Difference between revisions of "Team:Pasteur Paris/essai"

Line 8: Line 8:
 
   
 
   
 
}
 
}
−
h1{
+
html, body {
−
        font-family: 'Oswald', Arial, sans-serif;
+
  margin: 0;
−
padding:50px 30px 30px 30px;
+
  padding: 0;
−
font-size:42px;
+
  font-family: Helvetica, sans-serif;
−
color:#333;
+
−
text-align:left;
+
−
        text-align:center;
+
 
}
 
}
−
h2 {
+
body {
−
        display:block;
+
    background: #fafafa;
−
font-size: 27px;
+
 
−
        font-family: 'Oswald', Arial, sans-serif;
+
−
color:color:#333;
+
−
padding-top:20px;
+
 
}
 
}
 +
section#timeline {
 +
  width: 70%;
 +
  margin: 20px auto;
 +
  position: relative;
  
−
 
+
}
−
 
+
section#timeline:before {
−
div.lightbox {
+
  content: '';
−
   display: none;
+
   display: block;
−
   position: fixed;
+
   position: absolute;
−
   left: 0;
+
   left: 50%;
 
   top: 0;
 
   top: 0;
−
   width: 100%;
+
  margin: 0 0 0 -1px;
 +
   width: 2px;
 
   height: 100%;
 
   height: 100%;
 +
  background: #F0D423 ;
 
}
 
}
−
 
+
section#timeline article {
−
/*afficher le contenu des titres*/
+
−
div.lightbox:target {
+
−
  display: table;
+
−
    z-index:4001;
+
−
}
+
−
 
+
−
/*alignement du contenu pour tout navigateur*/
+
−
div.lightbox figure {
+
−
  display: table-cell;
+
−
  margin: 0;
+
−
  padding: 0;
+
 
   width: 100%;
 
   width: 100%;
−
   height: 100%;
+
   margin: 0 0 20px 0;
−
   vertical-align: middle;
+
   position: relative;
 
}
 
}
−
 
+
section#timeline article:after {
−
/*arrière plan du contenu bleu claire font plus foncé*/
+
  content: '';
−
div.lightbox figure figcaption {
+
 
   display: block;
 
   display: block;
−
   z-index:4000;
+
   clear: both;
−
  margin: auto;
+
−
  padding: 8px;
+
−
  background-color: #e4f2f3 ;
+
−
  height: 500px;
+
−
  position: relative;
+
−
  overflow: auto;
+
−
  border: 1px #000000 solid;
+
−
  border-radius: 10px;
+
−
  text-align: justify;
+
−
  font-size: 14px;
+
 
}
 
}
−
 
+
section#timeline article div.inner {
−
 
+
  width: 40%;
−
div.lightbox figure .closemsg {
+
  float: left;
 +
  margin: 5px 0 0 0;
 +
  border-radius: 6px;
 +
}
 +
section#timeline article div.inner span.date {
 
   display: block;
 
   display: block;
−
   margin: auto;
+
   width: 60px;
−
   height: 0;
+
   height: 50px;
−
   overflow: visible;
+
  padding: 5px 0;
−
   text-align: right;
+
   position: absolute;
−
 
+
  top: 0;
−
   cursor: default;
+
  left: 50%;
 +
  margin: 0 0 0 -32px;
 +
  border-radius: 100%;
 +
  font-size: 12px;
 +
  font-weight: 900;
 +
   text-transform: uppercase;
 +
  background: #25303B;
 +
   color: rgba(255,255,255,0.5);
 +
  border: 2px solid rgba(255,255,255,0.2);
 +
  box-shadow: 0 0 0 7px #25303B;
 
}
 
}
−
 
+
section#timeline article div.inner span.date span {
−
div.lightbox figure .closemsg, div.lightbox figure figcaption {
+
  display: block;
−
   width: 70%;
+
  text-align: center;
−
 
+
}
 +
section#timeline article div.inner span.date span.day {
 +
   font-size: 10px;
 
}
 
}
−
 
+
section#timeline article div.inner span.date span.month {
−
/*croix pour fermer*/
+
  font-size: 18px;
−
.closemsg::after {
+
}
−
   content: "\00D7";
+
section#timeline article div.inner span.date span.year {
−
   display: inline-block;
+
  font-size: 10px;
 +
}
 +
section#timeline article div.inner h2 {
 +
   padding: 15px;
 +
   margin: 0;
 +
  color: #fff;
 +
  font-size: 20px;
 +
  text-transform: uppercase;
 +
  letter-spacing: -1px;
 +
  border-radius: 6px 6px 0 0;
 
   position: relative;
 
   position: relative;
−
  right: -20px;
+
}
−
   top: -10px;
+
section#timeline article div.inner h2:after {
−
 
+
   content: '';
−
   color: ##e4f2f3;
+
   position: absolute;
−
   border: 1px #ffffff solid;
+
   top: 20px;
−
   border-radius: 10px;
+
   right: -5px;
−
  width: 20px;
+
  width: 10px;  
−
  height: 20px;
+
  height: 10px;
−
   line-height: 18px;
+
   -webkit-transform: rotate(-45deg);
−
   text-align: center;
+
}
 +
section#timeline article div.inner p {
 +
   padding: 15px;
 
   margin: 0;
 
   margin: 0;
−
   background-color: #e4f2f3;
+
   font-size: 14px;
−
   font-weight: bold;
+
  background: #fff;
−
   cursor: pointer;
+
  color: #656565;
 +
   border-radius: 0 0 6px 6px;
 +
}
 +
section#timeline article:nth-child(2n+2) div.inner {
 +
   float: right;
 +
}
 +
section#timeline article:nth-child(2n+2) div.inner h2:after {
 +
  left: -5px;
 +
}
 +
section#timeline article:nth-child(1) div.inner h2 {
 +
  background: #17A3B5;
 +
}
 +
section#timeline article:nth-child(1) div.inner h2:after {
 +
  background: #17A3B5 ;
 +
}
 +
section#timeline article:nth-child(2) div.inner h2 {
 +
  background: #EDA194 ;
 +
}
 +
section#timeline article:nth-child(2) div.inner h2:after {
 +
  background: #EDA194 ;
 +
}
 +
section#timeline article:nth-child(3) div.inner h2 {
 +
  background: #DF5939 ;
 +
}
 +
section#timeline article:nth-child(3) div.inner h2:after {
 +
  background: #DF5939 ;
 +
}
 +
section#timeline article:nth-child(4) div.inner h2 {
 +
  background: #A8D5D8 ;
 +
}
 +
section#timeline article:nth-child(4) div.inner h2:after {
 +
  background: #A8D5D8 ;
 +
}
 +
section#timeline article:nth-child(5) div.inner h2 {
 +
  background: #efb521 ;
 +
}
 +
section#timeline article:nth-child(5) div.inner h2:after {
 +
  background:#efb521 ;
 
}
 
}
−
 
  
  
Line 111: Line 151:
  
  
−
 
+
<section id="timeline">
−
 
+
  <article>
−
<div id="week1">
+
     <div class="inner">
−
<p><h2><B>June 6, 2016:</B></h2></p>
+
      <span class="date">
−
     <p>
+
        <span class="day">30<sup>th</sup></span>
−
        <a href="#exp1">Transformation of pSB1C3 and pET43.1a(+)</a></br>  
+
        <span class="month">Jan</span>
−
 
+
        <span class="year">2014</span>
−
<p><h2><B>June 7, 2016: </B></h2></p>
+
      </span>
−
    <p>
+
      <h2>The Title</h2>
−
        <a href="#exp2">Harvest the culture with Midiprep</a></br>  
+
      <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean quis rutrum nunc, eget dictum massa. Nam faucibus felis nec augue adipiscing, eget commodo libero mattis.</p>
−
    </p>
+
    </div>
−
 
+
   </article>
−
<p><h2><B>June 8, 2016: </B></h2></p>
+
   <article>
−
    <p>
+
    <div class="inner">
−
      <a href="#exp3">Extraction of plasmid DNA</br>
+
      <span class="date">
−
    </p>
+
        <span class="day">26<sup>th</sup></span>
−
 
+
        <span class="month">Jan</span>
−
<p><h2><B>June 9, 2016: </B></h2></p>
+
        <span class="year">2014</span>
−
      <p>
+
      </span>
−
          <a href="#exp4">Digestion of the plasmid pSB1C3 and pET43.1a(+)</a></br>  
+
      <h2>The Title</h2>
−
    </p>
+
      <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean quis rutrum nunc, eget dictum massa. Nam faucibus felis nec augue adipiscing, eget commodo libero mattis.</p>
−
 
+
    </div>
−
<div class="lightbox" id="exp1">
+
   </article>
−
  <figure>
+
  <article>
−
    <a href="#" class="closemsg"></a>
+
    <div class="inner">
−
    <figcaption> <p><U> Aim:</U> To increase the amount of plasmid by transformation in competent cells. </br>
+
      <span class="date">
−
The amount of plasmid supplied is insufficient to perform all our future experiments. Therefore, we need to amplify the amount of plasmids. </br></br>
+
        <span class="day">26<sup>th</sup></span>
−
 
+
        <span class="month">Jan</span>
−
<U> Protocol:</U> follow in this link
+
        <span class="year">2014</span>
−
</br></br>
+
      </span>
−
<U>What we did in the lab:</U>
+
      <h2>The Title</h2>
−
</br>
+
      <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean quis rutrum nunc, eget dictum massa. Nam faucibus felis nec augue adipiscing, eget commodo libero mattis.</p>
−
<U>Materials:</U>
+
    </div>
−
</br>
+
   </article>
−
&bull;  subcloning competent cells</br>
+
  <article>
−
&bull; PSB1C3 plasmid (from shipped BioBrick-competent cells testing kit), chloramphenicol resistance</br>
+
    <div class="inner">
−
&bull; pET43.1a (GE health care), ampicillin resistance (or carbenicillin) </br>
+
      <span class="date">
−
&bull; SOC (Super optimal Broth) media</br>
+
        <span class="day">26<sup>th</sup></span>
−
&bull; LB (lysogeny Luria broth) Agar plates containing 50 g/ml carbenicillin or 34 g/ml chloramphenicol</br>
+
        <span class="month">Jan</span>
−
&bull; Microbiology equipment (type of incubator, Bunsen burner, water bath, etcfollow this link)
+
        <span class="year">2014</span>
−
</br></br>
+
       </span>
−
<U>Method:</U></br>
+
       <h2>The Title</h2>
−
1.Thaw cells from -80°C on ice, thaw plasmid at 37°C and store on ice, aliquot cells for 50 &#181;l/vial.</br>
+
       <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean quis rutrum nunc, eget dictum massa. Nam faucibus felis nec augue adipiscing, eget commodo libero mattis.</p>
−
2. Add 50 pg plasmids to each 50 &#181;L of competent cells vial and tap gently.</br>
+
     </div>
−
3. Place on ice for 30 min. Meanwhile, warm LB agar plates from cold room in 37°C non shaking incubator.</br>
+
   </article>
−
4. Place cells in 42°C water bath for exactly 40 seconds and then place immediately on ice for at least 3 min.</br>
+
   <article>
−
5. Add 500 &#181;L of SOC in each tube and place them to shaking incubator (incline tube for better shaking efficiency).</br>
+
     <div class="inner">
−
6. Grow for 40 minutes at 37°C in shaking incubator at 150 rpm.</br>
+
       <span class="date">
−
7. Near Bunsen burner flame, add 45 &#181;L of competent cells +135&#181;L   of SOC or 200 &#181;L aliquots of culture and streak plate with sterile rake on LB-agar plates containing the appropriate antibiotic.</br>
+
        <span class="day">26<sup>th</sup></span>
−
8. Place plates inverted in the static 37°C incubator overnight. </br>
+
        <span class="month">Jan</span>
−
</p>
+
        <span class="year">2014</span>
−
</figcaption>
+
       </span>
−
   </figure>
+
       <h2>The Title</h2>
−
</div>
+
       <p>Lorem ipsum dolor sit amet, consectetur adipiscing elit. Aenean quis rutrum nunc, eget dictum massa. Nam faucibus felis nec augue adipiscing, eget commodo libero mattis.</p>
−
 
+
    </div>
−
<div class="lightbox" id="exp2">
+
   </article>
−
  <figure>
+
</section>
−
    <a href="#" class="closemsg"></a>
+
−
    <figcaption><p>
+
−
<U> Aim:</U> To start a culture for Miniprep. In order to obtain a large amount of plasmid, we need to grow the bacteria overnight.
+
−
</br></br>
+
−
 
+
−
 
+
−
<U> Protocol:</U> follow in this link
+
−
</br></br>
+
−
 
+
−
<U>What we did in the lab:</U>
+
−
</br>
+
−
<U>Materials:</U>
+
−
&bull; Microbiology equipement </br>
+
−
&bull; 25 ml flasks<br>
+
−
&bull; Carbenicillin 50 mg/ml</br>
+
−
&bull; Chloramphenicol 34 mg/ml</br>
+
−
&bull; LB medium</br>
+
−
</br>
+
−
<U>Method:</U></br>
+
−
1. One colony is picked from the plates and shaken in 25 ml of LB supplemented with Carbenicillin or Chloramphenicol at 50 g/ml or 34 g/ml respectively. </br>
+
−
2. The flask is placed in a shaking incubator at 37°C, 150 rpm overnight. </br>
+
−
</p>
+
−
</figcaption>
+
−
   </figure>
+
−
</div>
+
−
 
+
−
<div class="lightbox" id="exp3">
+
−
  <figure>
+
−
    <a href="#" class="closemsg"></a>
+
−
    <figcaption><p>
+
−
<U> Aim:</U> To perform a midiprep to isolate plasmid DNA of pSB1C3 and pET43.1a. </br>
+
−
The amplification method to increase the amount of plasmid is called Mini or Midiprep.
+
−
 
+
−
</br></br>
+
−
 
+
−
 
+
−
<U> Protocol:</U> follow in this link
+
−
</br></br>
+
−
 
+
−
<U>What we did in the lab:</U>
+
−
</br>
+
−
<U>Materials:</U>
+
−
&bull; 50 ml Falcon tube</br>
+
−
&bull; Shaking incubator (INFORS HT)<br>
+
−
&bull; Carbenicillin 50 mg/ml</br>
+
−
&bull; Swing bucket centrifuge (JOUAN GR41)</br>
+
−
&bull; QIAGEN Midiprep kit 2016 (QiaFilter, Cat No.ID: 28704)</br>
+
−
</br>
+
−
 
+
−
<U>Method:</U></br>
+
−
The protocol in step 1 ask for spinning at 6000g but we can only achieve 3500 g so we used 3500 g for 20 minutes. We will follow most of the protocol of QIAGEN Midiprep 2016 except for a few modifications, which we describe, therefore, below. </br>
+
−
 
+
−
1. Use culture from overnight (17 hr) step on June 7, 2016 </br>
+
−
2. Pour culture in 50 ml Falcon nd centrifuge (15 min, 3500g, 4°C) </br>
+
−
3. Discard the supernatant (in biological waste) and add 4 ml of Buffer P1 (stored on ice) to the pellet </br>
+
−
4. Add 4 ml of Buffer P2 (for cell lysis) and mix by inverting the Falcon a few times. Wait 5 min at 22°C (room temperature: RT, EU). Note: The color of the solution will change to blue. </br>
+
−
5. Prepare syringes with their cap and the reservoir (on 50 ml Falcon) </br>
+
−
6. Add 4 ml of Buffer P3 (for neutralization) to the Falcon and mix by inverting the tube a few times. Note: The color of the solution changes to white. </br>
+
−
7. Pour the content of the Falcon in the syringes and let it sit for 10 min. In the meanwhile, equilibrate the provided columns with 4 ml of OBT (equilibration buffer) </br>
+
−
8. Transfer the contents from the syringe to the column and wash with 2 X 10 ml of QC buffer</br>
+
−
9. Prepare 10 tubes of 2 ml to aliquot pET43.1 and pSB1C3. </br>
+
−
Because we have only bench microfuges, we need to dispense our volume in smaller fractions. </br>
+
−
10. Elution of DNA with 5 ml of QF and aliquot in 2 ml tubes</br>
+
−
11. Centrifuge (30 min, 15000g, room temperature) </br>
+
−
12. Add 3.5 ml of isopropanol, mix to precipitate the DNA</br>
+
−
13. Centrifuge (30 min, 15 000g, at RT) </br>
+
−
14. Remove isopropanol with pipet without taking DNA and place into chemical waste container</br>
+
−
15. Add 1 ml of 70% ethanol, centrifuge again (15 min, 15 000g, RT) and let air dry</br>
+
−
16. Resuspend in 50 L of Tris 10 mM pH 8.0, EDTA, 1 mM (TE) and store at -20°C</br>
+
−
</p>
+
−
</figcaption>
+
−
   </figure>
+
−
</div>
+
−
 
+
−
<div class="lightbox" id="exp4">
+
−
  <figure>
+
−
    <a href="#" class="closemsg"></a>
+
−
    <figcaption><p>
+
−
<U> Aim:</U> To linearize the different plasmids with appropriate enzymes. </br>
+
−
We perform restriction enzyme digestion in order to recover linear backbones of the plasmids. We choose appropriate restriction sites based on the host plasmids.
+
−
 
+
−
</br></br>
+
−
 
+
−
 
+
−
<U> Protocol:</U> follow in this link
+
−
</br></br>
+
−
 
+
−
<U>What we did in the lab:</U>
+
−
</br>
+
−
<U>Materials:</U></br>
+
−
&bull; Restriction enzymes: XbaI, HindIII, SpeI, BamHI (New England Biolabs, NEB)</br>
+
−
&bull; Restriction enzyme buffers <br>
+
−
&bull; 37°C water bath</br>
+
−
&bull; UV spectrophotometer</br>
+
−
</br>
+
−
<U>Method:</U></br>
+
−
Measure the quantity of plasmid using a spectrophotometer (utrospec 3100 pro, Pharmacia GE health care)</br>
+
−
 
+
−
Results:
+
−
 
+
−
<table>
+
−
  <thead>
+
−
    <tr>
+
−
      <th>&#x3BB;= 260 nm</th>
+
−
      <th>pET43.1 a(+)</th>
+
−
      <th>pSB1C3</th>
+
−
    </tr>
+
−
  </thead>
+
−
  <tbody>
+
−
    <tr>
+
−
      <td><strong><p>A<sub>260</sub></p></strong></td>
+
−
      <td>0.008</td>
+
−
       <td>0.026</td>
+
−
    </tr>
+
−
    <tr>
+
−
       <td><strong>A<sub>280</sub></strong></td>
+
−
       <td>0.001</td>
+
−
      <td>0.014</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td><strong>A<sub>260</sub>/A<sub>280</sub></strong></td>
+
−
      <td>4</td>
+
−
      <td>1.85</td>
+
−
     </tr>
+
−
    <tr>
+
−
      <td><strong>C diluted</strong></td>
+
−
      <td>0.4 ng/&#181;l</td>
+
−
      <td>1.9 ng/&#181;l</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td><strong>C final</strong></td>
+
−
      <td>26.7 ng/&#181;l
+
−
</td>
+
−
      <td>86.7 ng/&#181;l
+
−
</em></td>
+
−
    </tr>
+
−
   </tbody>
+
−
</table>
+
−
Table 1
+
−
</br></br></br>
+
−
 
+
−
The measured concentration after dilution were too low. We eventually switched to a Nanodrop (Thermofisher) because the plastic uvette gave too much background. </br>
+
−
</br>
+
−
 
+
−
<U>Method for digestion by Restriction enzymes</U></br>
+
−
1. Mix all the reagents and let digest during 2 hr at 37°C </br>
+
−
Big volumes must be added first!</br>
+
−
 
+
−
&bull; pET43.1 a at 87.7 ng/ will be digested by BamHI and Hind III (NEB)</br>
+
−
&bull; pSB1C3 at 26.7  ng/ will be digested by SpeI and XbaI (NEB)</br>
+
−
 
+
−
2. We began the digestion 17h20. Here we digest 400 ng of DNA. We doubled Hind III volumes because this enzyme has only 50% of efficiency in Custmart (NEB) buffer.</br>
+
−
<table>
+
−
   <thead>
+
−
     <tr>
+
−
       <th></th>
+
−
      <th>pSB1C3 (26.7 ng/&#181;l)</th>
+
−
      <th>pET43.1 a (86.7 ng/&#181;l)</th>
+
−
    </tr>
+
−
  </thead>
+
−
  <tbody>
+
−
    <tr>
+
−
      <td><strong><p>Vol SpeI </p></strong></td>
+
−
      <td>1 &#181;l</td>
+
−
       <td>0 &#181;l</td>
+
−
    </tr>
+
−
    <tr>
+
−
       <td><strong>Vol XbaI </strong></td>
+
−
       <td>1 &#181;l</td>
+
−
      <td>0 &#181;l</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td><strong>Vol HindIII </strong></td>
+
−
      <td>0 &#181;l </td>
+
−
      <td>2 &#181;l</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td><strong>Vol BamHI </strong></td>
+
−
      <td>0 &#181;l</td>
+
−
      <td>1 &#181;l</td>
+
−
    </tr>
+
−
    <tr>
+
−
      <td><strong>Vol H2O</strong></td>
+
−
      <td>28 &#181;l</td>
+
−
      <td>37.4 &#181;l</em></td>
+
−
<tr>
+
−
      <td><strong>Vol buffer 10X (Cutsmart) </strong></td>
+
−
      <td>5 &#181;l</td>
+
−
      <td>5 &#181;l</td>
+
−
    </tr>
+
−
<td><strong>Vol TOTAL </strong></td>
+
−
      <td>50 &#181;l</td>
+
−
      <td>50 &#181;l</td>
+
−
    </tr>
+
−
    </tr>
+
−
  </tbody>
+
−
</table>Table 2
+
−
 
+
−
3. Store at -20°C</br>
+
−
 
+
−
</p>
+
−
</figcaption>
+
−
   </figure>
+
−
</div>
+
−
</div>
+
−
 
+
  
 
</body>
 
</body>
 
<html>
 
<html>

Revision as of 11:59, 3 October 2016