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      <h1  class="text-center">Notebook</h1>
     <h2 class="text-center" id="h2-calendar"> Calendars 2018</h2>
     <div class="container" >
        <div class="row text-center text-xs-center text-sm-center text-md-center align-items-center justify-content-center" >
      <!-- -------------------------------------------- June---------------------------------------------------------- -->     
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					<td><b>M</b></td>
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					<td>1</td>
					<td>2</td>
					<td>3</td>
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				<tr>
					<td>4</td>
					<td>5</td>
					<td>6</td>
					<td>7</td>
					<td>8</td>
					<td>9</td>
					<td>10</td>
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					<td>11</td>
					<td class="date"><a href="#jun12" class="pointer" title="Tuesday 12th June">12</a></td>
					<td class="date"><a href="#jun13" class="pointer" title="Wednesday 13th June">13</a></td>
					<td class="date"><a href="#jun14" class="pointer" title="Thurday 14th June">14</a></td>
					<td>15</a></td>
					<td>16</td>
					<td>17</td>
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					<td>18</td>
					<td>19</td>
					<td class="date"><a href="#jun20" class="pointer" title="Wednesday 20th June">20</a></td>
					<td >21</td>
					<td >22</td>
					<td >23</td>
					<td >24</td>
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					<td >25</td>
					<td>26</td>
					<td class="date"><a href="#jun27" class="pointer" title="Wednesday 27th June">27</a></td>
					<td>28</td>
					<td class="date"><a href="#jun29" class="pointer" title="Friday 29th June">29</a></td>
					<td>30</td>
					<td id="j1">1</td>
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            <!-- -------------------------------------------------.\ June------------------------------------------- --> 
              <!-- ------------------------------------------------July------------------------------------------- -->     
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					<td class="date"><a href="#jul2" class="pointer" title="Monday 2nd July">2</a></td>
					<td>3</td>
					<td>4</td>
					<td class="date"><a href="#jul5" class="pointer" title="Thurday 5th July">5</a></td>
					<td>6</td>
					<td>7</td>
					<td>8</td>
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					<td class="date"><a href="#jul9" class="pointer" title="Monday 9th July">9</a></td>
					<td class="date"><a href="#jul10" class="pointer" title="Tuesday 10th July">10</a></td>
					<td>11</td>
					<td>12</td>
					<td class="date"><a href="#jul13" class="pointer" title="Friday 13th Jult">13</a></td>
					<td>14</td>
					<td>15</td>
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					<td class="date"><a href="#jul16" class="pointer" title="Monday 16th July">16</a></td>
					<td class="date"><a href="#jul16" class="pointer" title="Tuesday 17th July">17</a></td>
					<td>18</td>
					<td class="date"><a href="#jul19" class="pointer" title="Thurday 19th July">19</a></td>
					<td>20</td>
					<td>21</td>
					<td>22</td>
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					<td class="date"><a href="#jul23" class="pointer" title="Monday 23th July">23</a></td>
					<td class="date"><a href="#jul24" class="pointer" title="Tuesday 24th July">24</a></td>
					<td class="date"><a href="#jul25" class="pointer" title="Wedneday 25th July">25</a></td>
					<td class="date"><a href="#jul26" class="pointer" title="Thurday 26th July"> 26</a></td>
					<td class="date"><a href="#jul27" class="pointer" title="Friday 27th July">27</a></td>
					<td>28</td>
					<td>29</td>
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					<td>30</td>
					<td class="date"><a href="#jul31" class="pointer" title="Tuesday 31th July">31</a></td>
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					<td ></td>
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					<td><b>S</b></td>
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					<td class="date"><a href="#a1" class="pointer" title="Wednesday 1st  August">1</a></td>
					<td class="date"><a href="#a2" class="pointer" title="Thurday 2nd August">2</a></td>
					<td class="date"><a href="#a3" class="pointer" title="Friday 3th August">3</a></td>
					<td>4</td>
					<td>5</td>
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					<td class="date"><a href="#a6" class="pointer" title="Monday 6th August">6</a></td>
					<td class="date"><a href="#a7" class="pointer" title="Tuesday 7th August">7</a></td>
					<td>8</td>
					<td>9</td>
					<td>10</td>
					<td>11</td>
					<td>12</td>
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					<td>13</td>
					<td>14</td>
					<td>15</td>
					<td>16</td>
					<td>17</td>
					<td>18</td>
					<td>19</td>
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					<td>20</td>
					<td class="date"><a href="#a21" class="pointer" title="Tuesday 21th August">21</a></td>
					<td class="date"><a href="#a22" class="pointer" title="Tuesday 22th August">22</a></td>
					<td>23</td>
					<td>24</td>
					<td>25</td>
					<td>26</td>
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					<td>27</td>
					<td class="date"><a href="#a28" class="pointer" title="Tuesday 28th August">28</a></td>
					<td class="date"><a href="#a29" class="pointer" title="Tuesday 29th August">29</a></td>
					<td class="date"><a href="#a30" class="pointer" title="Tuesday 30th August">30</a></td>
					<td>31</td>
					<td></td>
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					<td class="date"><a href="#s3" class="pointer" title="Monday 3th September">3</a></td>
					<td class="date"><a href="#s4" class="pointer" title="Tuesday 4th September">4</a></td>
					<td class="date"><a href="#s5" class="pointer" title="Wednesday 5th September">5</a></td>
					<td class="date"><a href="#s6" class="pointer" title="Thurday 6th September">6</a></td>
					<td class="date"><a href="#s7" class="pointer" title="Fridat 7th September">7</a></td>
					<td>8</td>
					<td>9</td>
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					<td class="date"><a href="#s10" class="pointer" title="Monday 10th September">10</a></td>
					<td class="date"><a href="#s11" class="pointer" title="Tuesday 11th September">11</a></td>
					<td class="date"><a href="#s12" class="pointer" title="Wednesday 12th September">12</a></td>
					<td class="date"><a href="#s13" class="pointer" title="Monday 13th September">13</a></td>
					<td>14</td>
					<td>15</td>
					<td>16</td>
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					<td class="date"><a href="#s17" class="pointer" title="Monday 17th September">17</a></td>
					<td>18</td>
					<td>19</td>
					<td>20</td>
					<td>21</td>
					<td>22</td>
					<td>23</td>
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					<td>24</td>
					<td>25</td>
					<td>26</td>
					<td>27</td>
					<td>28</td>
					<td>29</td>
					<td>30</td>
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					<td>1</td>
					<td>2</td>
					<td>3</td>
					<td>4</td>
					<td>5</td>
					<td>6</td>
					<td>7</td>
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					<td>8</td>
					<td>9</td>
					<td>10</td>
					<td>11</td>
					<td>12</td>
					<td>13</td>
					<td>14</td>
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					<td>15</td>
					<td>16</td>
					<td>17</td>
					<td>18</td>
					<td>19</td>
					<td>20</td>
					<td>21</td>
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					<td>22</td>
					<td>23</td>
					<td>24</td>
					<td>25</td>
					<td>26</td>
					<td>27</td>
					<td>28</td>
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					<td>29</td>
					<td>30</td>
					<td></td>
					<td></td>
					<td></td>
					<td></td>
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            <!-- -----------------------------------------.\ September---------------------------------------- -->
             
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     <br> <br> <br>
     
     <div class="container-fluid">
<div class="d-flex justify-content-center"><h2 class="text-center" style="background:#e6e6e6;  width: 300px; border-radius: 10px;border-bottom: none;"> June</h2></div>
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            <h2 class="text-center" id="jun12"> Tuesday 12th June 2018</h2> 
               <h4 text-xs-left text-sm-left>PCR of rubisco intron, paromomycin CDS and pSAD promoter </h4>
               <h6>Institut de biologie physico-chimique (IBPC) </h6>
            <div class="d-flex justify-content-xs-start justify-content-sm-start  justify-content-md-start justify-content-lg-end">
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  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Aurélie
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  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Jimmy
</div> </div> </div>
<p Primers Ig5/Ig6/Ig3/Ig4/Ig11/Ig12 diluted at 10µM. DNA templates diluted at 2ng/µL.</p>

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   <th  class="th-notebook"></th>
   <th  class="th-notebook">Quantity in µL</th>
   <th  class="th-notebook"> Mix*8</th>
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   <td class="td-notebook">Plasmid DNA</td>
   <td class="td-notebook">1</td>
   <td class="td-notebook"></td>
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  <tr >
   <td class="td-notebook">Phusion enzyme</td>
   <td class="td-notebook">0.25</td>
   <td class="td-notebook">2
   </td>
  </tr>
  <tr>
   <td class="td-notebook">HF buffer (5X)</td>
   <td class="td-notebook">5</td>
   <td class="td-notebook"">40</td>
  </tr>
  <tr>
   <td class="td-notebook">dNTPs (10µM)</td>
   <td class="td-notebook">0.5</td>
   <td class="td-notebook">4</td>
  </tr>
  <tr>
   <td class="td-notebook">Fw Primer (10µM)</td>
   <td class="td-notebook" >1.25</td>
   <td class="td-notebook"> </td>
  </tr>
  <tr>
   <td class="td-notebook"> Rv Primer (10µM) </td>
    <td class="td-notebook">1.25</td>
   <td class="td-notebook"></td>
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  <tr>
   <td class="td-notebook">H20</td>
   <td class="td-notebook">15.75</td>
   <td class="td-notebook">126</td>
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<br>
<p clas="text-notebook"> 21.5µL of mix by tube + 1µL of DNA + 2*1.25µL of primer.</p>
<p>pSAD promoter : primers Ig3/Ig4, template p0-07 : Expected length : 807 pb </p>
<p>Paromomycin CDS : primers Ig11/Ig12, template p0-12: Expected length : 1652 pb <br>
<p>Rubisco intron : primers Ig5/Ig6, template p0-02: Expected length : 177 pb </p>

<ol>
<li>negative control : puc 19 + Ig5/Ig6  </li>
<li>Rubisco intron : p0-02 + Ig5/Ig6 </li>
<li> negative control : puc 19 + Ig3/Ig4 </li>
<li> pSAD promoter : p0-07 + Ig3/Ig4</li>
<li>negative control : puc 19 + Ig11/Ig12 </li>
<li> paromomycin CDS : p0-12 + Ig11/Ig12</li>
<li>negative control : H20 + Ig5/Ig6 </li>
<li> negative control : H20 + Ig11/Ig12</li>
</ol>
<br>
<p> <span style="font-weight:bold;">Cycle </span> : 98°C, 35*(98°C - 1min, 50°C - 2min, 72°C - 3min), 72°C - 3min, 12°C - hold </p>
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  <!-- ----------------------------------- Wednesday 13th June ------------------------------------------------------- -->
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        <div class="note"> 
 <h2 class="text-center" id="jun13"> Wednesday 13th June</h2>
 
               <h4> Migration and purification of PCR product <br> “paromomycin”, “pSAD”, “rubisco intron”  </h4>
              <h6>Institut de biologie physico-chimique (IBPC) </h6>
          <div class="d-flex justify-content-end">  <div class="chip-icone"> <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
Aurélie
</div>
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  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Soukeyna
</div> </div> </div>
<p class="text-notebook">+ 5µL of loading buffer 6X in PCR product <br>
15 µL/well, 1% agarose gel <br>
Migration : 100V, 20 min 
</p>
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   <th class="th-notebook">Position</th>
   <th class="th-notebook">1</th>
   <th class="th-notebook">2</th>
    <th class="th-notebook">3</th>
    <th class="th-notebook">4</th>
    <th class="th-notebook">5</th>
    <th class="th-notebook">6</th>
    <th class="th-notebook">7</th>
    <th class="th-notebook">8</th>
    <th class="th-notebook">9</th>
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  <tr>
   <td class="td-notebook">Content</td>
   <td class="td-notebook">Ladder</td>
   <td class="td-notebook">puc19 <br>Ig5/Ig6<br> rubisco</td>
      <td class="td-notebook">p0-2 <br>Ig5/Ig6 <br>  rubisco</td>
      <td class="td-notebook">puc19 <br>Ig3/Ig4 <br> pSAD</td>
      <td class="td-notebook">p0-7 <br>Ig3/Ig4<br> pSAD</td>
      <td class="td-notebook">puc19 <br>Ig11/Ig12<br>paromycine R</td>
      <td class="td-notebook">p0-12 <br>Ig11/Ig12<br> paromycine R</td>
      <td class="td-notebook">H2O <br>Ig5/Ig6</td>
      <td class="td-notebook">H2O <br>Ig11/Ig12</td>
  </tr>
  <tr >
   <td class="td-notebook">Expected lenght (pb)</td>
   <td class="td-notebook"></td>
   <td class="td-notebook">0</td>
    <td class="td-notebook">177</td>
    <td class="td-notebook">0</td>
    <td class="td-notebook">807</td>
    <td class="td-notebook">0</td>
    <td class="td-notebook">1 652</td>
     <td class="td-notebook">0</td>
    <td class="td-notebook">0</td>
  </tr>
  </tbody> 
</table>
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<br> 
            
<p> <h5>Gel analysis </h5>
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    <p><span style="font-weight: bold;">Figure 1 : </span>Gel electrophoresis of PCR products  rubisco (2&amp;3) intron,pSAD (4&amp;5), paromomycin(6&amp;7)   </p>
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<br>Rubisco and pSAD : Ok for rubisco intron and pSAD, visible bands <br>
Paromycin: the main band does not have the expected length, the band at 1.6 kb is still purifed<br>
Cutting of the bands corresponding to the expected bands (photobenchling)<br><br>

<h5> DNA purification </h5>
            Kit used: Nucleospin Gel 2 PCR Clean up- gel extraction (NT1= 800µl)<br>
            Elution in 15µl , mesure of the DNA extracts concentration  using a nanodrop<br>
</p>

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   <th class="th-notebook">Fragment</th>
   <th class="th-notebook">Notation</th>
   <th class="th-notebook">Measured concentration (ng/µL)</th>

  </tr>
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  <tbody>      
  <tr>
   <td class="td-notebook">Rubisco intron</td>
   <td class="td-notebook">i0- IGEM1</td>
   <td class="td-notebook">26,9</td>

  </tr>
  <tr >
   <td class="td-notebook">pSAD</td>
   <td class="td-notebook"> i0-IGEM3</td>
   <td class="td-notebook">19,3</td>
  </tr>
        <tr >
   <td class="td-notebook">paromycin</td>
   <td class="td-notebook">i0-ApHVIII</td>
   <td class="td-notebook">7,8</td>
  </tr>
  </tbody> 
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</div>
<br>
<p> <span style="font-weight: bold;">Note</span> : We should re-do the PCR of “ paromycine” </p>
<br>
<h5> Launch of  2 bacterial cultures  overnight (4ml with spectinomycine)</h5>
<p> - p0universal = pAGM9121 <br>
- p0B3 pICH41258
</p>
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 <!-- -----------------------------------  Wednesday 14th June ------------------------------------------------------- -->
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        <div class="note"> 
 <h2 class="text-center" id="jun14"> Wednesday 14th June</h2>
 
               <h4> Plasmid purification &amp; Reaction of ligation/digestion  </h4>
             <h6>Institut de biologie physico-chimique (IBPC) </h6>
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Victor
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 Aurélie
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Soukeyna
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<p class="text-notebook"> <span style="font-weight:bold">Aim</span> : Extraction of plasmides from <a href="#jun13" style="text-decoration: none;">yesterday’s cultures </a> and reactions of digestion/ligation p0universal/ pSAD and p0-B3/intron rubisco.</p>
<h5>Plasmid purification </h5>
<p (p0universel=p0universel-pAGM9121)<br>
(p0- B3 = p0B3- pICH41258)</p>
<p class="text-notebook">  Purification of p0universel and p0- B3 plasmides  (kit used: Nucleopsin Plasmid) : Elution volume= 40µL</p>
<p class="text-notebook">Measure of DNA extracts concentration using Nanodrop (2 extracts per plasmid)<br>
- p0-universel= 71,5 ng/µl ,  64,9ng/µl <br>
- p0-B3= 92,8ng/µl, 90,7ng/µL
</p>

<h5> Reaction of ligation/digestion </h5>
<p class="text-notebook"> <u>Mix DNA</u> </p>
  <div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="tablenotebook text-center">
    <thead>    
  <tr>
   <th class="th-notebook">Tube number</th>
   <th class="th-notebook">2</th>
   <th class="th-notebook">6</th>
    <th class="th-notebook">1</th>
      <th class="th-notebook">5</th>

  </tr>
</thead>   
  <tbody>      
  <tr>
   <td class="td-notebook">µl plasmid</td>
   <td class="td-notebook">0,68</td>
   <td class="td-notebook">0,68</td>
    <td class="td-notebook">0,97</td>
      <td class="td-notebook">0,97</td>

  </tr>
  <tr >
   <td class="td-notebook">µl insert </td>
   <td class="td-notebook"> 2,733</td>
   <td class="td-notebook"></td>
    <td class="td-notebook">1,98</td>
      <td class="td-notebook"></td>
  </tr>
        <tr >
   <td class="td-notebook">µl H2O</td>
   <td class="td-notebook">10,56</td>
   <td class="td-notebook">13,32</td>
   <td class="td-notebook">11,06</td>
   <td class="td-notebook">13,07</td>         
  </tr>
         <tr >
   <td class="td-notebook"></td>
   <td class="td-notebook">pSAD</td>
   <td class="td-notebook">pSAD</td>
   <td class="td-notebook">Rubisco</td>
   <td class="td-notebook">Rubisco</td>         
  </tr>   
  </tbody> 
</table>
</div>
    </div>    

<p class="text-notebook"> <span style="font-weight:bold;">2</span>: plasmide uni+ pSAD+ enzymes <br>
<span style="font-weight:bold;">6</span>: plasmide uni + enzymes <br>
<span style="font-weight:bold;">1</span>: plasmides B3+ introns enzymes <br>
<span style="font-weight:bold;">5</span>: plasmides B3+ enzymes
</p> 

<p class="text-notebook"> <u>Mid enzyme</u> (6µl/tube) number of ligations : 4.5 </p>
    <div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="tablenotebook text-center">
       <thead>    
  <tr>
   <th class="th-notebook">Reaction mix</th>
   <th class="th-notebook">µL</th>
   <th class="th-notebook">master mix</th>
  </tr>
</thead> 
  <tbody>      
  <tr>
   <td class="td-notebook"> BS/HF (µl)</td>
   <td class="td-notebook">1</td>
   <td class="td-notebook">4,5</td>


  </tr>
  <tr >
   <td class="td-notebook">T4 ligase(µl)</td>
   <td class="td-notebook"> 1</td>
   <td class="td-notebook">4,5</td>

  </tr>
        <tr >
   <td class="td-notebook">Buffer NEB CS (µl)</td>
   <td class="td-notebook">2</td>
   <td class="td-notebook">9</td>
         
  </tr>
         <tr >
   <td class="td-notebook"> ATP 10mM(µl)</td>
   <td class="td-notebook">2</td>
   <td class="td-notebook">9</td>         
  </tr>   
  </tbody> 
</table>
</div>
    </div> 

<p>Cycle: <br>
- 37°C- 10min * 3 <br>
- 16°C- 10min *3 <br>
- 37°C- 10min <br>
- 65°C- 20min <br>
- 16°C- ∞ 
</p>
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<!-- ----------------------------------- Wednesday 20th June ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="jun20"> Wednesday 20th June</h2>
            
               <h4> PCR of the “ paromycine” fragment <br>  Purification of “p0- IGEM1” and “p0- IGEM3” plasmides  </h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
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 Aurélie
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 Ursula
</div> </div> </div>

<h5> PCR reactions</h5>
 <div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="text-center tablenotebook">
    <thead>    
  <tr>
   <th class="th-notebook">Product</th>
   <th class="th-notebook">Quantity (µL) </th>
   <th class="th-notebook"> Quantity (µL)</th>
  </tr>
</thead>   
  <tbody>      
  <tr>
   <td class="td-notebook">Plasmide de p0-12</td>
   <td class="td-notebook">1</td>
   <td class="td-notebook">↗</td>
  </tr>
  <tr >
   <td class="td-notebook">Enzyme Phusion</td>
   <td class="td-notebook">0.25</td>
   <td class="td-notebook">2
   </td>
  </tr>
  <tr>
   <td class="td-notebook">HF buffer (5X)</td>
   <td class="td-notebook">5</td>
   <td class="td-notebook">40</td>
  </tr>
  <tr>
   <td class="td-notebook">dNTPs (10mM)</td>
   <td class="td-notebook">0.5</td>
   <td class="td-notebook">4</td>
  </tr>
  <tr>
   <td class="td-notebook">Primer F Ig11 (10µM)</td>
   <td class="td-notebook" >1.25</td>
   <td class="td-notebook">10 </td>
  </tr>
  <tr>
   <td class="td-notebook"> Primer F Ig12 (10µM) </td>
    <td class="td-notebook">1.25</td>
   <td class="td-notebook">10</td>
  </tr>
          <tr>
   <td class="td-notebook">DNA</td>
   <td class="td-notebook">0 </td>
   <td class="td-notebook">1</td>
  </tr>
      <tr>
   <td class="td-notebook">H20</td>
   <td class="td-notebook">15.75</td>
   <td class="td-notebook">126</td>
  </tr>
     <tr>
   <td class="td-notebook">Total quantity</td>
   <td class="td-notebook">25 </td>
   <td class="td-notebook">25</td>
  </tr> 
  </tbody> 
</table>
</div>
</div> 
<br>
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   <th class="th-notebook"></th>
   <th class="th-notebook">DNA (template) </th>
   <th class="th-notebook"> Hybridation temperature</th>
  </tr>
</thead>   
  <tbody>      
  <tr>
   <td class="td-notebook">Tube 1</td>
   <td class="td-notebook">p⦽-12</td>
   <td class="td-notebook">50°C</td>
  </tr>
  <tr >
   <td class="td-notebook">Tube 2</td>
   <td class="td-notebook">p⦽-12</td>
   <td class="td-notebook">52°C</td>
  </tr>
  <tr>
   <td class="td-notebook">Tube 3</td>
   <td class="td-notebook">p⦽-12</td>
   <td class="td-notebook">53°C</td>
  </tr>
  <tr>
   <td class="td-notebook">Tube 4</td>
   <td class="td-notebook">p⦽-12</td>
   <td class="td-notebook">54°C</td>
  </tr>
  <tr>
   <td class="td-notebook">Tube 5</td>
   <td class="td-notebook" >p⦽-12</td>
   <td class="td-notebook">55°C </td>
  </tr>
  <tr>
   <td class="td-notebook"> Tube 6 </td>
    <td class="td-notebook">p⦽-12</td>
   <td class="td-notebook">157°C</td>
  </tr>
          <tr>
   <td class="td-notebook">Controle E </td>
   <td class="td-notebook">p⦽-19 </td>
   <td class="td-notebook">50°C</td>
  </tr>

  </tbody> 
</table>
</div>
</div>
<p class="text-notebook">Cycle: 
98°C <br>
98°C     - 1min    *30<br>
50-57°C- 2 min   *30 <br>
72°C     - 3 min   *30<br>
72°C     - 3 min <br>
12°C     - ∞
</p>
<p class="text-notebook">- Addition of  5µl loading buffer 6* (5*µL in 25µl)<br>
- Deposit of 30µl of extract on 1% agarose gel<br>
- Migration at 90V for 40 min<br><br></p> 
<p class="text-notebook"> Missing: PCR photo <br>
- PCR ok with paromycine at 800pb different from the expected 1657 pb expected before <br>
- Insert Paro stocked at -20°C <br> <br>
</p>

<h5> Purification of “p⦽-IGEM1” and “p⦽-IGEM3” plasmids </h5>
<p class="text-notebook">Kit used: Gene JET Plasmid-MiniprepKit- Thermo Scientific <br>
Centrifugation 13000 RPM <br>
Digestion to check the size of the inserts (using BsaI) <br>
 </p>

    
 <div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="text-center tablenotebook">
    <thead>    
  <tr>
   <th class="th-notebook">Plasmid(µl)</th>
   <th class="th-notebook">5 </th>
   <th class="th-notebook">5</th>
  </tr>
</thead>   
  <tbody>      
  <tr>
   <td class="td-notebook">Cut Smart Buffer (10X) (µl)</td>
   <td class="td-notebook">11</td>
   <td class="td-notebook">9C</td>
  </tr>
  <tr >
   <td class="td-notebook">Enzyme Bsa  (µl)</td>
   <td class="td-notebook">0.5</td>
   <td class="td-notebook">4.5</td>
  </tr>
  <tr>
   <td class="td-notebook">Water (µl)</td>
   <td class="td-notebook">3.5</td>
   <td class="td-notebook">31.5</td>
  </tr>


  </tbody> 
</table>
</div>
</div>

<p class="text-notebook"> Addition of 2µl of loading buffer 6* (2µl in 10µl)<br>
Deposit of 12µl on 1% agarose gel <br>
Migration at 90V, for 40min <br>
</p>
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    <p><span style="font-weight: bold;">Figure : </span>Gel electrophoresis  : Purification of “p⦽-IGEM1” and “p⦽-IGEM3” plasmids </p>
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<p ckass="text-notebook"> <br>We keep : 1a= rubisco intron= p⦽-IGEM1(1b too is conserved)<br>
                 2a= pSAD- p⦽-IGEM3<br>
Stock in glycerol￫ -30°C (DH5𝛂)mycologue <br> <br>
<span style="font-weight:bold;">Note</span>: Transplanting of the microalguae strain that was previously blocked by FedEX
</p>
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<!-- ----------------------------------- Wednesday 27th June ------------------------------------------------------- -->
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<h2 class="text-center" id="jun27"> Wednesday 27th June</h2>
            
               <h4>Bacterial culture </h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
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 Aurélie
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 Ursula
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<p class="text-notebook">Launch of 2* 3.5 mL of bacterial culture → +spectromycine  of pICH4 1264 which contains the destination plasmid of the “paromicycn fragment)
</p>
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<!-- ----------------------------------- Wednesday 29th June ------------------------------------------------------- -->
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<h2 class="text-center" id="jun29"> Wednesday 29th June</h2>
            
               <h4> Purification of pICH41264 plasmid using the Nucleospin Plasmid K <br> Plasmid concentration measures using nanodrop  </h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
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 Aurélie
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 Ursula
</div> </div> </div>
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    <table class="text-center tablenotebook">
  <tbody>      
  <tr>
   <td class="td-notebook">Culture 1</td>
   <td class="td-notebook">26,7ng/µl</td>
   <td class="td-notebook">1.92</td>
   <td class="td-notebook">1.73</td>   
  </tr>
  <tr >
   <td class="td-notebook">Culture  2 </td>
   <td class="td-notebook">25,7ng/µl</td>
   <td class="td-notebook">1.92</td>
    <td class="td-notebook">2.02</td>
  </tr>
  </tbody> 
</table>
</div>
</div>
<p class="text-notebook">The pICH41264 extracts are stocked at -20°C</p>

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<!-- ----------------------------------------------------------------July Month--------------------------------------  -->
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<div class="d-flex justify-content-center"><h2 class="text-center" style="background: #e6e6e6; width: 300px; border-radius: 10px; border-bottom: none;"> july</h2></div>
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            <h2 class="text-center" id="jul2"> Monday 2 July</h2>

               <h4 text-xs-left text-sm-left> Transformation of 𝝱-10 bacterias with  p⦽ pICH4 1264 + insert paroR <br>Production of p⦽ plasmids for 3’LTR and 5’LTR parts. </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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 Ursula
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 Dounia
</div> </div> </div>
<h5> Transformation of 𝝱-10 bacterias with  p⦽ pICH4 1264 + insert paroR</h5>
<p class="text-notebook">-𝝱-10 bacteria : 30µl <br>
Ligation product (paro): 4µl (insert or negative control)<br>
30min_ 4°C <br>
55 sec_ 42°C <br>
2 min_ 4°C <br>
+700µL of LB medium <br>
1h_ 37°C <br>
Staggering of 350 on LB+spec+Xgal dish <br>
37°C O/N <br></p>
<h5> Production of p⦽ plasmids for 3’LTR and 5’LTR parts. </h5>
<p clas="text-notebook">Resuspension of IDT1 3’LTR in 31,03µL <br>
Resuspension of IDT2 5’LTR in 27,56µL <br>
[C]= 100 fmol/µL <br>
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Sub cloning of 2 white colonies of the day before. These colonies are supposed to contain the pICH4 1264 plasmid and the “PARO” insert. <br>
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 <h2 class="text-center" id="jul5"> Thurday 5 July</h2>
 
               <h4> Transplanting of single clones from each subcloning of the 3rd of  july <br> Transformation of 𝝱-10 bacterias with  p⦽ pUniversal, containing either LTR5’ or LTR3’ as insert  </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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 Dounia
</div> </div> </div>
<p class="text-notebook">𝝱-10  Bacteria _ 30µL <br>
Ligation product_ 4µL (insert or control) <br>
      - 30 min_4°c <br>
     - 55 sec_ 42°C <br>
     - 2 min  _ 4°C <br>
  + 700µl of LB <br>
 Spreading of 250µl on a dish containing LB + SPEC+ Xgal at 37°C
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<!-- ----------------------------------- Thurday 9 July ------------------------------------------------------- -->
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<h2 class="text-center" id="jul9"> Thurday 9 July</h2>
            
               <h4> Lauch of miniprep <br>Verifcation of Paro1 and Paro 2 clones <br> Assembly of GAG-Pol   </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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Ursula
</div> </div> </div>
<h5> Lauch of miniprep of 4 clones of each LTD in media spec+ Xgal </h5>
<div class="d-flex justify-content-center">
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  <tbody>      
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   <td class="td-notebook">LTR3’1</td>
   <td class="td-notebook">LTR5’1</td>  
  </tr>
  <tr >
   <td class="td-notebook">LTR3’2 </td>
   <td class="td-notebook">2LTR5’2</td>
  </tr>
       <tr >
   <td class="td-notebook">LTR3’3 </td>
   <td class="td-notebook">LTR5’3</td>
  </tr>
       <tr >
   <td class="td-notebook">LTR3’4 </td>
   <td class="td-notebook">LTR5’4</td>
  </tr>
  </tbody> 
</table>
</div>
</div>

<h5>Verifcation of Paro1 and Paro 2 clones  </h5>
<p class="text-notebook">Purification of plasmids (methode for digestion of verification)*? , using Nucleospin Plasmid Kit</p>

<h5> Assembly of GAG-Pol ⇨ 6 parts</h5>
<p class="text-notebook">1) Dilution at 10fmol/µl of each fragment received </p>
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  <tbody>      
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   <td class="td-notebook">IDT3_L0_B1</td>
   <td class="td-notebook">1578/10 ⇒ 157,8</td>  
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   <td class="td-notebook">IDT4_L0_B2 </td>
   <td class="td-notebook">3216/10⇒321,6</td>
  </tr>
       <tr>
   <td class="td-notebook">IDT6_L0_B3 </td>
   <td class="td-notebook">900/10 ⇒ 90</td>
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   <td class="td-notebook">IDT4_L0_A3</td>
   <td class="td-notebook">1098/10⇒109,8</td>
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   <td class="td-notebook">IDT4_L0_A1 </td>
   <td class="td-notebook">2078/10⇒207,8</td>
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   <td class="td-notebook">IDT4_L0_A2</td>
   <td class="td-notebook">1171/10⇒ 117,1</td>
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<p class="text-notebook">2) Reaction of digestion/ligation </p>
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<!-- ----------------------------------- Thurday 10th July ------------------------------------------------------- -->
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<h2 class="text-center" id="jul10"> Thurday 10th July</h2>
               <h4> Transformation of 𝝱10 with digestion/ligation products  of GAG POL <br> (<a href="#jul9">experiences of 07/09/18</a>) </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie</div>
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Ursula
</div> </div> </div>
<p class="text-notebook">- 30µl of  𝝱+ 4µl of ligation product <br>
- 30min at 4°C <br>
- 55 sec at 42°C <br>
- 2 min at 4°C <br>
- + 700µL of LB <br> <br<
1H at 37°c <br><br>
Spreading of 250µL on 2  petri dishes  Xgal+ Spec : control/ Gagpol<br>
Incubation at 30°C over night</p>
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<!-- ----------------------------------- Friday 13th July ------------------------------------------------------- -->
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<h2 class="text-center" id="jul13"> Friday 13th July</h2>
            
               <h4> Launch of minipreps for 6 clones of GAG-Pol <br> </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Ursula
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 Asmaa
</div> </div> </div>
<h5> Launch of minipreps for 6 clones of GAG-Pol in Xgal+spec media GAG_Pol 1, GAG_Pol 2, GAG_Pol 3, GAG_Pol 4 , GAG_Pol 5, GAG_Pol 6</h5>
<p class="text-notebook">Purification of PAGM9121 plasmide using Nucleopsin Plasmid Kit</p>
<h5>Measure of DNA concentration at Nanodrop  </h5>
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   <th class="th-notebook">Fragment</th>
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    <th class="th-notebook"> </th>
    <th class="th-notebook"> </th>  
  </tr>
</thead>    
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   <td class="td-notebook">LTR3’1</td>
   <td class="td-notebook">42,2</td> 
    <td class="td-notebook">1.97</td>
   <td class="td-notebook">1.85</td>    
  </tr>
  <tr>
   <td class="td-notebook">LTR3’2</td>
   <td class="td-notebook">38,4</td>
    <td class="td-notebook">1.98</td>
   <td class="td-notebook">1.86</td>    
  </tr>
       <tr>
   <td class="td-notebook">LTR3’3 </td>
   <td class="td-notebook">37,7</td>
    <td class="td-notebook">1.95</td>
   <td class="td-notebook">1.64</td>         
  </tr>
       <tr>
   <td class="td-notebook">LTR3’4</td>
   <td class="td-notebook">94,1</td>
    <td class="td-notebook">1.91</td>
   <td class="td-notebook">2.06</td>         
  </tr>
            <tr>
   <td class="td-notebook">LTR5’1 </td>
   <td class="td-notebook">48,4</td>
    <td class="td-notebook">1.95</td>
   <td class="td-notebook">1.86</td>              
  </tr>
            <tr >
   <td class="td-notebook">LTR5’3</td>
   <td class="td-notebook">120</td>
    <td class="td-notebook">1.89</td>
   <td class="td-notebook">1.94</td>              
  </tr>
                <tr >
   <td class="td-notebook">Paromycin 1</td>
   <td class="td-notebook">55,8</td>
    <td class="td-notebook">1.91</td>
   <td class="td-notebook">1.89</td>              
  </tr>
                <tr >
   <td class="td-notebook">Paromycin 2</td>
   <td class="td-notebook">49,1</td>
    <td class="td-notebook">1.91</td>
   <td class="td-notebook">1.87</td>              
  </tr>
                <tr >
   <td class="td-notebook">GAG_Pol 1</td>
   <td class="td-notebook">78,7</td>
    <td class="td-notebook">1.89</td>
   <td class="td-notebook">1.92</td>              
  </tr>
                <tr >
   <td class="td-notebook">GAG_Pol 2</td>
   <td class="td-notebook">25,3</td>
    <td class="td-notebook">1.97</td>
   <td class="td-notebook">2.85</td>              
  </tr>
                <tr >
   <td class="td-notebook">GAG_Pol 3</td>
   <td class="td-notebook">49,0</td>
    <td class="td-notebook">1.94</td>
   <td class="td-notebook">2.62</td>              
  </tr>
                <tr >
   <td class="td-notebook">GAG_Pol 4</td>
   <td class="td-notebook">17,7</td>
    <td class="td-notebook">2.00</td>
   <td class="td-notebook">3.90</td>              
  </tr>
                <tr >
   <td class="td-notebook">GAG_Pol 5</td>
   <td class="td-notebook">44,0</td>
    <td class="td-notebook">1.97</td>
   <td class="td-notebook">2.68</td>              
  </tr>
                <tr >
   <td class="td-notebook">GAG_Pol 6</td>
   <td class="td-notebook">21,2</td>
    <td class="td-notebook">2.01</td>
   <td class="td-notebook">3.65</td>              
  </tr>
  </tbody> 
</table>
</div>
</div>
<h5>Digestion to check the inserts size ( using BsaI) </h5>
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   <th class="th-notebook"> 10 µl reaction </th>  
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   <td class="td-notebook">DNA</td>
   <td class="td-notebook">200 ng (except for GAG pol 4 & 6 : DNA=150ng)</td>    
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   <td class="td-notebook">10X CutSmart Buffer</td>
   <td class="td-notebook">1µl</td>   
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   <td class="td-notebook">BsaI </td>
   <td class="td-notebook"> 0.2µ</td>         
  </tr>
       <tr>
   <td class="td-notebook">Nuclease-free Water</td>
   <td class="td-notebook">	to 50 µl</td>        
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<!-- ----------------------------------- Monday 16th July ------------------------------------------------------- -->
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<h2 class="text-center" id="jul16"> Monday 16th July</h2>
             
               <h4>  Migration of digestion reaction products </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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 Anaïs
</div> </div> </div>
<h5> Migration of digestion reaction products from the day before </h5>
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    <p><span style="font-weight: bold;">Figure : </span>Gel electrophoresis of digestion reaction products to check the inserts size  </p>
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<p class="text-notebook">Agarose gel 1% <br>
2µL loading buffer 6X per 10µl sample + BET <br>
Migration 100V, 20 min 
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<!-- ----------------------------------- Tuesday 17th July ------------------------------------------------------- -->
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<h2 class="text-center" id="jul17"> Tuesday 17th July</h2>
             
               <h4> Arrangement in the box iGEM 2018 <br> Preparation of all the inserts in order to send them to sequencing </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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Aurélie
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 Anaïs
</div> </div> </div>
<h5> Arrangement in the box iGEM 2018  </h5>
<h5>Preparation of all the inserts in order to send them to sequencing </h5>
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<p class="text-notebook">[C] Nanodrop <br>
PSAD 2a: 107.9 ng/µL <br>
1.97 <br>
3.81 <br>
PSAD 2d: 150.6 ng/µl <br>
1.94 <br>
3.12 <br>
Intron Rb= 103 ng/µL <br>
1.99 <br>
3.82 <br><br>

[AMG 9121]: 71.5 ng/µl <br>
Samples sent to sequencing <br>
</p>
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<!-- ----------------------------------- Thurday 19th July ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="jul19"> Thursday 19th July</h2>
             
               <h4> Analysis of sequencing results </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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 Anaïs
</div> </div> </div>
<div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="text-center tablenotebook">
       <thead>    
  <tr>
   <th class="th-notebook">Fragment</th>
   <th class="th-notebook">Comments </th>
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">Paromycine</td>
   <td class="td-notebook">ok</td> 
  </tr>
  <tr>
   <td class="td-notebook">Gagpol (GG40 and GG41) <br> Rubisco intron <br>
pSAD
</td>
   <td class="td-notebook">Absurd results <br>
Re-do the digestion reaction whith different  BsaI enzymes
</td>
  </tr>
  </tbody> 
</table>
</div>
</div>
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<!-- ----------------------------------- Monday 23th July ------------------------------------------------------- -->
     <!-- One day of the notebook-->
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        <div class="note"> 
<h2 class="text-center" id="jul23"> Monday 23th July</h2>
             
               <h4>Transformation to redo the stock of each plasmid</h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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 Anaïs
</div> </div> </div>
<p class="text-notebook"> - 10µL of  𝝱10 bacterias <br>
- Gag Pol 5 : 1.5 µL <br>
            Gag Pol 3 : 0.5 µL<br>
            Igem 2, 4 and 6: 2µL<br>
            Igem 3, 2 : 2.5 µL<br>
            ↳ plasmids to transfect <br><br>
- 30 min at 4°C <br>
- 55 sec at 42°C<br>
- 2 min at 4°C <br>
+ 700 µl of LB <br><br>
 Spreading of 250µL on plates containing LB+ spec
</p>
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<!-- ----------------------------------- Thurday 24th July ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="jul24"> Thurday 24th July</h2>
             
               <h4> Transplanting of a clamydomonas clone <br> Plasmid extraction from 𝝱 bacterias transformed <br> Concentration measurements </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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 Dounia
</div> </div> </div>
<h5>1. Transplanting of a clamydomonas clone in 3ml of LB+ Spec </h5>
<h5>2. Plasmid extraction from 𝝱 bacterias transformed <a href="#jul23">the day before</a> ( directly from dish).</h5>
<p class="text-notebook">Kit used = Nucleopsin Plasmid <br>
Elution volum= 40µL <br>
</p>
<h5>3. Concentration measurements using Nanodrop </h5>

    
<div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="text-center tablenotebook">
       <thead>    
  <tr>
   <th class="th-notebook">Fragment</th>
   <th class="th-notebook">Concentration(ng/µl) </th>
    <th class="th-notebook"> </th>
    <th class="th-notebook"> </th>  
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">LTR3’2</td>
   <td class="td-notebook">241.3</td> 
    <td class="td-notebook">1.90</td>
   <td class="td-notebook">2.17</td>    
  </tr>
  <tr>
   <td class="td-notebook">ParoII</td>
   <td class="td-notebook">196.0</td>
    <td class="td-notebook">1.90</td>
   <td class="td-notebook">2.09</td>    
  </tr>
 <tr>
   <td class="td-notebook">LTR5’</td>
   <td class="td-notebook">166.2</td>
    <td class="td-notebook">1.89</td>
   <td class="td-notebook">2.15</td>    
  </tr>
  <tr>
   <td class="td-notebook">ParoI</td>
   <td class="td-notebook">160.9</td>
    <td class="td-notebook">1.91</td>
   <td class="td-notebook">2.11</td>    
  </tr>
  <tr>
   <td class="td-notebook">LTR3’4</td>
   <td class="td-notebook">232.8</td>
    <td class="td-notebook">1.89</td>
   <td class="td-notebook">2.21</td>    
  </tr>
  <tr>
   <td class="td-notebook">Gag5</td>
   <td class="td-notebook">393.6</td>
    <td class="td-notebook">1.91</td>
   <td class="td-notebook">2.16</td>    
  </tr>     
 <tr>
   <td class="td-notebook">Rubisco</td>
   <td class="td-notebook">31.3</td>
    <td class="td-notebook">2.04</td>
   <td class="td-notebook">2.09</td>    
  </tr>
 <tr>
   <td class="td-notebook">pSAD</td>
   <td class="td-notebook">133.8</td>
    <td class="td-notebook">1.90</td>
   <td class="td-notebook">2.12</td>    
  </tr>
 <tr>
   <td class="td-notebook">Gag3</td>
   <td class="td-notebook">370.6</td>
    <td class="td-notebook">1.91</td>
   <td class="td-notebook">2.11</td>    
  </tr>

  </tbody> 
</table>
</div>
</div>

<h5>4. Replanting of Chlamydomonas  </h5>
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<!-- ----------------------------------- Wednesday 25th July ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="jul25"> Wednesday 25th July</h2>
             
               <h4> Plasmid extraction from bacteria transformed  <br> Concentration measurement <br> Digestion </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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 Anais
</div> </div> </div>

<h5>Plasmid extraction from <a href="#jul24">bacteria transformed</a>  </h5>
<p class="text-notebook"> Put on liquid media the 24/07 <br> kit used : Nucleopsin plasmid  
</p>

<h5>Concentration measurement using Nanodrop  </h5>
    
<div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="text-center tablenotebook">
       <thead>    
  <tr>
   <th class="th-notebook">Fragment</th>
   <th class="th-notebook">Concentration(ng/µl) </th>
    <th class="th-notebook"> </th>
    <th class="th-notebook"> </th>  
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">Paro II</td>
   <td class="td-notebook">110.4</td> 
    <td class="td-notebook">1.89</td>
   <td class="td-notebook">2.21</td>    
  </tr>
  <tr>
   <td class="td-notebook">PSAD</td>
   <td class="td-notebook">62.1</td>
    <td class="td-notebook">1.89</td>
   <td class="td-notebook">2.21</td>    
  </tr>
 <tr>
   <td class="td-notebook">GagPol 5</td>
   <td class="td-notebook">231.6</td>
    <td class="td-notebook">1.89</td>
   <td class="td-notebook">2.21</td>    
  </tr>
  <tr>
   <td class="td-notebook">ParoI</td>
   <td class="td-notebook">96.4</td>
    <td class="td-notebook">1.89</td>
   <td class="td-notebook">2.18</td>    
  </tr>
  <tr>
   <td class="td-notebook">LTR5’</td>
   <td class="td-notebook">65.4</td>
    <td class="td-notebook">1.90</td>
   <td class="td-notebook">2.17</td>    
  </tr>
  <tr>
   <td class="td-notebook">LTR3’2</td>
   <td class="td-notebook">102.3</td>
    <td class="td-notebook">1.91</td>
   <td class="td-notebook">2.26</td>    
  </tr>     
 <tr>
   <td class="td-notebook">LTR3’4</td>
   <td class="td-notebook">111.0</td>
    <td class="td-notebook">1.88</td>
   <td class="td-notebook">2.29</td>    
  </tr>
 <tr>
   <td class="td-notebook">Gag Pol3</td>
   <td class="td-notebook">189.8</td>
    <td class="td-notebook">1.90</td>
   <td class="td-notebook">2.21</td>    
  </tr>
 <tr>
   <td class="td-notebook">Intron Rubisco</td>
   <td class="td-notebook">73.9</td>
    <td class="td-notebook">1.89</td>
   <td class="td-notebook">2.19</td>    
  </tr>
  </tbody> 
</table>
</div>
</div>

<h5>Digestion</h5> 
<div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="text-center tablenotebook">
       <thead>    
  <tr>
   <th class="th-notebook"></th>
   <th class="th-notebook">Intron </th>
    <th class="th-notebook">PSAD</th>
    <th class="th-notebook">LTR5’1</th>  
        <th class="th-notebook">LTR3’4 </th>
    <th class="th-notebook"> LTR3’2</th>
        <th class="th-notebook">Uni </th>
    <th class="th-notebook">GagPol3 </th>  
        <th class="th-notebook">GagPol5 </th>  
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">Enzyme</td>
   <td class="td-notebook">0.1</td> 
    <td class="td-notebook">0.1</td>
   <td class="td-notebook">0.1</td> 
   <td class="td-notebook">0.1</td>
   <td class="td-notebook">0.1</td>
   <td class="td-notebook">0.1</td>
   <td class="td-notebook">0.2</td>
   <td class="td-notebook">0.2</td>
  </tr>
  <tr>
   <td class="td-notebook">Buffer</td>
   <td class="td-notebook">1</td>
    <td class="td-notebook">1</td>
   <td class="td-notebook">1</td> 
    <td class="td-notebook">1</td>
   <td class="td-notebook">1</td>
    <td class="td-notebook">1</td>
   <td class="td-notebook">1</td>
    <td class="td-notebook">1</td>
  </tr>
 <tr>
   <td class="td-notebook">ADN</td>
   <td class="td-notebook">2</td>
    <td class="td-notebook">2</td>
   <td class="td-notebook">4</td>  
    <td class="td-notebook">2</td>
   <td class="td-notebook">5</td>
          <td class="td-notebook">3</td>
   <td class="td-notebook">3</td>
     <td class="td-notebook">3</td>
  </tr>
  <tr>
   <td class="td-notebook">H2O</td>
   <td class="td-notebook">6.8</td>
    <td class="td-notebook">6.8</td>
   <td class="td-notebook">4.9</td>  
   <td class="td-notebook">6.8</td>
   <td class="td-notebook">3.9</td>
          <td class="td-notebook">5.9</td>
   <td class="td-notebook">5.9</td>
      <td class="td-notebook">5.9</td>
  </tr>
  <tr>
   <td class="td-notebook"></td>  
   <td class="td-notebook">BstEII <br> SaeI</td>
   <td class="td-notebook">BstEI <br> StuI</td>
    <td class="td-notebook">BstEII <br> BamHI</td>
   <td class="td-notebook">BstEII <br> BamHI</td>  
    <td class="td-notebook">BstEII <br> BamHI</td>
   <td class="td-notebook">BstEII <br> BamHI</td>
          <td class="td-notebook">BstEII</td>
   <td class="td-notebook">BstEII</td>
  </tr>
  </tbody> 
</table>
</div>
</div>
<p class="text-notebook">Digestion at 37°C/ 1H , conservation at -20°C overnight</p> </div>  
            </div>    
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<!-- ----------------------------------- Thurday 26th July ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="jul26"> Thurday 26th July</h2>
             
               <h4> Migration of digestion samples <br> Digestion <br> Migration on gel electrophoresis </h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
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 Aurélie
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 Anais
</div> </div> </div>
<h5>Migration of digestion samples from the day before 
 </h5>
<p class="text-notebook">1% agarose gel, deposit of 10µ+2µL of loading buffer 6X, ladder “2log” <br> Gel plan</p>

<div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="text-center tablenotebook">
       <thead>    
  <tr>
   <th class="th-notebook">Rb intron</th>
   <th class="th-notebook">pSAD </th>
    <th class="th-notebook">LTR3’2</th>
    <th class="th-notebook">LTR3’4</th>  
        <th class="th-notebook">LTR5’4 </th>
    <th class="th-notebook"> 9121</th>
        <th class="th-notebook">Gag3 </th>
    <th class="th-notebook">Gag5 </th>    
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">1 2 3</td>
   <td class="td-notebook">1 2 3</td> 
    <td class="td-notebook">1 2 3</td>
   <td class="td-notebook">1 2 3</td> 
   <td class="td-notebook">1 2 3</td>
   <td class="td-notebook">1</td>
   <td class="td-notebook">1 3</td>
   <td class="td-notebook">1 3</td>
  </tr>
  <tr>
   <td class="td-notebook">3 bands <br> 1714 pb <br> 620 pb <br> 66 pb</td>
   <td class="td-notebook">2 bands <br> 1662 pb <br> 1182 pb </td>
    <td class="td-notebook" colspan="2">1 band <br>2000-3000pb <br> (2270 pb)</td> 
    <td class="td-notebook">1 band <br> 2270 pb</td>
   <td class="td-notebook">2 band <br> 11831pb <br>828 pb</td>
    <td class="td-notebook" colspan="2">2 bands <br> 4242 pb <br> 2260 pb</td>

  </tr>
  </tbody> 
</table>
</div>
</div>
<br> <br>
    <!-- --------------------------------------------FIGURE CAPTION A--------------------------------------------- -->
 <a  class="pointer" title="Click on the image for a larger view " data-toggle="modal" data-target=".Migration-180726"><div class="container-figurecaption">
  <img   class="img-figurecaption" src="https://static.igem.org/mediawiki/2018/f/fd/T--Sorbonne_U_Paris--M180726Migrationsample.jpg" alt="Notebook">
  <div class="content-figurecaption">
    <p><span style="font-weight: bold;">Figure : </span>Gel electrophoresis of digestion reaction products to check the inserts size  </p>
  </div>
</div></a>
<div class="modal fade Migration-180726" tabindex="-1" role="dialog" aria-labelledby="myLargeModalLabel" aria-hidden="true">
  <div class="modal-dialog modal-lg">
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</div>
<!-- --------------------------------------------FIGURE CAPTION--------------------------------------------- -->
<p class="text-notebook">There seems to be no inserts</p> 
<h5>Digestion </h5>
<p class="text-notebook">- NDE I, 1h, 37°C <br>
- Tampon 10X : 1µl <br>
- ADN : 3µL <br>
- Nde I : 0.2 µL <br>
- H2O : 5.8 µ <br>
</p> 

<h5> Migration on gel</h5>

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<div class="table-responsive-sm">
    <table class="text-center tablenotebook">
       <thead>    
  <tr>
   <th class="th-notebook">Rb Intron</th>
   <th class="th-notebook">pSAD </th>
    <th class="th-notebook">LTR3’2</th>
    <th class="th-notebook">LTR3’4</th>  
        <th class="th-notebook">LTR5’1 </th>
    <th class="th-notebook"> GagP3</th>
        <th class="th-notebook">Gag P5 </th>
    <th class="th-notebook">Paro I </th>  
     <th class="th-notebook">Paro II </th>
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">1 2 </td>
   <td class="td-notebook">1 2 </td> 
    <td class="td-notebook">1 2 </td>
   <td class="td-notebook">1 2 </td> 
   <td class="td-notebook">1 2 </td>
   <td class="td-notebook">1</td>
   <td class="td-notebook">1 </td>
   <td class="td-notebook">1 2</td>
    <td class="td-notebook">1 2</td>  
  </tr>
  </tbody> 
</table>
</div>
</div>
<p class="text-notebook">Non informative, forgotten time of incubation </p> 
</div>  
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<!-- ----------------------------------- Friday 27th July ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="jul27"> Friday 27th July</h2>
             
               <h4> Enzymatic digestion reaction  </h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
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Aurélie
</div>
 </div> </div>

<h5>Enzymatic digestion reaction ( fast digest enzyme)</h5>
<div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="text-center tablenotebook">
       <thead>    
  <tr>
   <th class="th-notebook"></th>
   <th class="th-notebook">1 reaction </th>
    <th class="th-notebook">mix *20</th>
 
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">Bcl Enzyme  </td>
   <td class="td-notebook">0.2 µL </td> 
    <td class="td-notebook">4 µL </td>  
  </tr>
   <tr>
   <td class="td-notebook">Buffer FD (10X) </td>
   <td class="td-notebook">1  µL </td> 
    <td class="td-notebook">20µL </td>  
  </tr>
    <tr>
   <td class="td-notebook">DNA  </td>
   <td class="td-notebook">3  µL </td> 
    <td class="td-notebook">↗ </td>  
  </tr>
     <tr>
   <td class="td-notebook">H2O </td>
   <td class="td-notebook">5.8 µL  </td> 
    <td class="td-notebook">116 µL </td>  
  </tr>   
  </tbody> 
</table>
</div>
</div>
<p class="text-notebook">↪7µl mix/ per tube <br>
 + 3µL  DNA <br>
+ 0.2 µl XmaII in pSAD  <br> <br>

Incubation 2H at 37°C<br>
Migration in 0.1 % agarose gel, ladder “2log”, deposit of 10µL of reaction sample + 2µl de loading buffer 6X<br>
</p>

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<!-- ----------------------------------- Tuesday 31th July ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="jul31"> Tuesday 31th July</h2>
             
               <h4> DNA sent to sequencage <br> Digestion reaction using BSaI <br> </h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
     <div class="d-flex justify-content-end">  <div class="chip-icone"> <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
Aurélie
</div>
 </div> </div>
<h5>1. DNA sent to sequencage </h5>
<p class="text-notebook">pSAD <br> AYL0014041 ← GG40 <br> 042 ← GG41 </p>

<h5>Digestion reaction using BSaI </h5>

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   <th class="th-notebook"></th>
   <th class="th-notebook">1 reaction </th>
    <th class="th-notebook">mix *12</th>
 
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">Bcl Enzyme  </td>
   <td class="td-notebook">0.2 µL </td> 
    <td class="td-notebook">24 µL </td>  
  </tr>
   <tr>
   <td class="td-notebook">Buffer Cutsmart (10X) </td>
   <td class="td-notebook">1  µL </td> 
    <td class="td-notebook">12µL </td>  
  </tr>
    <tr>
   <td class="td-notebook">DNA  </td>
   <td class="td-notebook">4  µL </td> 
    <td class="td-notebook">↗ </td>  
  </tr>
     <tr>
   <td class="td-notebook">H2O mq</td>
   <td class="td-notebook">4.8 µL  </td> 
    <td class="td-notebook">57.6µL </td>  
  </tr>   
  </tbody> 
</table>
</div>
</div>
<p class="text-notebook">Incubation 3h 37°C <br>
Migration on 1% agarose gel of 10µl sample + 2µL loading buffer  <br>
Ladder 2Log  <br>
90V, 20 minutes <br>
</p>  
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<p class="text-notebook"> OK for gag pol 3’ and 5’ LTR, Rb+ paro Sequencing  <br> 
pSAD not ok → replanting of colonies </p></div> 
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<!-- ----------------------------------------------------------------August Month--------------------------------------  -->
     <div class="container-fluid">
<div class="d-flex justify-content-center"><h2 class="text-center" style="background: #e6e6e6; width: 300px; border-radius: 10px; border-bottom: none;"> August</h2></div>
     </div>
<!-- ----------------------------------- Wednesday 1st Augus ------------------------------------------------------- -->
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        <div class="note"> 
            <h2 class="text-center" id="a1"> Wednesday 1st August</h2>
               <h4 text-xs-left text-sm-left> Sending samples to sequencing <br> Cloning of pSAD</h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
                  <div class="d-flex justify-content-end">      <div class="chip-icone"> <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Aurélie
</div>
   </div> </div>
<h5>1. Sending samples to sequencing  </h5>
<p class="text-notebook">- GAG 3 (10µL DNA+ 10µL H2O) <br>
- GAG 5 (10µL DNA+ 10µL H2O) <br>
- 3’ LTR (3’4) (10µL DNA+ 5 µL H2O) <br>
- 5’ LTR (10µL DNA+ 5 µL H2O) <br>
- Rb (10µL DNA+ 5 µL H2O) <br>
- GG40 (50µL) and GG41 (10µL) <br>
-Ig18 (20µL), Ig19, Ig20 (10µL) <br>
Stock from 07/25/18
</p>
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<h5>Cloning of pSAD from 07/23/2018 experiment, in solid LB+ spec </h5>
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<!-- -----------------------------------Thurday 2nd August------------------------------------------------------- -->
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        <div class="note"> 
 <h2 class="text-center" id="a2"> Thurday 2nd August</h2>

               <h4> Replanting of PSAD and Rb intron</h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
  <div class="d-flex justify-content-end"><div class="chip-icone"> <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Aurélie
</div>
  </div> </div>
<p class="text-notebook">Replanting of PSAD and Rb intron in liquid media+ spec .</p>
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<!-- ----------------------------------- Friday 3th August ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="a3"> Friday 3th August</h2>
           
               <h4> Miniprep of PSAD and Rb</h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
    <div class="d-flex justify-content-end">          <div class="chip-icone"> <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Aurélie
</div>
  </div> </div>
<p class="text-notebook">Miniprep of PSAD and Rb (5 clones of each) <br>
Kit used: Nucleopsin Plasmid 
</p> </div>  
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<!-- ----------------------------------- Monday 6th August------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="a6"> Monday 6th August</h2>
             
               <h4> Digestion of the 5 new clones of pSAD and Rb  </h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
   <div class="d-flex justify-content-end"><div class="chip-icone"> <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Aurélie
</div>
  </div> </div>
<div class="d-flex justify-content-center">
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    <table class="text-center tablenotebook">
       <thead>    
  <tr>
   <th class="th-notebook"></th>
   <th class="th-notebook">1 reaction </th>
    <th class="th-notebook">mix *12</th>
 
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">BSAI Enzyme </td>
   <td class="td-notebook">0.2 µL </td> 
    <td class="td-notebook">2.4 µL</td>  
  </tr>
   <tr>
   <td class="td-notebook">Buffer Cut Smart (10X) </td>
   <td class="td-notebook">1  µL </td> 
    <td class="td-notebook">12µL </td>  
  </tr>
    <tr>
   <td class="td-notebook">DNA  </td>
   <td class="td-notebook">4  µL </td> 
    <td class="td-notebook">↗ </td>  
  </tr>
     <tr>
   <td class="td-notebook">H2O mq</td>
   <td class="td-notebook">4.8 µL  </td> 
    <td class="td-notebook">57.6µL </td>  
  </tr>   
  </tbody> 
</table>
</div>
</div>
<p class="text-notebook">6µl of mix per tube + 4µl of DNA <br>
Digestion 2H at 37°C+ 2µL of loading buffer 6X, 4µL of 2log ladder <br>
Migration 90V, 40min <br>
</p>
    <!-- --------------------------------------------FIGURE CAPTION A--------------------------------------------- -->
 <a  class="pointer" title="Click on the image for a larger view " data-toggle="modal" data-target=".Migration-180806"><div class="container-figurecaption">
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  <div class="content-figurecaption">
    <p><span style="font-weight: bold;">Figure : </span>Gel electrophoresis of digestion reaction products to check the inserts size : 5 new clones of pSAD and Rb </p>
  </div>
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<!-- ----------------------------------- Tuesday 7th August ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="a7"> Tuesday 7th August</h2>
             
               <h4> Digestion/ ligation reaction of pSAD 2 Rb intron <br> Transformation <br> Preparation X-GAL </h4>
   <div class="d-flex justify-content-end">         <div class="chip-icone"> <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Aurélie
</div>
  </div> </div>
<h5>1. Digestion/ ligation reaction of pSAD 2 Rb intron </h5>
<p class="text-notebook"> <a href="#jun14">see 06/14/2018 protocol</a></p>
<h5>2. Transformation  </h5>
<p class="text-notebook">-90µl of DH5α +Ligation product +10µl CMR <br>
           30 min 4°C <br>
           90 sec at 42°C <br>
           2 min on ice <br>
700µL LB <br>
            1h at 37°C<br>
                  spreading on 2 dishes <br>
</p>
<h5> 3. Preparation X-GAL </h5>
<p class="text-notebook">40mg ⇨ 2mL DMSO</p>
 </div>  
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<!-- ----------------------------------- Tuesday 21th August ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="a21"> Tuesday 21th August</h2>
            
               <h4> New PCR of “pSAD” and “rubisco” intron  </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
    <div class="d-flex justify-content-end">         <div class="chip-icone"> <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Aurélie
</div>
  <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Ursula
</div> </div> </div>
<p class="text-notebook">Reaction Mix</p>
<div class="d-flex justify-content-center">
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       <thead>    
  <tr>
   <th class="th-notebook">Solutions</th>
   <th class="th-notebook">Volume for 1 tube (uL) </th>
    <th class="th-notebook">Volume for 7 tubes (uL)</th>
 
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">Plasmid DNA</td>
   <td class="td-notebook">1</td> 
    <td class="td-notebook">-</td>  
  </tr>
   <tr>
   <td class="td-notebook">Phusion Enzyme</td>
   <td class="td-notebook">0.25 </td> 
    <td class="td-notebook">1.75 </td>  
  </tr>
    <tr>
   <td class="td-notebook">HF Buffer (5X) </td>
   <td class="td-notebook">5 </td> 
    <td class="td-notebook">35 </td>  
  </tr>
     <tr>
   <td class="td-notebook">dNTP</td>
   <td class="td-notebook">0.5  </td> 
    <td class="td-notebook">3.5</td>  
  </tr> 
      <tr>
   <td class="td-notebook">Forward Primer</td>
   <td class="td-notebook">1.25  </td> 
    <td class="td-notebook">- </td>  
  </tr>
       <tr>
   <td class="td-notebook">Reverse Primer</td>
   <td class="td-notebook">1.25  </td> 
    <td class="td-notebook">- </td>  
  </tr>
      <tr>
   <td class="td-notebook">H2O</td>
   <td class="td-notebook">15.75 µL  </td> 
    <td class="td-notebook">110.25</td>  
  </tr>    
  </tbody> 
</table>
</div>
</div>

<p class="text-notebook">- 21.5 uL of Mix/Tube <br>
- 1 uL of DNA <br>
- 2 X 1.25 uL Primer <br><br>

pSAD = Ig3/Ig4 primers + template p0.07 → 807 bp <br>
Rb intron = Ig5/Ig6 primers + template 0.07 → 177 bp <br><br>

1- p007 + Ig3/Ig4 <br>
2- pUC19 + Ig3/Ig4 <br>
3- H2O + Ig3/Ig4 <br>
4- p0-02 + Ig5/Ig6 <br>
5- pUC19 + Ig5/Ig6 <br>
6- H2O + Ig5/Ig6 <br>
</p> </div>  
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<!-- ----------------------------------- Tuesday 22th August ------------------------------------------------------- -->
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<h2 class="text-center" id="a22"> Tuesday 22th August</h2>
            
               <h4> Migration of the PCR products and the fragments of GAG Pol <br> Purification of the product the gel of the 2 PCRs: <br> Cloning of pSAD </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
    <div class="d-flex justify-content-end">         <div class="chip-icone"> <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Aurélie
</div>
  <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Ursula
</div> </div> </div>
<h5>1. Migration of the PCR products and the fragments of GAG Pol sent by IDT </h5>
<p class="text-notebook">Products of the PCR à OK <br>
IDT4 L0A1 + IDT4 L0A2 + IDT4 L0A3 + IDT3 L0B1 à OK <br><br>
 
No bands for IDT4 L0B2                	Were the samples mixed during the loading?  <br>
Plusieurs bands for IDT6 L0B3 <br>
</p>
<h5>2.Purification of the product the gel of the 2 PCRs </h5>
<p class="text-notebook">Elution in 20 uL <br>
Used kit: Nucleospin Gel and PCR clean up <br> <br>
Concentration measurements using the Nanodrop: <br>
- PSAD= 39.8 ng/uL<br>
- Rb= 55.1 ng/uL<br>
</p>
<h5>3. Cloning of pSAD </h5>
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<h5>Intron Rubisco </h5>
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<h5> Ligation </h5>
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<h5> Cycle</h5> 
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<h2 class="text-center" id="a28"> Tuesday 28th August</h2>
            
               <h4> Plasmidic extraction of bacteria transformed <br> Measure of the concentration  <br> Digestion of the plasmid by BstEII and SalI <br> Preparation of DNA samples for sequencing  </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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Soukeyna
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<h5> Plasmidic extraction of bacteria transformed by plasmid containing “Rubisco intron”</h5>
<p class="text-notebook">Used kit : NucleoSpin Plasmid (Macherey-Nagel) </p>

<h5> Measure of the concentration at Nanodrop </h5>
<p class="text-notebook">Rb 1 : 109.9 ng/µl <br>
Rb 2 :  96.8 ng/µl <br>
Rb 3 :  77.2 ng/µl<br>
Rb 4 :  88.7 ng/µl<br>
</p> 

<h5> Measure of the concentration at Nanodrop </h5>
<p class="text-notebook">Digestion of the plasmid by BstEII and SalI <br>
Goal : Check if the plasmid effectively contain the insert “Rubisco intron” <br> <br>
Expected result : 
</p>

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    <p><span style="font-weight: bold;">Figure : </span>Gel electrophoresis of digestion reaction products to check the inserts size </p>
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<h5>Preparation of DNA samples for sequencing  </h5>
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<p class="text-notebook"> add 2 µl of primers at 10 µL for each tube  </p>
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<h5>Concentration measurement on Nanodrop of recieved parts for trehalose   </h5>
<p class="text-notebook"> Provider : DC biosciences  </p>
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   <td class="td-notebook">165.3 </td> 
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   <td class="td-notebook">103.9 </td>   
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<h2 class="text-center" id="a29"> Tuesday 29th August</h2>
            
               <h4>Sample preparation to send “rubisco intro” at sequencing </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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 Ursula
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<p class="text-notebook">Add 2µl of primers at 10µM</p>
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<!-- ----------------------------------- Tuesday 30th August ------------------------------------------------------- -->
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<h2 class="text-center" id="a30"> Tuesday 30th August</h2>
            
               <h4> Digestion/ligation reaction of otsA and otsB  <br> Transformation of B10 bacteria with ligation product <br> PlCH47811 plasmid extraction <br> Replanting of 𝝱10 clones transformed <br> Checking of DNA content of IDT4 sample for Gag Po </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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Soukeyna
</div> </div> </div>
<h5>1. Digestion/ligation reaction of otsA and otsB  </h5>
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<h5>2. Transformation of B10 bacteria with ligation product </h5>
<p class="text-notebook">- 25µl of B10 bacteria + 4µl of ligation product (4 different transformations) <br>
- 30 min at 4°C <br>
- 55 sec at 42°C <br>
- 2 min at 4°C <br>
+700µl of LB medium  <br>
1 h at 37°C  <br><br>
Spreading of 250 µl in plates containing LB+spectinomomycin+X-gal <br>
</p>

<h5>3. PlCH47811 plasmid extraction from overnight culture </h5>
<p class="text-notebook">kit used: Machinery Nagel easypure kit plasmid DNA extraction</p> 

<h5>4. PlCH47811 plasmid  DNA concentration measurement on Nanodrop </h5>
<p class="text-notebook">a :  52.8 ng/µl    1.91    1.98 <br>
b  : 39.8 ng/µl    1.90    1.84
</p> 

<h5>5. Replanting of 𝝱10 clones transformed the day before on LB media + spectinomycine </h5>
<p class="text-notebook">2 clones of OTSA : OTSA1  OTSA2 (one culture per clone)<br>
2 clones of OTSB : OTSB1  OTSB2 (one culture per clone)<br>
1 clone of  pGAM 1276<br>
1 clone of  pAGM1299<br> <br>

the 6 cultures previously described are placed on a will for over week/end culture

</p>
<h5>6. Checking of DNA content of IDT4 sample for Gag Pol</h5>
The tube is vortexed upside down then centrifuged (to check if the DNA is not stuck on the tube’s lid) <br>
DNA concentration measurement on nanodrop: <br>
(blank : H2O)<br>
1.5ng/µL<br>
1.6ng/µL<br>
1.5ng/µl<br>

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<!-- ----------------------------------------------------------------September Month--------------------------------------  -->
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<div class="d-flex justify-content-center"><h2 class="text-center" style="background: #e6e6e6; width: 300px; border-radius: 10px; border-bottom: none;"> September</h2></div>
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<h2 class="text-center" id="s3"> Monday 3th September</h2>
            
               <h4>Suspension of primers IDT-4L0-B2-F and IDT-4L0-B2-R <br> Miniprep of otsA, otsB and pAGM1276 and pAGM1299 <br>Concentration measurement <br> Level 0 ligation protocol for GAGPOL parts and Level 1 ligation protocol for the CARGO retrotransposon</h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
          <div class="d-flex justify-content-end">   <div class="chip-icone"> <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Aurélie
</div>
  </div> </div>
<h5>1. Suspension of primers IDT-4L0-B2-F and IDT-4L0-B2-R received today according to Eurofins instructions</h5>
<p class="text-notebook">Then, hybridation during 15 minutes (there are in fact, the missing DNA fragment of GAGPOL)
.</p>
<h5>2. Miniprep on the over week-end culture of otsA, otsB and pAGM1276 and pAGM1299</h5>
<p class="text-notebook">Used kit : NucleoSpin Plasmid (Macherey-Nagel)</p>
<h5>3. Concentration measurement at Nanodrop</h5>
<p class="text-notebook">- pAGM1276 : 56ng/µl <br>
- pAGM1299 : 76.6ng/µl <br>
- otsA 1 : 78.4ng/µl <br>
- otsA 2 : 75.6ng/µl <br>
- otsB 1 : 73.1ng/µL<br>
- otsB2 : 78.2ng/µl <br>
</p>
<h5>4. Level 0 ligation protocol for GAGPOL parts : <a href="jul9">(see 09/07/2018)</a></h5>
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<h5>5. Level 1 ligation protocol for the CARGO retrotransposon </h5>

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<!-- -----------------------------------Tuesday 4th September ------------------------------------------------------- -->
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<h2 class="text-center" id="s4"> Tuesday 4th September</h2>
            
               <h4>Digestion of otsA and otsB ligation reaction <br> Transformation of B10 bacteria with cloning reaction  </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
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Soukeyna
</div>
  </div> </div>
<h5>1. Digestion of otsA and otsB ligation reaction of <a href="#a3">03/08/2018</a></h5>
<p class="text-notebook">Digestion reaction of otsA : <br> <br>

- bstEII-HF : 0.2µl <br> 
- CutSmart buffer (10X) : 1 µl<br> 
- DNA plasmid : 3µl <br> 
- H20: 5.8µl <br> 
</p>
<p class="text-notebook">Digestion reaction of otsB : <br> <br>

- bstEII-HF : 0.1µl <br>
- NotI-HF : 0.1µl <br>
- CutSmart buffer (10X) : 1µl <br>
- DNA plasmid : 3µl  <br>
- H20 : 5.8µl <br> <br>
Electrophoresis on 0.1% agarose, with 2 log ladder and 12µl of reaction
</p>

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    <p><span style="font-weight: bold;">Figure : </span>Gel electrophoresis of digestion reaction products to check the inserts size </p>
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<p class="text-notebook">The band on the gel are correct but there is a lot od DNA on the top of the gel and the secnd band is very weak. Morover, the sequence we ordered for otsB is wrong, so we need to correct it by PCR. So we will ordered primers and we’ll send to sequence after the PCR. For otsA, we’ll make an other transformation to select only one pure clone
</p>

<h5>2. Transformation of B10 bacteria with cloning reaction of <a href="#s3">03/09 </a>: p1-iGEM1 and p0-iGEM5 </h5>
<p class="text-notebook">25µl of B10 bacteria + 4µl of ligation product (4 different transformations) <br>
30 min at 4°C <br>
55 sec at 42°C <br>
2 min at 4°C <br>
+700µl of LB medium <br>
1 h at 37°C <br> <br>

Spreading of 250 µl in plates containing LB+spectinomomycin+X-gal for p0-iGEM5 <br>
Spreading of 250 µl in plates containing LB+ampicilin+X-gal for p1-iGEM2<br>
</p> </div>  
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<!-- -----------------------------------Wednesday 5th September ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="s5"> Wednesday 5th September</h2>
            
               <h4>Transformation of B10 bacteria with cloning reaction p1-iGEM1 and p0-iGEM5<br> Replanting of white colonies from p0-iGEM-GAGPO <br> Re-do of L1 ligation  of p1-iGEM2 (Cargo) </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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Soukeyna
</div>
  </div> </div>
<h5>1. Transformation of B10 bacteria with cloning reaction of <a href="#s3">03/09 </a>: p1-iGEM1 and p0-iGEM5 </h5>
<p class="text-notebook">Observation of plates from the day before <br>
- p1-iGEM2 (Cargo) : only blue colonies for + and - controle <br>
- p0-iGEM5- GagPol: only blue colonies for the positive (+) experiment , and both white and blue colonies for the negative (-)  control
</p>
<h5>2. Replanting of white colonies from p0-iGEM-GAGPOL (-)on liquid media </h5>
<p class="text-notebook">Composition of the media :Amp+ Spec+ Xgal <br>
4 colonies were replanted 
</p> 
<h5>Re-do of L1 ligation  of p1-iGEM2 (Cargo) <a href="#s3"> (same experiment as the 09/03/2019)</a> </h5> </div>  
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<!-- -----------------------------------Tuesday 6th September ------------------------------------------------------- -->
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<h2 class="text-center" id="s6"> Tuesday 6th September</h2>
            
               <h4> PDNA extraction of 4 differents clones p0-iGEM5 <br> Concentration measurement <br> Verification digestion of p0-iGEM5 plasmid <br> Transformation of bacteria with the ligation produc </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
</div>
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  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Soukeyna
</div>
  </div> </div>
<h5>1. DNA extraction of 4 differents clones p0-iGEM5  </h5>
<p class="text-notebook">Kit utilisé : NucleoSpin Plasmid (Macherey-Nagel)</p>

<h5>2. Concentration measurement at Nanodrop  </h5>
<p class="text-notebook">p0-iGEM5-1 : 97.8ng/µl <br>
p0-iGEM5-2 : 89.5ng/µl <br>
p0-iGEM5-3 : 123.4ng/µl <br>
p0-iGEM5-4 : 67.6ng/µL <br>
</p>


<h5>3.Verification digestion of p0-iGEM5 plasmid </h5>
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       <thead>    
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   <th class="th-notebook"></th>
   <th class="th-notebook">Clone 1 and 3</th>
    <th class="th-notebook">Clone 2 and 4</th>  
 
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">bstEII enzyme</td>
   <td class="td-notebook">0.2µl </td> 
   <td class="td-notebook">0.2µl </td>   
  </tr>
   <tr>
   <td class="td-notebook">Plasmid DNA</td>
   <td class="td-notebook">2µl </td>
   <td class="td-notebook">3µ </td>    
  </tr>
    <tr>
   <td class="td-notebook">NEB cutsmart buffer (10X) </td>
   <td class="td-notebook">1µl </td>  
    <td class="td-notebook">1µ </td>    
  </tr>
     <tr>
   <td class="td-notebook">H20</td>
   <td class="td-notebook">6.8µl </td> 
   <td class="td-notebook">5.8µl </td>      
  </tr>     
  </tbody> 
</table>
</div>
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  <div class="content-figurecaption">
    <p><span style="font-weight: bold;">Figure : </span>Gel electrophoresis of digestion reaction products to check the inserts size </p>
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<h5>4. Transformation of bacteria with the ligation product of the 05/09 </h5>
<p class="text-notebook">25µl of bacteria + 3µl of ligation product <br>
30min at 4°C <br>
55sec at 42°C <br>
2min at 4°C <br>
+700µl of LB medium on each tube <br>
1h at 37°C <br>
Spreading of 200µl of each transformation on LB + Xgal + amp + IPTG plate<br>

Oovernight at 37°C

</p>
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<!-- -----------------------------------Friday 7th September ------------------------------------------------------- -->
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<h2 class="text-center" id="s7"> Friday 7th September</h2>
            
               <h4> Ligation reaction of p0-iGEM5 (GAGPOL) <br> otsB PCR </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
</div>
  </div> </div>
<h5>1. Ligation reaction of p0-iGEM5 (GAGPOL)</h5>
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<h5>2. otsB PCR </h5>
<p class="text-notebook">- p0-iGEM8 (otsB) : 1µl at 2ng/µl <br>
- Enzyme Phusion : 0.25µl <br> 
- HF Buffer (5X) : 5 µl <br>
- dNTP : 0.5µl  <br>
- Primer OTSB-v2-F : 1.25 µl <br>
- Primer OTSB-v2-R : 1.25µl <br>
-H20 : 15.75 µl <br>
</p>
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   <th class="th-notebook" colspan="3">*30</th> 
      <th class="th-notebook"></th> 
      <th class="th-notebook"></th> 
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">98°C</td>
   <td class="td-notebook">98°C </td> 
   <td class="td-notebook">50°C </td>
   <td class="td-notebook">72°C</td>
   <td class="td-notebook">72°C </td> 
   <td class="td-notebook">12°C</td>   
  </tr>
   <tr>
   <td class="td-notebook">1min</td>
   <td class="td-notebook">1 min </td>
   <td class="td-notebook">2 min </td>
    <td class="td-notebook">3 min</td>
   <td class="td-notebook">3 min </td>
   <td class="td-notebook">Hold </td>    
  </tr>     
  </tbody> 
</table>
</div>
</div>
<h5>3. 6 colonies <a href="s6">from yesterday</a> transformation p1igem2 in LB medium + amp </h5>

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<!-- -----------------------------------.\ One day of the notebook ------------------------------------------------------- -->
<!-- -----------------------------------Monday 10th September ------------------------------------------------------- -->
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<h2 class="text-center" id="s10"> Monday 10th September</h2>
            
               <h4>DNA extraction of the 6 bacteria culture <br> Concentrations measurement <br> Digestion reaction to check p1-iGEM2 cloning reaction <br> Transformation of B10 bacteria  </h4>
<h6>Institut de biologie physico-chimique (IBPC) </h6>
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 Aurélie
</div>
  </div> </div>
<h5>1. DNA extraction of the 6 bacteria culture prepare on <a href="#s7">7/09</a></h5>
<p class="text-notebook">Used kit : NucleoSpin Plasmid (Macherey-Nagel)</p>
<h5>2. Concentrations measurement at Nanodrop </h5>
<p class="text-notebook">#1 : 101.5ng/µl <br>
#2 : 90.0ng/µl <br>
#3 : 97.7ng/µL <br>
#4 : 75.1ng/µl <br>
#5 : 61.5ng/µl <br>
#6 : 115.6ng/µl <br>
</p> 

<h5>3. Digestion reaction to check p1-iGEM2 cloning reaction </h5> 
<div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="text-center tablenotebook">
       <thead>    
  <tr>
   <th class="th-notebook">NcoI</th>
   <th class="th-notebook">0.1µl</th> 
      <th class="th-notebook">6.5µl</th> 
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">NdeI</td>
   <td class="td-notebook">0.1µl </td> 
   <td class="td-notebook">6.5µL </td>   
  </tr>
   <tr>
   <td class="td-notebook">NotI</td>
   <td class="td-notebook">0.1µl</td>
   <td class="td-notebook">6.5µl </td>   
  </tr>
        <tr>
   <td class="td-notebook">Cutsmart buffer 10X</td>
   <td class="td-notebook">1µl</td>
   <td class="td-notebook">6.5µl </td>   
  </tr>
        <tr>
   <td class="td-notebook">ADN</td>
   <td class="td-notebook">2.5µl</td>
   <td class="td-notebook"></td>   
  </tr>
        <tr>
   <td class="td-notebook">H2O</td>
   <td class="td-notebook">6.2µl</td>
   <td class="td-notebook">40.3µl </td>   
  </tr>
  </tbody> 
</table>
</div>
</div>
<p class="text-notebook">Used kit : NucleoSpin Plasmid (Macherey-Nagel)</p>
<h5>Concentrations measurement at Nanodrop </h5>
<p class="text-notebook">7.5µl of mix by tube + 2.5µl of DNA <br><br>
On 1% agarose gel, with otsB PCR from <a href="#s7">7/09</a> : expected result : >800bp
</p> 
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<h5> 4. Transformation of B10 bacteria with ligation product from <a href="#s7">07/09</a> </h5>
<p class="text-notebook">B10 bacteria + 3.5µl of ligation product <br>
- 30 min at 4°C <br>
- 55sec at 42°C <br>
- 2min at 4°C <br>
- +700µl of LB medium <br>
- 1h at 37°C <br>
- Spreading of 200µl on plate with LB+spec+Xgal  <br>
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<!-- -----------------------------------.\ One day of the notebook ------------------------------------------------------- -->
<!-- -----------------------------------Tuesday 11th September ------------------------------------------------------- -->
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        <div class="note"> 
<h2 class="text-center" id="s11"> Tuesday 11th September</h2>
            
               <h4>Digestion of p1-IGEM2 with bbsI <br> Transformation of bacteria with Gagpol </h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
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Sara
</div>
  </div> </div>
<h5>1. Digestion of p1-IGEM2 with bbsI</h5>
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       <thead>    
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   <th class="th-notebook">Components</th>
   <th class="th-notebook">1 reaction</th> 
      <th class="th-notebook">Mix (*6)</th> 
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">bbsI</td>
   <td class="td-notebook">0.5µL </td> 
   <td class="td-notebook">3 µL</td>   
  </tr>
   <tr>
   <td class="td-notebook">Cutsmart buffer (10X)</td>
   <td class="td-notebook">1 µL</td>
   <td class="td-notebook">6 µL </td>   
  </tr>
        <tr>
   <td class="td-notebook">DNA</td>
   <td class="td-notebook">2.5 µL</td>
   <td class="td-notebook">6.5µl </td>   
  </tr>
        <tr>
   <td class="td-notebook">H2O</td>
   <td class="td-notebook">6 µL</td>
   <td class="td-notebook">36µL</td>   
  </tr>
  </tbody> 
</table>
</div>
</div>


<h5>2. Transformation of bacteria with Gagpol </h5>
<p class="text-notebook">2 white  colonies were observed on the plate containing bacterias transformed with GAGPOL <br>
- Replanting on liquid media <br>
- Replanting on plate (not isolable) <br>
</p> 
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<!-- -----------------------------------Wednesday 12th September ------------------------------------------------------- -->
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<h2 class="text-center" id="s12"> Wednesday 12th September</h2>
            
               <h4>Migration of the digestion products from the day before (pI-IGEM2)
 <br> Plasmid extraction from the GAG clone culture <br> Purification of DNA contained in the migration gel for OtsB PCR products <br> Digestion reaction for Gag POL <br> Level 0 Otsb (digestion/ligation reaction)  </h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
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  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Sara
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Victor
</div>
  </div> </div>
<h5>1. Migration of the digestion products from the day before (pI-IGEM2)</h5>
<p class="text-notebook"> (0.1% agarose gel, 2-log ladder, 100V)<br>
We were expecting a 4.5kb band for the plasmid and a 2.6kb band for the insert. <br>
We do have the expected band for the plasmid but, we observe a band at 0.6kb for the insert (Lac Z maybe?)</p> 

<h5>2. Plasmid extraction from the GAG clone culture launched the day before </h5>
<p class="text-notebook">Kit used Nucleospin Plasmid </p> 

<h5>3. Purification of DNA contained in the migration gel for OtsB PCR products <a href="#s7">( from 09/07/2018)</a>
 </h5>
<p class="text-notebook">Kit used : Nucleospin gel to PCR clean up </p> 

<h5>4. DNA concentration measurement on Nanodrop </h5>
<p class="text-notebook">Insert OtsB= 4ng/µL <br>
GAG palamsid = 48.1 ng/µl
 </p>

<h5>5. DNA concentration measurement on Nanodrop </h5>
<p class="text-notebook">Insert OtsB= 4ng/µL <br>
GAG palamsid = 48.1 ng/µl
 </p>

<h5>6. Level 0 Otsb (digestion/ligation reaction) </h5>
<p class="text-notebook">→ purification of PCR band and Launch of miniprep <br>
(To do next level 1 pI-Igem2)</p>
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<!-- -----------------------------------Tuesday 13th September ------------------------------------------------------- -->
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<h2 class="text-center" id="s13"> Tuesday 13th September</h2>
            
               <h4>Transformation of 𝛃10 bacterias with OtsB ligation products <br> DNA plamsid extraction of pO-IGEM5 <br> Digestion reaction  </h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
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 Sara
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Victor
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  </div> </div>
<h5>1. Transformation of 𝛃10 bacterias with OtsB ligation products obtained the day before </h5>
<p class="text-notebook"> 𝛃10+ 4µL of  OtsB ligation reaction products <br>
- 30min at 4°C <br>
- 55 sec at 42°C <br>
- 2 min at 4°C <br>
- Addition of 70µL of LB <br> <br>
1 Hour at 37°C <br>
Spreading of 200µL on LB+Xgal + Spec plate <br>
37°C O/N
</p> 

<h5>2. DNA plamsid extraction of pO-IGEM5 launch on liquid media yesterday
 </h5>
<p class="text-notebook">Kit used Nucleospin Plasmid </p> 

<h5>3. Nanodrop measurement for p0-IGEM5 </h5>
<p class="text-notebook">26.4 ng/µL </p> 

<h5>4.Digestion reaction </h5>
<p class="text-notebook">bbsI =0.5µL <br>
buffer= 2µL<br>
DNA =10µL <br>
H2O= 6.5µL <br>
Expected result: 4,4kb + (?)</p>

<h5>5. Gel migration of digestion reaction products </h5>
<p class="text-notebook">ast well = digestion reaction product of GagPOL from the day before 
→ Does not correspond to the expected lenght </p>

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<!-- -----------------------------------Monday 17th September ------------------------------------------------------- -->
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<h2 class="text-center" id="s17"> Monday 17th September</h2>
            
               <h4>Ligation <br> Migration of  the Gag Pol sample <br> Preculture of OTSB (7 white colonies)
  </h4>
<h6> Muséum national d'histoire naturelle (MNHN) </h6>
          <div class="d-flex justify-content-end">   <div class="chip-icone"> <div class="chip">
  <img src="https://static.igem.org/mediawiki/2018/1/1d/T--Sorbonne_U_Paris--Claude_avatar.png" alt="Person" width="96" height="96">
 Sara
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Victor
</div>
  </div> </div>
<h5>1. Ligation </h5>
<p class="text-notebook">3 ligation tubes are prepared , T-, G, pre-mix of E, T- <br>
- Tube 1 : T-  1.2µl of blackbone +12.8 µL of H2O (14µL in total) <br>
- Tube 2 : G  2 µL of the 6 sub-parts for GagPol)+.0.8µl of H2O +1.2µL of backbone ( 14µl in total)
Tube 3: Master mix of enzymes 
</p> 
<div class="d-flex justify-content-center">
<div class="table-responsive-sm">
    <table class="text-center tablenotebook">
       <thead>    
  <tr>
   <th class="th-notebook">Components</th>
   <th class="th-notebook">1 reaction</th> 
  </tr>
</thead>    
  <tbody>      
  <tr>
   <td class="td-notebook">ATP</td>
   <td class="td-notebook">4 µL </td>   
  </tr>
   <tr>
   <td class="td-notebook">Cutsmart buffer (10X)</td>
   <td class="td-notebook">4 µL</td>  
  </tr>
        <tr>
   <td class="td-notebook">T4 ligase</td>
   <td class="td-notebook">2 µL</td>  
  </tr>
        <tr>
   <td class="td-notebook">bbstI</td>
   <td class="td-notebook">2 µL</td>   
  </tr>
  </tbody> 
</table>
</div>
</div>
<p class="text-notebook"> 12µL of mix in total dispatched in tubes 1 and 2 (6µL each)</p>

<h5>2. 1% agarose gel preparation, in order to migrate the Gag Pol sample  </h5>
<p class="text-notebook">- 0.5g of agarose <br>
- 50ml of TEA 1X <br>
- Cyber green <br>

Gel plan:  well 1 : 3µl of DNA ladder, well 2 and 3 10µl of Gagpol
 </p> 

<h5>Preculture of OTSB (7 white colonies) </h5>
<p class="text-notebook">For each : <br>
- 4ml of LB media <br>
- 2µl of spectinomycin (100mg/ml) (in order to obtain 10µg/ml) <br> 
-  1 white clone  <br>
Incubation at 37°C overnight

 </p> 

<h5>4. To do next</h5>
<p class="text-notebook">
- Otsb miniprep + digestion <br>
- Gag digestion <br>
- Gag transfo)
</p>



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