Difference between revisions of "Team:Pasteur Paris/Microbiology week2"

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  <a href="https://2016.igem.org/Team:Pasteur_Paris/Microbiology"><h2><B>Microbiology Notebook</B></h2><a/>
 +
 +
 +
<div id="week2">
 +
<p><h5><B>Week 2</B></h5></p>
 +
<p><h3><B> June 13, 2016:</B></h3></p>
 +
    <p>
 +
<a href="#exp1"><h4> 5. Analysis and storage of the transformation done on June 6, 2016 </h4></a></br> 
 +
<a href="#exp2"><h4> 6. Electrophoresis of pET43.1 and pSB1C3 </h4></a></br>
 +
<a href="#exp3"><h4> 7. Preculture of pET43.1a(+) DH5&alpha; to make stab culture </h4></a></br> 
 +
 +
 +
<p><h3><B> June 14, 2016:</B></h3></p>
 +
    <p>
 +
<a href="#exp4"><h4> 8. Make a culture of DH5&alpha; pET43.1(a+) to monitor the growth curve </h4></a></br>
 +
    </p>
 +
 +
<p><h3><B> June 15, 2016:</B></h3></p>
 +
    <p>
 +
<a href="#exp5"><h4> 9. Make a growth curve of bacteria culture </h4></a></br> 
 +
<a href="#exp6"><h4> 10. Stab culture</h4></a></br>
 +
<a href="#exp7"><h4> 11. Resuspension of C1 and C2 and all inserts synthesized by iDT</h4></a></br>
 +
    </p>
 +
 +
<p><h3><B> June 16, 2016:</B></h3></p>
 +
      <p>
 +
<a href="#exp8"><h4> 12. Extraction of pET43.1a(+) and pSB1C3 DNA from agarose gel </h4></a></br>
 +
<a href="#exp9"><h4> 13. Digestion of inserts (C1 and C2)</h4></a></br>
 +
<a href="#exp10"><h4> 14. Dephosphorylation </h4></a></br> 
 +
<a href="#exp11"><h4> 15. Ligation of C1 and C2 with pET43.1a(+) </h4></a></br>
 +
    </p>
 +
 +
 +
 +
 +
 +
 +
<div class="lightbox" id="exp1">
 +
  <figure>
 +
      <a href="# exp1" class="closemsg"></a>
 +
          <figcaption>
 +
            <p>
 +
            <h6><U>Aim:</U></h6> To check the transformation and the digestion of the plasmids by electrophoresis on agarose gel.</br>
 +
Seed pET43.1 DH5&alpha; in LB + carbenicillin media to make stab culture. Indeed, the validity of transformants needs to be verified by checking if an insert is present in the transformed plasmid. Secondly, verified plasmid-DH5&alpha combinations need to be stored beyond the lifetime of plate colonies. </br></br><br />
 +
            <h6><U>What we did in the lab:</U></h6></br>Counting of colonies on petri dish: overnight grown plates at 37°C, supplemented with carbenicillin 50 &#181;g/ml (LB + CB50) or with chloramphenicol 34 &#181;g/ml (LB + CM34) were counted for the presence of individual distinct colonies. </br></br>LB + CB50</br></br>
 +
 +
<table>
 +
<caption align="bottom" align="center"><i><p><U>Table 3 :</U> LB + CM34</p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th>Box 1</th>
 +
      <th>Box 2</th>
 +
      <th>Box 3</th>
 +
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong><p>pET43.1a(+) DH5&alpha;</p></strong></td>
 +
      <td align = "center"; valign = "center">0</td>
 +
      <td align = "center"; valign = "center">2</td>
 +
      <td align = "center"; valign = "center">23</td>
 +
 +
    </tr>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>pET43.1a(+) DH5&alpha;</br> (different batch)</p></strong></td>
 +
      <td align = "center"; valign = "center">Uncountable (Overgrown)</td>
 +
      <td align = "center"; valign = "center">2</td>
 +
      <td align = "center"; valign = "center">23</td>
 +
    </tr>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong><p>pET43.1a(+) DH5&alpha;</br> (different batch)</p></strong></td>
 +
      <td align = "center"; valign = "center">Uncountable </br>(Overgrown)</td>
 +
      <td align = "center"; valign = "center">15</td>
 +
      <td align = "center"; valign = "center">23</td>
 +
    </tr>
 +
</tbody>
 +
</table>
 +
</br><br />
 +
 +
 +
<table>
 +
<caption align="bottom" align="center"><i><p><U>Table 4</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th>Box 1</th>
 +
      <th>Box 2</th>
 +
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong><p>pSB1C3 DH5&alpha;</p></strong></td>
 +
      <td align = "center"; valign = "center">1</td>
 +
      <td align = "center"; valign = "center">78</td>
 +
 +
    </tr>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>pSB1C3 DH5&alpha;</br> (different batch)</p></strong></td>
 +
      <td align = "center"; valign = "center">38 </td>
 +
      <td align = "center"; valign = "center"> Uncountable (Overgrown) </td>
 +
    </tr>
 +
</tbody>
 +
</table></br>
 +
</br><br /><br />
 +
</p>
 +
        </figcaption>
 +
    </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp2">
 +
  <figure>
 +
    <a href="# exp2" class="closemsg"></a>
 +
      <figcaption><p>
 +
<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><br />
 +
<h6><U>What we did in the lab:</U></h6></br>
 +
          <h6><U>Materials:</U></h6>
 +
&bull; 1X TAE buffer </br>
 +
&bull; Agarose </br>
 +
&bull; Microwave oven </br>
 +
&bull; Distilled water</br>
 +
&bull; Ethidium Bromide</br>
 +
&bull; Gel caster, and power supply</br>
 +
&bull; Precision balance </br></br>
 +
          <h6><U>Method:</U></h6>
 +
1. Buffer solution: we have 1.0X stock TAE (Tris Acetate EDTA) solution and we prepared 1.0X TAE gels with 0.5X TAE running buffer. </br>
 +
2. Agarose gel: We prepared 0.7% w/v agarose in 1X TAE and allow it to set.</br></br>
 +
 +
<B> For agarose gel electrophoresis experiment:</B></br></br>
 +
<I>Samples:</I></br>
 +
&bull; pSB1C3 plasmid (obtained with Midiprep on June 8, 2016)</br>
 +
&bull; pSB1C3 plasmid (digested by SpeI/XbaI)<br />
 +
&bull; pET43.1a plasmid (obtained with Midiprep on June 8, 2016)< /br>
 +
&bull; pET43.1a plasmid digested by BamH I/Hind III</br>
 +
&bull; gel 0.7% agarose</br>
 +
&bull; TAE 0.5X buffer</br>
 +
&bull; Electrophoresis power supply</br>
 +
&bull; DNA ladder (Thermofisher Gene ruler 1kb)</br>)</br>
 +
 +
<I> Method:</I></br>
 +
1. After filling the electrophoresis chamber with TAE 0.5X buffer, samples were loaded in 1X gel loading buffer in the wells, according to the following lane pattern.</br>
 +
 +
 +
<table>
 +
<caption align="bottom" align="center"><i><p><U>Table 5</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th>Lanes</th>
 +
      <th>L1</th>
 +
      <th>L2</th>
 +
      <th>L3</th>
 +
      <th>L4-7</th>
 +
      <th>L9-8</th>
 +
      <th>L10-13</th>
 +
      <th>L14</th>
 +
      <th>L15</th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong><p>Sample </p></strong></td>
 +
      <td align = "center"; valign = "center">Molecular </br> weight</td>
 +
      <td align = "center"; valign = "center">pET43.1a(+)</br> uncut</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center">pET43.1a(+)</br> B-H</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center">pBS1C3</br> S-X</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center">pBS1C3</br> uncut</td>
 +
    </tr>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong> </strong></td>
 +
      <td align = "center"; valign = "center">DNA (&micro;l)</td>
 +
      <td align = "center"; valign = "center"> 5</td>
 +
      <td align = "center"; valign = "center"> 2</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center"> 2</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center"> 4</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center"> 4</td>
 +
    </tr>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong> </strong></td>
 +
      <td align = "center"; valign = "center">H<sub>2</sub>0</td>
 +
      <td align = "center"; valign = "center"> 0</td>
 +
      <td align = "center"; valign = "center"> 8</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center">8</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center">6</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center">6</td>
 +
    </tr>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong> </strong></td>
 +
      <td align = "center"; valign = "center">6X</br>Loading</br>buffer</td>
 +
      <td align = "center"; valign = "center"> 0</td>
 +
      <td align = "center"; valign = "center"> 2</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center">2</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center">2</td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center">2</td>
 +
    </tr>
 +
 +
</tbody>
 +
</table></br>
 +
</br></br> 
 +
<h6><U>Results</U></h6></br>
 +
The digestion has worked since the plasmids digested have moved faster. </br></br>
 +
&emsp; NB: the gel suffered from overheating. </br>
 +
</br></br></br>
 +
        </p>
 +
      </figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp3">
 +
  <figure>
 +
    <a href="# exp3" class="closemsg"></a>
 +
        <figcaption><p>
 +
              <h6><U>Aim:</U></h6> Repeat Midiprep for pET43.1a(+). </br>Since we need a lot of pET43.1a(+) we have to repeat the Midiprep.</br></br>
 +
            <h6><U> Protocol:</U></h6> follow in this <a href="https://2016.igem.org/Team:Pasteur_Paris/Science">link</a></br></br><br />
 +
              <h6><U>What we did in the lab:</U></h6></br>
 +
              <h6><U>Materials:</U></h6>
 +
&bull; Falcon of 50 ml</br>
 +
&bull; Petri dish with DH5&alpha; transformed with pET43.1a(+)<br>
 +
&bull; LB (Luria Broth) medium autoclaved (5 ml) </br>
 +
&bull; Shaking incubator (INFORS HT)</br>
 +
&bull; Antibiotic: carbenicillin 100 mg/ml<br />
 +
&bull; Pipetman P10 and 5 ml graduated pipet</br>
 +
&bull; Bunsen burner </br>
 +
&bull; Cones for P10<br />
 +
&bull; Propipet</br>
 +
&bull; Rack for 50 ml tubes </br></br>
 +
              <h6><U>Method:</U></h6>
 +
 +
1. Pick one colony and put it into 5 ml of LB medium + 2.5 &#181;l of carbenicillin stock, to obtain 50 &micro;g/&micro;l.
 +
</br>
 +
2. Grow overnight at 37°C in a shaking incubator at 150 rpm.</br>
 +
<br /><br /><br />
 +
</p>
 +
      </figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
 +
 +
<div class="lightbox" id="exp4">
 +
  <figure>
 +
    <a href="# exp4" class="closemsg"></a>
 +
        <figcaption>
 +
            <p>
 +
              <h6><U> Aim:</U></h6> Use the preculture of June 13, 2016 to start the new culture.</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 />
 +
              <h6><U>What we did in the lab:</U></h6></br>
 +
              <h6><U>Materials:</U></h6>
 +
&bull; Falcon of 50 ml</br>
 +
&bull; LB autoclaved 500 ml<br />
 +
&bull; 3 precultures performed on June 13, 2016 (redundant cultures in case colonies don’t grow)</br>
 +
&bull; carbenicillin 100 mg/ml</br>
 +
&bull; swing bucket centrifuge (JOUAN GR4i)</br></br>
 +
              <h6><U>Method:</U></h6>
 +
1. Get back preculture performed on the June 13, 2016, put it in the swing bucket centrifuge during 7 minutes (3500 RPM) and remove the supernatant. </br>
 +
2. Add 5ml of fresh LB, resuspend the pellet and centrifuge again at 3500 rpm during 7 minutes. </br>
 +
3. Repeat one more time step 2.</br>
 +
4. Resuspend the pellet into 5 ml of fresh LB+ 2.5 &micro;l of carbenicillin 100 mg/ml.</br>
 +
5. Grow in the shaking incubator (T= 37 °C / 130 rpm) overnight.</br>
 +
</br></br>
 +
 +
We overshot the 0.7 OD, set point therefore we continued overnight. </br>
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
</div>
 +
 +
 +
 +
 +
<div class="lightbox" id="exp5">
 +
  <figure>
 +
    <a href="# exp5" class="closemsg"></a>
 +
        <figcaption>
 +
            <p>
 +
              <h6><U> Aim:</U></h6> To make a stab culture we need to capture the bacteria growing at the exponential phase. </br>
 +
In order to do that we need to take optical density at 600<sub>nm</sub> (OD600<sub>nm</sub>).</br></br>
 +
            <h6><U> Protocol:</U></h6> follow in this <a href="https://static.igem.org/mediawiki/2016/b/b1/Stab_culture_Pasteur_Paris_2016.pdf">link</a></br></br><br />
 +
              <h6><U>What we did in the lab:</U></h6></br>
 +
              <h6><U>Materials:</U></h6>
 +
&bull; Falcon of 50 ml </br>
 +
&bull; LB autoclaved 500 ml<br>
 +
&bull; LB (Luria Broth) autoclaved (500 ml) </br>
 +
&bull Precultures performed on the June 14, 2016</br>
 +
&bull; Antibiotic: carbenicillin 100 mg/ml</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> Before starting the culture we need to have molten LB-agar so we need to place the solid LB agar on a hot plate with stirring. </br>
 +
1. During growth of the bacterial culture, take one ml aliquot and measure the absorbance at OD600<sub>nm</sub> every hour for the first three hours, then every 20 minutes onwards. We recorded OD600<sub>nm</sub> for this specific experiment after 3 hours.</br></br>
 +
 +
<table>
 +
<caption align="bottom" align="center"><i><p><U>Table 6</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th>Time</th>
 +
      <th>Absorbance A’</th>
 +
      <th>Absorbance A’’</th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong><p>12h37 </p></strong></td>
 +
      <td align = "center"; valign = "center"></td>
 +
      <td align = "center"; valign = "center">0.013</td>
 +
      <td align = "center"; valign = "center">0.015</td>
 +
 +
    </tr>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>13h07</p></strong></td>
 +
      <td align = "center"; valign = "center">0.023</td>
 +
      <td align = "center"; valign = "center"> 0.027</td>
 +
    </tr>
 +
 +
  <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>13h37</p></strong></td>
 +
      <td align = "center"; valign = "center">0.063</td>
 +
      <td align = "center"; valign = "center"> 0.059 </td>
 +
    </tr>
 +
  <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>14h07</p></strong></td>
 +
      <td align = "center"; valign = "center">0.110 </td>
 +
      <td align = "center"; valign = "center"> 0.105</td>
 +
    </tr>
 +
  <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>14h27</p></strong></td>
 +
      <td align = "center"; valign = "center">0.172 </td>
 +
      <td align = "center"; valign = "center"> 0.170</td>
 +
    </tr>
 +
  <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>14h47</p></strong></td>
 +
      <td align = "center"; valign = "center">0.265 </td>
 +
      <td align = "center"; valign = "center"> 0.256</td>
 +
    </tr>
 +
  <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>15h07</p></strong></td>
 +
      <td align = "center"; valign = "center">0.384 </td>
 +
      <td align = "center"; valign = "center"> 0.377 </td>
 +
    </tr>
 +
  <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>15h27</p></strong></td>
 +
      <td align = "center"; valign = "center">0.491 </td>
 +
      <td align = "center"; valign = "center"> 0.482</td>
 +
    </tr>
 +
  <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>15h47</p></strong></td>
 +
      <td align = "center"; valign = "center">0.567 </td>
 +
      <td align = "center"; valign = "center"> 0.557</td>
 +
    </tr>
 +
  <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>16h06</p></strong></td>
 +
      <td align = "center"; valign = "center">0.697</td>
 +
      <td align = "center"; valign = "center"> 0.608</td>
 +
    </tr>
 +
  <tr>
 +
      <td align = "center"; valign = "center"><strong> <p>16h29</p></strong></td>
 +
      <td align = "center"; valign = "center">0.816</td>
 +
      <td align = "center"; valign = "center"> 0.755</td>
 +
    </tr>
 +
<tr>
 +
      <td align = "center"; valign = "center"><strong> <p>17h03</p></strong></td>
 +
      <td align = "center"; valign = "center">0.954 </td>
 +
      <td align = "center"; valign = "center"> 0.909 </td>
 +
    </tr>
 +
<tr>
 +
      <td align = "center"; valign = "center"><strong> <p>17h20</p></strong></td>
 +
      <td align = "center"; valign = "center">0.954</td>
 +
      <td align = "center"; valign = "center"> 0.909</td>
 +
    </tr>
 +
<tr>
 +
      <td align = "center"; valign = "center"><strong> <p>17h40</p></strong></td>
 +
      <td align = "center"; valign = "center">1.061</td>
 +
      <td align = "center"; valign = "center"> 1.029</td>
 +
    </tr
 +
<tr>
 +
      <td align = "center"; valign = "center"><strong> <p>18h00</p></strong></td>
 +
      <td align = "center"; valign = "center">1.092</td>
 +
      <td align = "center"; valign = "center"> 1.082</td>
 +
    </tr
 +
<tr>
 +
      <td align = "center"; valign = "center"><strong> <p>18h00</p></strong></td>
 +
      <td align = "center"; valign = "center">1.122</td>
 +
      <td align = "center"; valign = "center"> 1.132</td>
 +
    </tr>
 +
 +
</tbody>
 +
</table>
 +
</br><br /><br />
 +
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp6">
 +
  <figure>
 +
    <a href="# exp6" class="closemsg"></a>
 +
        <figcaption>
 +
            <p>
 +
           
 +
<h6><U> What we did on the lab:</U></h6></br>
 +
<h6><U> Method:</U></h6>
 +
1. 20 ml of warm LB + agar is placed in a 50 ml Falcon and 20 &micro;l of carbenicillin 50 mg/ml added, swirl to mix.</br>
 +
2. Aliquot 1 ml of LB + agar in 2 ml sterile tubes (10 tubes per each culture, so 20 tubes total). </br>
 +
3. When the mixture solidified, soak a toothpick in bacterium culture and stab the LB agar several times.</br>
 +
4. Grow overnight at 37 °C and next day place it at 4°C.</br>
 +
<br /><br /><br />
 +
</p>
 +
      </figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp7">
 +
  <figure>
 +
    <a href="# exp7" class="closemsg"></a>
 +
        <figcaption>
 +
            <p>
 +
            <h6><U> Aim:</U></h6> We received our gene block synthesized DNA constructs in lyophilized form. In order to ligate them in our plasmid, we need to resuspend them in buffer. </br></br>
 +
<h6><U>Method:</U></h6> Add TE (Tris 10 mM pH 8.0 EDTA 1 mM) buffer in each tubes and refer to the next table for volumes.
 +
</br></br>
 +
 +
 +
<table>
 +
<caption align="bottom" align="center"><i><p><U>Table 7</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th>Name </th>
 +
      <th>Weight (mg)</th>
 +
      <th>Cfinal (ng/&micro;l)</th>
 +
      <th>V(TE) (&micro;l)</th>
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong><p>A1 </p></strong></td>
 +
      <td align = "center"; valign = "center">500</td>
 +
      <td align = "center"; valign = "center">10</td>
 +
      <td align = "center"; valign = "center">50</td>
 +
</tr>
 +
    <tr>
 +
    <td align = "center"; valign = "center"><strong><p>A2 </p></strong></td>
 +
      <td align = "center"; valign = "center">500</td>
 +
      <td align = "center"; valign = "center">10</td>
 +
      <td align = "center"; valign = "center">50</td>
 +
</tr>
 +
<tr>
 +
    <td align = "center"; valign = "center"><strong><p>C1 </p></strong></td>
 +
      <td align = "center"; valign = "center">1000</td>
 +
      <td align = "center"; valign = "center">10</td>
 +
      <td align = "center"; valign = "center">100</td>
 +
    </tr>
 +
    <tr>
 +
    <td align = "center"; valign = "center"><strong><p>C2 </p></strong></td>
 +
      <td align = "center"; valign = "center">1000</td>
 +
      <td align = "center"; valign = "center">10</td>
 +
      <td align = "center"; valign = "center">100</td>
 +
    </tr>
 +
    <tr>
 +
  <td align = "center"; valign = "center"><strong><p>D1 </p></strong></td>
 +
      <td align = "center"; valign = "center">500</td>
 +
      <td align = "center"; valign = "center">10</td>
 +
      <td align = "center"; valign = "center">50</td>
 +
    </tr>
 +
    <tr>
 +
  <td align = "center"; valign = "center"><strong><p>D2 </p></strong></td>
 +
      <td align = "center"; valign = "center">500</td>
 +
      <td align = "center"; valign = "center">10</td>
 +
      <td align = "center"; valign = "center">50</td>
 +
    </tr>
 +
    <tr>
 +
  <td align = "center"; valign = "center"><strong><p>E1 </p></strong></td>
 +
      <td align = "center"; valign = "center">500</td>
 +
      <td align = "center"; valign = "center">10</td>
 +
      <td align = "center"; valign = "center">50</td>
 +
    </tr>
 +
    <tr>
 +
  <td align = "center"; valign = "center"><strong><p>E2 </p></strong></td>
 +
      <td align = "center"; valign = "center">500</td>
 +
      <td align = "center"; valign = "center">10</td>
 +
      <td align = "center"; valign = "center">50</td>
 +
    </tr>
 +
    <tr>
 +
  <td align = "center"; valign = "center"><strong><p>B2 </p></strong></td>
 +
      <td align = "center"; valign = "center">1000</td>
 +
      <td align = "center"; valign = "center">10</td>
 +
      <td align = "center"; valign = "center">100</td>
 +
    </tr>
 +
</tbody>
 +
</table></br>
 +
</br><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp8">
 +
  <figure>
 +
    <a href="# exp8" class="closemsg"></a>
 +
        <figcaption>
 +
            <p>
 +
            <h6><U> Aim:</U></h6> We want to have only the DNA backbone of pET43.1a(+) and pSB1C3, not the inserts that lie between the restriction sites. Spe I/ Xba I or BamH I/Hind III respectively, so we have to extract the plasmid backbone on out of an agarose gel. Furthermore, to clean the pET43.1a(+) and pSB1C3 backbones we use the gel extraction kit of QIAGEN.  Afterwards we ligated our plasmid with the inserts. In this experiment we use inserts C1 and C2.
 +
</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><br />
 +
            <h6><U>What we did in the lab:</U></h6></br>
 +
            <h6><U>Material:</U></h6>
 +
&bull; NEB extraction kit. Monarch DNA Gel Extraction Kit (/#T1020G). </br></br>
 +
<h6><U>Method:</U></h6>
 +
 +
1. Excise the DNA fragment from the agarose gel and transfer to 1 ml Eppendorf tube. </br>
 +
2. Add 4 volumes of gel dissolving buffer (NT1) to the gel slice. For NT1 volumes, refer to the Table 8: </br>
 +
 +
 +
<table>
 +
<caption align="bottom" align="center"><i><p><U>Table 8</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th> </th>
 +
      <th></th>
 +
      <th>Empty</br>Eppendorf (g)</th>
 +
      <th>Eppendorf</br>+ gelf (g)</th>
 +
      <th>Final weight (mg)</th>
 +
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong><p>pET43.1a(+)</p></strong></td>
 +
      <td align = "center"; valign = "center">m1</td>
 +
      <td align = "center"; valign = "center">0.9</td>
 +
      <td align = "center"; valign = "center">1.3</td>
 +
      <td align = "center"; valign = "center">329.6</td>
 +
 +
</tr>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong><p>pET43.1a(+)</p></strong></td>
 +
      <td align = "center"; valign = "center">m2</td>
 +
      <td align = "center"; valign = "center">0.9</td>
 +
      <td align = "center"; valign = "center">1.3</td>
 +
      <td align = "center"; valign = "center">265.3</td>
 +
 +
    </tr>
 +
<tr>
 +
    <td align = "center"; valign = "center"><strong><p>pSB1C3 </p></strong></td>
 +
      <td align = "center"; valign = "center">m1</td>
 +
      <td align = "center"; valign = "center">0.9</td>
 +
      <td align = "center"; valign = "center">1.2</td>
 +
      <td align = "center"; valign = "center">251.1</td>
 +
 +
    </tr>
 +
    <tr>
 +
    <td align = "center"; valign = "center"><strong><p> pSB1C3 </p></strong></td>
 +
      <td align = "center"; valign = "center">m2</td>
 +
      <td align = "center"; valign = "center">1.0</td>
 +
      <td align = "center"; valign = "center">1.2</td>
 +
      <td align = "center"; valign = "center">185.6</td>
 +
    </tr>
 +
</tbody>
 +
</table>
 +
</br></br>
 +
3. Incubate the samples between 37-55°C, vortexing periodically until the gel slice is completely dissolved (/ ~ 10 min). </br>
 +
4. Load sample onto the column. Close the cap, spin for 1 minute then discard flow through.</br>
 +
5. Re insert column into collection tube. Add 200 &micro;µl DNA wash buffer and spin for one minute. Discard flow-through.</br>
 +
6. Repeat step 5.</br>
 +
7. Transfer column to a clean 1.5 ml microfuge tube.</br>
 +
8. Add 6 &micro;l of DNA Elution buffer to the center of the matric. Wait for 1 minute and spin for 1 minute to elute DNA.</br>
 +
<br /><br /><br />
 +
</p>
 +
</figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp9">
 +
  <figure>
 +
    <a href="# exp9" class="closemsg"></a>
 +
        <figcaption>
 +
            <p>
 +
            <h6><U> Aim:</U></h6> After digestion of the plasmid backbone we now proceed with the digestion of the inserts C1 and C2.  </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><br />
 +
            <h6><U>What we did in the lab:</U><h6></br>
 +
 +
<h6><U>Method:</U></h6>
 +
1. Add all reagents in 1 ml Eppendorf. </br>
 +
2. Digest during 2 h at 37 °C. </br>
 +
3. For the reagent volumes, refer to the Table 9. Total volume = 50 &micro;l.</br>
 +
 +
 +
<table>
 +
<caption align="bottom" align="center"><i><p><U>Table 9</U></p></i></caption>
 +
  <thead>
 +
    <tr>
 +
      <th></th>
 +
      <th>C1</th>
 +
      <th>C2</th>
 +
 +
    </tr>
 +
  </thead>
 +
  <tbody>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong><p>DNA (&micro;l)</p></strong></td>
 +
      <td align = "center"; valign = "center">20</td>
 +
      <td align = "center"; valign = "center">20</td>
 +
   
 +
</tr>
 +
    <tr>
 +
      <td align = "center"; valign = "center"><strong><p>BamH I (&micro;l)</p></strong></td>
 +
      <td align = "center"; valign = "center">1</td>
 +
      <td align = "center"; valign = "center">1</td>
 +
</tr>
 +
 +
<tr>
 +
    <td align = "center"; valign = "center"><strong><p>Hind III (&micro;l)</ </p></strong></td>
 +
            <td align = "center"; valign = "center">2</td>
 +
      <td align = "center"; valign = "center">2</td
 +
    </tr>
 +
</tr>
 +
    <tr>
 +
    <td align = "center"; valign = "center"><strong><p> H<sub>2</sub>0 (&micro;l)</ </p></strong></td>
 +
      <td align = "center"; valign = "center">22</td>
 +
      <td align = "center"; valign = "center">22</td>
 +
</tr>
 +
    <tr>
 +
    <td align = "center"; valign = "center"><strong><p> CutSmart(&micro;l)</ </p></strong></td>
 +
      <td align = "center"; valign = "center">5</td>
 +
      <td align = "center"; valign = "center">5</td>
 +
</tr>
 +
 +
</tbody>
 +
</table>
 +
</br></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> Dephosphorylate our inserts</br></br>
 +
 +
            <U> Protocol:</U> follow in this <a href="https://static.igem.org/mediawiki/2016/c/c0/T--Pasteur_Paris--dephosphorylation_protocol.pdf">link</a></br></br>
 +
<br /><br /><br />
 +
          </p>
 +
      </figcaption>
 +
  </figure>
 +
</div>
 +
 +
 +
 +
<div class="lightbox" id="exp11">
 +
  <figure>
 +
    <a href="# exp11" class="closemsg"></a>
 +
        <figcaption>
 +
            <p>
 +
            <h6><U> Aim:</U></h6> Resuspend C1, C2 and all inserts synthesized by iDT. <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>
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      </figcaption>
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Latest revision as of 21:35, 19 October 2016