +
                            <td align="center"; valign="center"> 15 </td>
 +
                            <td align="center"; valign="center"> 15 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> dNTPs (&#181;l) </p></strong></td>
 +
                            <td align="center"; valign="center"> 15 </td>
 +
                            <td align="center"; valign="center"> 15 </td>
 +
                            <td align="center"; valign="center"> 15 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> Buffer (&#181;l) </p></strong></td>
 +
                            <td align="center"; valign="center"> 30 </td>
 +
                            <td align="center"; valign="center"> 30 </td>
 +
                            <td align="center"; valign="center"> 30 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> H<sub>2</sub>O (&#181;l) </p></strong></td>
 +
                            <td align="center"; valign="center"> 216 </td>
 +
                            <td align="center"; valign="center"> 222 </td>
 +
                            <td align="center"; valign="center"> 222 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> Total (&#181;l) </p></strong></td>
 +
                            <td align="center"; valign="center"> 294 </td>
 +
                            <td align="center"; valign="center"> 294 </td>
 +
                            <td align="center"; valign="center"> 294 </td>
 +
                          </tr>
 +
                      </tbody>
 +
                  </table><br/>
 +
                  2. For each mix, spread 49 &#181;l of it in the samples. <br />
 +
    3. Add 1 &#181;l of DNA following the number of tubes : <br /><br />
 +
                  <table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 115</U></p></i></caption>
 +
                    <thead>
 +
                        <tr>
 +
                            <th> </th>
 +
                            <th> Mix with two primers </th>
 +
                            <th> Mix with primer S only </th>
 +
                            <th> Mix with primer AS only </th>
 +
                        </tr>
 +
                  </thead>
 +
                    <tbody>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> A1 </p></strong></td>
 +
                            <td align="center"; valign="center"> tube 1 </td>
 +
                            <td align="center"; valign="center"> tube 2 </td>
 +
                            <td align="center"; valign="center"> tube 3 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> A2 </p></strong></td>
 +
                            <td align="center"; valign="center"> tube 4 </td>
 +
                            <td align="center"; valign="center"> tube 5 </td>
 +
                            <td align="center"; valign="center"> tube 6 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> D1 </p></strong></td>
 +
                            <td align="center"; valign="center"> tube 7 </td>
 +
                            <td align="center"; valign="center"> tube 8 </td>
 +
                            <td align="center"; valign="center"> tube 9 </td>
 +
                          </tr>
 +
                          <tr>
 +
                            <td align="center"; valign="center"><strong><p> D2 </p></strong></td>
 +
                            <td align="center"; valign="center"> tube 10 </td>
 +
                            <td align="center"; valign="center"> tube 11 </td>
 +
                            <td align="center"; valign="center"> tube 12 </td>
 +
                          </tr>
 +
                      </tbody>
 +
                  </table><br />
 +
    And tube 13 is the one without DNA<br />
 +
    4. Launch the process of PCR.<br />
 +
    5. Do an electrophoresis with the results of PCR <br />
 +
<br /><br /> <br />
 +
        </p>
 +
      </figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp5">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
      <figcaption>
 +
        <p>
 +
            <h6><U> Aim :</U></h6> get back the maximum quantity of DNA.<br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4e/T--Pasteur_Paris--Gel_extraction_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab:</U></h6><br />
 +
<h6><U>Materials :</U></h6>
 +
&bull; Gel of A1&#8260;A2&#8260;D1&#8260;D2 <br />
 +
&bull; QIAGEN Extraction gel kit<br /><br />
 +
<h6><U>Method :</U></h6>
 +
Follow the Qiagen gel extraction kit steps with the gel :<br />
 +
&emsp; A1 : m  &#61; 122 mg<br />
 +
&emsp; A2 : m  &#61; 153 mg<br />
 +
&emsp; D1 : m  &#61; 120 mg<br />
 +
&emsp; D2 : m  &#61; 152 mg<br /><br />
 +
<h6><U>Results :</U></h6><br /><br />
 +
<center><img src = "https://static.igem.org/mediawiki/2016/b/bb/Week_10_154Gel_extraction_of_A1-A2-D1-D2.jpg"; alt "Extraction gel of A1-A2-D1-D2"/>
 +
<i><p> <U>Figure 19 :</U> Extraction gel of A1-A2-D1-D2 </p></i></center>
 +
<br /><br /> <br />
 +
        </p>
 +
      </figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp6">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Get back purified DNA.<br /><br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4e/T--Pasteur_Paris--Gel_extraction_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab:</U></h6><br />
 +
<h6><U>Materials:</U></h6>
 +
&bull; Gel of B2/E1/E2 <br />
 +
&bull; QIAGEN Extraction gel kit<br /><br />
 +
<h6><U>Method:</U></h6>
 +
Follow the Qiagen Extraction gel kit steps with :<br /><br />
 +
<table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 116</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> DNA </th>
 +
      <th> Colonies </th>
 +
      <th> Weight of the gel (mg) </th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td rowspan = 3; align="center"; valign="center"><strong><p> B2 </p></strong></td>
 +
      <td align="center"; valign="center"> Colony 4 </td>
 +
      <td align="center"; valign="center"> 367 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"> Colony 7 </td>
 +
      <td align="center"; valign="center"> 432 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"> Colony 9 </td>
 +
      <td align="center"; valign="center"> 269 </td>
 +
    </tr>
 +
    <tr>
 +
      <td rowspan = 7; align="center"; valign="center"><strong><p> E2 </p></strong></td>
 +
      <td align="center"; valign="center"> Colony 1 </td>
 +
      <td align="center"; valign="center"> 300 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"> Colony 2 </td>
 +
      <td align="center"; valign="center"> 355 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"> Colony 3 </td>
 +
      <td align="center"; valign="center"> 354 </td>
 +
      </tr>
 +
      <tr>
 +
      <td align="center"; valign="center"> Colony 4 </td>
 +
      <td align="center"; valign="center"> 314 </td>
 +
      </tr>
 +
      <tr>
 +
      <td align="center"; valign="center"> Colony 5 </td>
 +
      <td align="center"> 299 </td>
 +
      </tr>
 +
      <tr>
 +
      <td align="center"; valign="center"> Colony 6 </td>
 +
      <td align="center"; valign="center"> 275 </td>
 +
      </tr>
 +
      <tr>
 +
      <td align="center"; valign="center"> Colony 7 </td>
 +
      <td align="center"; valign="center"> 277 </td>
 +
      </tr>
 +
      <tr>
 +
      <td rowspan = 2; align="center"; valign="center"><strong><p> E1 </p></strong></td>
 +
      <td align="center"; valign="center"> Colony 1 </td>
 +
      <td align="center"; valign="center"> 404 </td>
 +
      </tr>
 +
      <tr>
 +
      <td align="center"; valign="center"> Colony 2 </td>
 +
      <td align="center"; valign="center"> 321 </td>
 +
      </tr>
 +
  </tbody>
 +
</table>
 +
<br /><br /><br />
 +
        </p>
 +
      </figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp7">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Storage of the inserts.<br />
 +
<h6><U>What we did in the lab :</U></h6><br />
 +
<h6><U>Materials :</U></h6>
 +
&bull; NaAc <br />
 +
&bull; Ethanol 70%; <br />
 +
&bull; Inserts B2/E1/E2 <br /><br />
 +
<h6><U>Method :</U></h6>
 +
1. Use 1/10 volume of NaAc and 2.5 colume of ethanol for each insert : <br /><br />
 +
<table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 117</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th> B2 </th>
 +
      <th> E1 </th>
 +
      <th> E2 </th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> NaAc (&#181;l) </p></strong></td>
 +
      <td align="center"; valign="center"> 15 </td>
 +
      <td align="center"; valign="center"> 10 </td>
 +
      <td align = “center”; valign="center"> 35 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> Ethanol (&#181;l) </p></strong></td>
 +
      <td align="center"; valign="center"> 875 </td>
 +
      <td align = “center”; valign="center"> 250 </td>
 +
      <td align = “center”; valign="center"> 375 </td>
 +
    </tr>
 +
</tbody>
 +
</table>
 +
<br />
 +
2. Resuspend B2&#8260;E1&#8260;E2 in 15 &#181;l of H<sub>2</sub>O each. <br />
 +
      3. We estimated the weight of each inserts : <br />
 +
            &emsp; m(B2) &#61; 240 ng <br />
 +
            &emsp; m(E1) &#61; 60 ng <br />
 +
            &emsp; m(E2) &#61; 420 ng <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
 +
 +
<div class="lightbox" id="exp8">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Prepare the transformation. <br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/6f/T--Pasteur_Paris--Ligation_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab:</U></h6><br />
 +
<h6><U>Materials :</U></h6>
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; Inserts B2/E1/E2 <br />
 +
&bull; pET 43.1(a+) <br />
 +
&bull; TOPO vector <br />
 +
&bull; Distilled water <br />
 +
&bull; Ligase <br /><br />
 +
<h6><U>Method :</U></h6><br />
 +
Use the following volumes : <br />
 +
<table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 118</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th> E1 </th>
 +
      <th> E2 </th>
 +
      <th> B2 </th>
 +
      <th> pET 43.1(a+) </th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> E1 (&#181;l) </p></strong></td>
 +
      <td align="center"; valign="center"> 15 </td>
 +
      <td align="center" ; valign="center"> &#216; </td>
 +
      <td align = "center"; valign="center"> &#216; </td>
 +
      <td align = "center"; valign="center"> &#216; </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> E2 (&#181;l) </p></strong></td>
 +
      <td align="center"; valign="center"> &#216; </td>
 +
      <td align = "center"; valign="center"> 15 </td>
 +
      <td align = "center"; valign="center"> &#216; </td>
 +
      <td align = "center"; valign="center"> &#216; </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> B2 (&#181;l) </p></strong></td>
 +
      <td align="center"; valign="center"> &#216; </td>
 +
      <td align = "center"; valign="center"> &#216; </td>
 +
      <td align = "center"; valign="center"> 15 </td>
 +
      <td align = "center"; valign="center"> &#216; </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> pET 43.1(a+) (&#181;l) </p></strong></td>
 +
      <td align="center"; valign="center"> 4 </td>
 +
      <td align = "center"; valign="center"> 4 </td>
 +
      <td align = "center"; valign="center"> 4 </td>
 +
      <td align = "center"; valign="center"> 4 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> Ligase (&#181;l) </p></strong></td>
 +
      <td align="center"; valign="center"> 1 </td>
 +
      <td align = "center"; valign="center"> 1 </td>
 +
      <td align = "center"; valign="center"> 1 </td>
 +
      <td align = "center"; valign="center"> 1 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> TOP0  (&#181;l) </p></strong></td>
 +
      <td align="center"; valign="center"> 2.2 </td>
 +
      <td align = "center"; valign="center"> 2.2 </td>
 +
      <td align = "center"; valign="center"> 2.2 </td>
 +
      <td align = "center"; valign="center"> 2.2 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> H<sub>2</sub>O (&#181;l) </p></strong></td>
 +
      <td align="center"; valign="center"> &#216; </td>
 +
      <td align = "center"; valign="center"> &#216; </td>
 +
      <td align = "center"; valign="center"> &#216; </td>
 +
      <td align = "center"; valign="center"> 15 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> V<sub>total</sub> (&#181;l) </p></strong></td>
 +
      <td align="center"; valign="center"> 22.2 </td>
 +
      <td align = "center"; valign="center"> 22.2 </td>
 +
      <td align = "center"; valign="center"> 22.2 </td>
 +
      <td align = "center"; valign="center"> 22.2 </td>
 +
    </tr>
 +
</tbody>
 +
</table>
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp9">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Increase the quantity of DNA. <br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/d/d5/T--Pasteur_Paris--Miniprep_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab :</U></h6><br />
 +
<h6><U>Materials :</U></h6>
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; Qiagen Miniprep kit <br />
 +
&bull; Digestion enzyme Xba I and Hind III <br />
 +
&bull; Digestion buffer 2.1 <br />
 +
&bull; 1.5 ml Eppendorfs <br />
 +
&bull; Electrophoresis chamber <br />
 +
&bull; Distilled water
 +
<br /><br />
 +
<h6><U>Method :</U></h6>
 +
1. Use the Qiagen kit for our cultures from the 8<sup>th</sup> of August : <br />
 +
&emsp; 13 Eppendorfs of B1 v2. <br />
 +
&emsp; 20 Eppendorfs of C2 v2. <br />
 +
2. Digest the plasmid with the following volumes for each sample : <br />
 +
<table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 119</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th> Volumes (&#181;l) </th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> DNA </p></strong></td>
 +
      <td align="center"; valign="center"> 5 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> Xba I </p></strong></td>
 +
      <td align="center"; valign="center"> 1 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> Hind III </p></strong></td>
 +
      <td align="center"> 1 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> Buffer 2.1 </p></strong></td>
 +
      <td align="center"> 2 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> H<sub>2</sub>O </p></strong></td>
 +
      <td align="center"; valign="center"> 11 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> Total </p></strong></td>
 +
      <td align="center"; valign="center"> 20 </td>
 +
    </tr>
 +
</tbody>
 +
</table>
 +
<br />
 +
3. Analyze the digestion by electrophoresis on an agarose gel prepared with 1.05 g of agarose in 125 ml of TAE 1X. <br />
 +
4. Launch the electrophoresis, following the deposit table :<br /><br />
 +
C2 &#124; Ladder &#124; 1 &#124; 2 &#124; 3 &#124; 4 &#124; 5 &#124; 6 &#124; 7 &#124; 8 &#124; 9 &#124; 10 &#124; 11 &#124; 12 &#124; 13 &#124; 14 &#124; 15 &#124; 16 &#124; 17 &#124; 18 &#124; 19 &#124; Ladder <br /><br />
 +
B1 &#124; ladder &#124; 1 &#124; 2 &#124; 3 &#124; 4 &#124; 5 &#124; 6 &#124; 7 &#124; 8 &#124; 9 &#124; 10 &#124; 11 &#124; 12 &#124; 13 &#124; &#216; &#124; &#216; &#124; &#216; &#124; 20 &#124; ladder<br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp10">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Have different clones to know which contain the insert. <br />
 +
<h6><U> Protocol :</U><h6> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4b/T--Pasteur_Paris--Bacterial_culture_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab:</U></h6><br />
 +
<h6><U>Materials :</U></h6>
 +
&&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; Carbenicillin at 50 mg/ml <br />
 +
&bull; Digestion enzyme Xba I and Hind III <br />
 +
&bull; LB medium <br />
 +
&bull; pET43.1(a+) <br />
 +
&bull; C1 v2 colonies <br />
 +
&bull; Shaking incubator (INFORS HT)<br /><br />
 +
<h6><U>Method :</U></h6>
 +
1. Prepare 20 ml of LB with 20 &#181;l of carbenicillin. <br />
 +
2. Put 1 ml of this mix in twenty 1.5 ml Eppendorfs. <br />
 +
      3. Take 20 colonies of the petri dish C1 v2 and put them in the previous Eppendorfs. <br />
 +
      4. Let incubate overnight at 37°C and 150 rpm. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp11">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Create a stock of antibiotic. <br />
 +
<h6><U>What we did in the lab :</U></h6><br />
 +
<h6><U>Materials :</U></h6>
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; Carbenicillin at 50 mg/ml <br />
 +
&bull; 1.5 ml Eppendorfs <br />
 +
&bull; 15 ml Falcon <br />
 +
&bull; Distilled water <br /><br />
 +
<h6><U>Method :</U></h6>
 +
1. Prep: Put 500 mg of carbenicillin in a 15 ml Falcon and 10 ml of distilled water. Then, put the Falcon on ice. <br />
 +
2. Aliquot the mix in 10 Eppendorfs of 1.5 ml. <br />
 +
      3. Store at -20°C <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp12">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Check if the PCR works. <br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br /><br />
 +
<h6><U>Results :</U></h6><br /><br />
 +
<center><img src = "https://static.igem.org/mediawiki/2016/a/ad/Week_10_161Electrophoresis_of_the_PCR_done_on_the_8th_of_August_with_A1-A2-D1-D2.jpg" ; alt "Electrophoresis gel of the PCR"/>
 +
<i><p> <U>Figure 20 :</U> Electrophoresis gel of the PCR</p></i></center><br />
 +
The PCR works properly since we noticed significant bands at the expected level.<br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp13">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> After their ligation in TOPO cloning vector, they will be transformed into TOP 10 cells. <br />
 +
<h6><U>Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab :</U></h6><br />
 +
<h6><U>Materials</U></h6>
 +
&bull; Ligation’s products of A1/A2/D1/D2<br />
 +
&bull ; TOP 10 competent cells <br />
 +
&bull ; SOC <br />
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; Xgal at 10 mg/ml
 +
<br /><br />
 +
<h6><U>Method</U></h6>
 +
1. Add 6 &#181;l of ligation product in 50 &#181;l of competent cells.<br />
 +
2. Put the samples 30 minutes on ice, then 40 seconds at 42°C.<br />
 +
3. Put the samples 3 minutes on ice.<br />
 +
4. Add 150 &#181;l of SOC.<br />
 +
5. Let incubate 40 minutes at 37°C and 150 rpm.<br />
 +
6. Take LB with carbenicillin petri plate and add Xgal to reach 40 &#181;g/ml. As the volume is about 25 ml, add 1 mg of Xgal and as the stock solution is 10 mg/ml, spread 100 &#181;l on the plate.<br />
 +
7. Spread bacteria on four distincts petri dishes (one for each insert).<br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp14">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> After their ligation we must transform the inserts into bacteria. <br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab :</U></h6><br />
 +
<h6><U>Method</U></h6>
 +
Add 10 &#181;l of ligation product (to have 100 mg) at the beginning. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp15">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Get back the DNA. <br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/d/d5/T--Pasteur_Paris--Miniprep_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab : </U><h6><br/>
 +
<h6><U>Method</U></h6>
 +
We use a final volume of 50 &#181;l. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
 +
<div class="lightbox" id="exp16">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Split the insert and the plasmid. <br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab:</U></h6><br />
 +
<h6><U>Materials:</U></h6>
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; Qiagen Miniprep kit <br />
 +
&bull; Digestion enzyme Xba I and Hind III <br />
 +
&bull; Digestion buffer 2 X <br />
 +
&bull; 1.5 ml Eppendorfs <br />
 +
&bull; Distilled water <br />
 +
&bull; Shaking incubator (INFORS HT)<br /><br />
 +
<h6><U>Method:</U></h6>
 +
1. Realize a master mix with : <br /><br />
 +
<table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 120</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th> Volumes (&#181;l) </th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> Xba I </p></strong></td>
 +
      <td align="center"; valign="center"> 20 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> Hind III </p></strong></td>
 +
      <td align="center"; valign="center"> 20 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> Buffer 2X </p></strong></td>
 +
      <td align="center"; valign="center"> 40 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> H<sub>2</sub>O </p></strong></td>
 +
      <td align="center"; valign="center"> 220 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> Total </p></strong></td>
 +
      <td align="center"; valign="center"> 300 </td>
 +
    </tr>
 +
</tbody>
 +
</table>
 +
<br />
 +
2. Put &#181;l of the master mix in each of the twenty 1.5 ml Eppendorfs and add 5 &#181;l of DNA.<br/>
 +
3. Let incubate one hour at 37°C and 150 rpm, then 5 minutes at 65°C<br /><br />
 +
<h6><U>Results</U></h6> The 10<sup>th</sup> of August, we realize that we forgot to put Xgal, so we re-spreaded the previous mix on petri dishes with Xgal.<br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
 +
<div class="lightbox" id="exp17">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> We want to produce 5 &#181;g of dephosphorylated pET 43.1(a+) from pET 43.1(a+) at 400 ng/ml and we start with the digestion. <br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab :</U></h6><br />
 +
<h6><U>Materials :</U></h6>
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; Qiagen Miniprep kit <br />
 +
&bull; Digestion enzyme Xba I and Hind III <br />
 +
&bull; CutSmart buffer<br />
 +
&bull; 1.5 ml Eppendorfs <br />
 +
&bull; Distilled water <br />
 +
&bull; Shaking incubator (INFORS HT)<br /><br />
 +
<h6><U>Method :</U></h6>
 +
1. Put all the following reactants in a 1.5 ml Eppendorf and let digest one hour at 37°C : <br />
 +
<table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 121</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th> Volumes (&#181;l) </th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> DNA </p></strong></td>
 +
      <td align="center"; valign="center"> 12.5 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> Xba I </p></strong></td>
 +
      <td align="center"; valign="center"> 2 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> Hind III </p></strong></td>
 +
      <td align="center"; valign="center"> 4 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> Buffer CutSmart </p></strong></td>
 +
      <td align="center"; valign="center"> 5 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> H<sub>2</sub>O </p></strong></td>
 +
      <td align="center"; valign="center"> 26.5 </td>
 +
    </tr>
 +
<tr>
 +
      <td align="center"; valign="center"><strong><p> Total </p></strong></td>
 +
      <td align="center"; valign="center"> 50 </td>
 +
    </tr>
 +
</tbody>
 +
</table>
 +
<br/>
 +
2. Inactivate the enzymes 5 minutes at 65°C. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
 +
<div class="lightbox" id="exp18">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Get back the digested and purified plasmid before dephosphorylation. <br /><br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab :</U></h6><br />
 +
<h6><U>Materials :</U></h6>
 +
&bull; Products from the digestion of pET 43.1(a+) with Hind III and XbaI <br />
 +
&bull; Agarose<br />
 +
&bull; Electrophoresis chamber <br />
 +
<h6><U>Method :</U></h6>
 +
1. Make a 0.7% agarose gel <br />
 +
2. Prepare the chamber to do the migration at 100 V with two wells for pET43.1(a+) and one for the ladder. <br />
 +
3. Take the results and follow the kit steps of Qiagen extraction kit.<br /><br />
 +
<h6><U>Results :</U></h6>
 +
We notice two bands for digested pET43.1(a+), one at 6000 bp and another between 1000 and 1500 bp. We extract the bands at 6000 bp.<br />
 +
We obtained : <br />
 +
&emsp; m1 = 0.4079 g<br />
 +
&emsp; m2 = 0 .3720 g<br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp19">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Check if the digestion works properly and if we have inserts. <br /><br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab:</U></h6><br />
 +
<h6><U>Materials :</U></h6>
 +
&bull; Products from the digestion of C1 <br />
 +
&bull; Agarose<br />
 +
&bull; Electrophoresis chamber <br />
 +
<h6><U>Method :</U></h6>
 +
1. Take the 20 &#181;l of each sample from the digestion and add 4 &#181;l of loading buffer 6X. <br />
 +
2. Do the electrophoresis, following the deposit table : <br /><br />
 +
Ladder &#124; &#216; &#124; 1 &#124; 2 &#124; 3 &#124; 4 &#124; 5 &#124; 6 &#124; 7 &#124; 8 &#124; 9 &#124; 10 &#124; 11 &#124; 12 &#124; 13 &#124; 14 &#124; 15 &#124; 16 &#124; 17 &#124; &#216; &#124; Ladder <br /><br />
 +
Ladder &#124; &#216; &#124; 17 &#124; 18 &#124; 19 &#124; 20 <br /><br />
 +
<h6><U>Results</U></h6>
 +
There is no insert. The colonies do not contain our insert, we must redo the experiment with new colonies. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp20">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Transform the bacterias with our recombined plasmid. <br /><br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab :</U></h6><br />
 +
<h6><U>Method :</U></h6>
 +
The volumes of insert are too small, we add 5 &#181;l of H<sub>2</sub>O and diluted at 1/100. <br />
 +
We performed a transformation in DH5&alpha; with 1 &#181;l of DNA and 50 &#181;l of competent cells. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp21">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Have more antibodies. <br /><br />
 +
<h6><U>Results :</U></h6><br/> We obtained 30 &#181;/aliquot. <br /><br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp22">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Increase the quantity of colonies containing inserts. <br /><br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4b/T--Pasteur_Paris--Bacterial_culture_protocol.pdf">link</a><br /><br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp23">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Ligate the insert and the plasmid. <br /><br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/6f/T--Pasteur_Paris--Ligation_protocol.pdf">link</a><br /><br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
 +
<div class="lightbox" id="exp24">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Transform our inserts in TOP 10 competent cells. <br /><br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br /><br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
 +
<div class="lightbox" id="exp25">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Have bacteria with the right plasmid to produce our protein. <br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab:</U></h6><br />
 +
<h6><U>Materials:</U></h6>
 +
&bull; Digested B1 and C2 <br />
 +
&bull; BL21DE3 competent cells<br />
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; Shaking incubator (INFORS HT)<br />
 +
&bull; SOC <br />
 +
<h6><U>Method:</U></h6>
 +
1. Take 3 samples of C2 v2 (10, 13 and 16) and 2 samples of B1 v2 (6 and 8).<br />
 +
2. Put 1 &#181;l of DNA in 99 &#181;l of H<sub>2</sub>O. Then, put 1 &#181;l of DNA (B1 v2 or C2 v2) in 50 &#181;l of BL21DE3 competent cells.<br />
 +
3. Put the samples 30 minutes on ice and then 40 seconds at 42°C.<br />
 +
4. Put the samples 3 minutes on ice. <br />
 +
5. Add 150 &#181;l of SOC and let incubate 30 minutes at 37°C and 150 rpm.<br />
 +
6. Spread the mix on a petri dish with LB and carbenicillin.<br />
 +
7. Let incubate overnight at 37°C and 150 rpm.<br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp26">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Have the concentration of digested pET 43.1(a+) <br />
 +
<h6><U>Results</U></h6> Measure the concentration of digested pET 43.1(a+) with a Nanodrop. For the first tube, we find a concentration of 6.8 ng/&#181;l in 46 &#181;l. For the second tube, we find a concentration of 8.8 ng/&#181;l in 46 &#181;l. Then, store the samples at -20°C. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp27">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Transform C2 v2 and B1 v2 in pET43.1a(+) and DH&alpha; . <br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br /><br />
 +
<h6><U>Results</U></h6> For B2 (4, 7, 9), E1 (1, 2) and E2 (2, 3, 4, 5, 6, 7) we see small colonies so we let and 25 &#181;l of carbenicillin.<br />
 +
For B1 v2 and C2 v2 in pET43.1(a+), transformations were successful.<br />
 +
For A1, A2 and D1, D2 there were too many bacteria growing so we took a colony, diluted it in LB and then spread it on petri dish with LB, Xgal and carbenicillin. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp28">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> To produce proteins. <br />
 +
<h6><U>What we did in the lab:</U></h6><br />
 +
<h6><U>Materials:</U></h6>
 +
&bull; Spectrophotometer Ultrospec 3100<br />
 +
&bull; iPTG at 0.5 M
 +
<br /><br />
 +
<h6><U>Method:</U></h6>
 +
1. Make two measurements separated of 30 minutes : <br />
 +
<table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 122</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> Sample </th>
 +
      <th> 1 </th>
 +
      <th> 2 </th>
 +
      <th> 3 </th>
 +
      <th> 4 </th>
 +
      <th> 5 </th>
 +
      <th> 6 </th>
 +
      <th> Time of addition of iPTG </th>
 +
      <th> Concentration (ng/&#181;l)</th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> B1 (1) </p></strong></td>
 +
      <td align="center"; valign="center"> 0.022 </td>
 +
      <td align="center"; valign="center"> 0.062 </td>
 +
      <td align="center"; valign="center"> 0.152 </td>
 +
      <td align="center"; valign="center"> 0.344 </td>
 +
      <td align="center"; valign="center"> 0.557 </td>
 +
      <td align="center"; valign="center"> 0.694 </td>
 +
      <td align="center"; valign="center"> 16 h 55 </td>
 +
      <td align="center"; valign="center"> 0.859 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> B1 (2) </p></strong></td>
 +
      <td align="center"; valign="center"> 0.185 </td>
 +
      <td align="center"; valign="center"> 0.417 </td>
 +
      <td align="center"; valign="center"> 0.693 </td>
 +
      <td align="center"; valign="center">  </td>
 +
      <td align="center"; valign="center">  </td>
 +
      <td align="center"; valign="center">  </td>
 +
      <td align="center"; valign="center"> 15 h 25 </td>
 +
      <td align="center"; valign="center"> 0.956 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> B1 (3) </p></strong></td>
 +
      <td align="center"; valign="center"> 0.075 </td>
 +
      <td align="center"; valign="center"> 0.211 </td>
 +
      <td align="center"; valign="center"> 0.413 </td>
 +
      <td align="center"; valign="center"> 0.688 </td>
 +
      <td align="center"; valign="center">  </td>
 +
      <td align="center"; valign="center">  </td>
 +
      <td align="center"; valign="center"> 15 h 55 </td>
 +
      <td align="center"; valign="center"> 0.920 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> C2 (1) </p></strong></td>
 +
      <td align="center"; valign="center"> 0.060 </td>
 +
      <td align="center"; valign="center"> 0.166 </td>
 +
      <td align="center"; valign="center"> 0.350 </td>
 +
      <td align="center"; valign="center"> 0.627 </td>
 +
      <td align="center"; valign="center"> 0.699 </td>
 +
      <td align="center"; valign="center">  </td>
 +
      <td align="center"; valign="center"> 16 h 25 </td>
 +
      <td align="center"; valign="center"> 0.905 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> C2 (2) </p></strong></td>
 +
      <td align="center"; valign="center"> 0.044 </td>
 +
      <td align="center"; valign="center"> 0.119 </td>
 +
      <td align="center"; valign="center"> 0.296 </td>
 +
      <td align="center"; valign="center"> 0.510 </td>
 +
      <td align="center"; valign="center"> 0.698 </td>
 +
      <td align="center"; valign="center">  </td>
 +
      <td align="center"; valign="center"> 16 h 25 </td>
 +
      <td align="center"; valign="center"> 0.910 </td>
 +
    </tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> C2 (16) </p></strong></td>
 +
      <td align="center"; valign="center"> 0.080 </td>
 +
      <td align="center"; valign="center"> 0.230 </td>
 +
      <td align="center"; valign="center"> 0.445 </td>
 +
      <td align="center"; valign="center"> 0.689 </td>
 +
      <td align="center"; valign="center">  </td>
 +
      <td align="center"; valign="center">  </td>
 +
      <td align="center"; valign="center"> 15 h 55 </td>
 +
      <td align="center"; valign="center"> 0.907 </td>
 +
    </tr>
 +
</tbody>
 +
</table>
 +
<br/>
 +
2. When the OD<sub>600 nm</sub> reached 0.7 keep the last measurement and centrifuge for 3 minutes at 8000 g. <br />
 +
3. Throw away the supernatant and store at -20°C. <br />
 +
4. Add iPTG to reach 0.3 mM. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp29">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Make the future ligation easier. <br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/c0/T--Pasteur_Paris--dephosphorylation_protocol.pdf">link</a><br /><br />
 +
<h6><U>What we did in the lab :</U></h6><br />
 +
<h6><U>Materials :</U></h6>
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; rSAP <br />
 +
&bull; CutSmart buffer<br />
 +
&bull; 1.5 ml Eppendorfs <br />
 +
&bull; Distilled water <br />
 +
&bull; Shaking incubator (INFORS HT)<br /><br />
 +
<h6><U>Method :</U></h6>
 +
1. Start with tube 2 (8.6 ng/&#181;l in 46 &#181;l) and use the following mix : <br />
 +
<table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 123</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th> Volumes ( &#181;l) </th>
 +
</tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> DNA </p></strong></td>
 +
      <td align="center"; valign="center"> 46 </td>
 +
</tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> CutSmart </p></strong></td>
 +
      <td align="center"; valign="center"> 6 </td>
 +
</tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> H<sub>2</sub>O </p></strong></td>
 +
      <td align="center"; valign="center"> 6.7 </td>
 +
</tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> rSAP  </p></strong></td>
 +
      <td align="center"; valign="center"> 1.3 </td>
 +
</tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> Total </p></strong></td>
 +
      <td align="center"; valign="center"> 60 </td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<br/>
 +
2. Let incubate 30 minutes at 37°C then 5 minutes at 65°C. <br />
 +
3. Do the same for tube 1. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp30">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Get back the DNA. <br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/d/d5/T--Pasteur_Paris--Miniprep_protocol.pdf">link</a><br /><br />
 +
<h6><U> What we did in the lab </U></h6><br />
 +
<h6><U>Materials :</U></h6>
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; LB medium <br />
 +
&bull; Carbenicillin at 50 mg/ml <br />
 +
&bull; B1/E1/E2 in TOPO <br /><br />
 +
<h6><U>Method :</U></h6>
 +
We do 31 precultures with a mix with 1 ml of LB and 1 &#181;l of carbenicillin to have :<br />
 +
&emsp; 3 samples of E1 <br />
 +
&emsp; 14 samples of B2 <br />
 +
&emsp; 14 samples of E2 <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp31">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim :</U></h6> Send our insert for sequencing as the transformations in BL21DE3. <br />
 +
<h6><U> Protocol :</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/c/ca/T--Pasteur_Paris--Transformation_protocol.pdf">link</a><br /><br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp32">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Have precultures to redo the cultures in 4 x 1 l of LB as they grow well. <br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/4/4b/T--Pasteur_Paris--Bacterial_culture_protocol.pdf">link</a><br /><br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp33">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Increase the quantity of plasmid for the next ligation. <br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a><br /><br />
 +
<br/><br/><br/>
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp34">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Increase the quantity of DNA. <br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/d/d5/T--Pasteur_Paris--Miniprep_protocol.pdf">link</a><br /><br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp35">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Check if the colonies we took contain the insert. <br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/a/ab/T--Pasteur_Paris--Restriction_digestion_protocol.pdf">link</a><br /><br />
 +
<h6><U> What we did in the lab </U></h6><br />
 +
<h6><U> Materials </U></h6>
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; Digestion enzyme Xba I and Hind III <br />
 +
&bull; Digestion buffer 2.1 <br />
 +
&bull; 1.5 ml Eppendorfs <br />
 +
&bull; Distilled water <br />
 +
&bull; Shaking incubator (INFORS HT)<br />
 +
&bull; Inserts B2/E1/E2 <br/><br />
 +
<h6><U> Method </U></h6>
 +
1. In a 1.5 ml Eppendorf, put : <br />
 +
<table>
 +
<caption align="bottom" align="center"><i><p> <U>Table 124</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th> Volumes (&#181;l) </th>
 +
</tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> DNA </p></strong></td>
 +
      <td align="center"; valign="center"> 5 </td>
 +
</tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> Xba I </p></strong></td>
 +
      <td align="center"; valign="center"> 1 </td>
 +
</tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> H<sub>2</sub>O </p></strong></td>
 +
      <td align="center"; valign="center"> 11 </td>
 +
</tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> Hind III </p></strong></td>
 +
      <td align="center"; valign="center"> 1 </td>
 +
</tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> Buffer 2.1 </p></strong></td>
 +
      <td align="center"; valign="center"> 2 </td>
 +
</tr>
 +
    <tr>
 +
      <td align="center"; valign="center"><strong><p> Total </p></strong></td>
 +
      <td align="center"; valign="center"> 20 </td>
 +
</tr>
 +
</tbody>
 +
</table>
 +
<br />
 +
2. Let incubate one hour at 37°C , then 5 minutes at -20°C. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp36">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Produce the protein in higher quantity. <br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/a/a4/T--Pasteur_Paris--Protein_induction_protocol.pdf">link</a><br /><br />
 +
<h6><U> What we did in the lab </U></h6><br />
 +
<h6><U> Materials </U></h6>
 +
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etc… Follow this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a>) <br />
 +
&bull; iPTG <br />
 +
&bull; Carbenicillin at 50 mg/ml
 +
<br /><br />
 +
<h6><U> Method </U></h6>
 +
1. In a 2 l Erlenmeyer, put 1 l of LB and 1 ml of carbenicillin and incubate at 37°C and 150 rpm to warm the liquid.<br />
 +
2. For each preculture of 25 ml, put it in a 50 ml Falcon, centrifuge 5 minutes at 3500 rpm, throw away the supernatent and resuspend the pellet in 5 ml of clean LB.<br />
 +
3.Centrifuge 5 minutes at 3500 rpm, throw away the supernatent and resuspend the pellet in 20 ml of clean LB.<br />
 +
4. In each 2 l erlenmeyer, add 5 ml of our preculture : 4 erlenmeyer for C2 and 4 for B1. <br />
 +
5. Let incubate at 37 &#176;C ann 150 rpm and measure the DO. <br />
 +
6. Once the DO reaches 0.7, add 1 ml of iPTG.<br />
 +
7. Let incubate for 3 hours. <br />
 +
8. Centrifuge the cultures. <br />
 +
9. Resuspend the pellet in 10 ml of lysis buffer (See protein purification protocol) in a 50 ml Falcon of known weight.<br />
 +
10. Store at -20°C. <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp37">
 +
  <figure>
 +
    <a href="#" class="closemsg"></a>
 +
    <figcaption>
 +
 +
<p>
 +
<h6><U> Aim:</U></h6> Check if the digestion works. <br />
 +
<h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/6/60/T--Pasteur_Paris--Gel_electrophoresis_protocol.pdf">link</a><br /><br />
 +
<h6><U> What we did in the lab </U></h6><br />
 +
<h6><U> Materials </U></h6>
 +
&bull; Agarose <br />
 +
&bull; Qiagen kit <br />
 +
&bull; Nanodrop <br />
 +
&bull; Electrophoresis chamber <br />
 +
&bull; Loading buffer 6X
 +
<br /><br />
 +
<h6><U> Method </U></h6>
 +
1. Add 10 &#181;l of loading buffer 6X to reach 50 &#181;l for each sample.<br />
 +
2. Do an electrophoresis and extract the bands of the palsmid digested. We obtaines two tubes : <br />
 +
&emsp; Tube1 : m1 &#61; 1.276 &#8722;  0.9964 &#61; 131.2 mg <br />
 +
&emsp; Tube2 : m2 &#61; 1.1524 &#8722;  0.9994 &#61; 153.0 mg <br />
 +
3. Follow the Qiagen kit steps for a final volum of 50 &#181;l.<br />
 +
4. Measure the concentration with the Nanodrop to see the results.<br />
 +
5. Store at -20°C.<br /><br />
 +
<h6><U>Results</U></h6>
 +
Tube 1 : 20 .2 ng/&#181;l<br />
 +
Tube 2 : 24.8 ng/&#181;l <br />
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
</body>
 +
 +
</html>

Latest revision as of 02:20, 20 October 2016