Difference between revisions of "Team:Edinburgh/InterLab"

 
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{{Edinburgh_InterLab}}
 
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                   <a href="https://2015.igem.org/Team:Edinburgh">Home</a></li>
 
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Experiments">Experiments</a> </li>
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                    <!-- <li><a href="https://2015.igem.org/Team:Edinburgh/Experiments">Experiments</a> </li> -->
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Project/Protocols">Protocols</a> </li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/HeroinBiosensor">Heroin Biosensor</a></li>
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Results">Results</a></li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/DNPBiosensor">DNP Biosensor</a></li>
                       <li><a href="https://2015.igem.org/Team:Edinburgh/Design">Design</a></li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/PMABiosensor">PMA Biosensor</a></li>
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                       <li><a href="https://2015.igem.org/Team:Edinburgh/CBD">Making it Stick</a></li>           
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Results">Limits of Detection</a></li>
 
                     </ul>
 
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                     <a href="#" class="dropdown-toggle" data-toggle="dropdown" role="button" aria-expanded="false">Parts<span class="caret"></span></a>
 
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                     </ul>
 
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              </div>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/PMADetection">PMA Detection</a> </li>
          </nav>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/DNPDetection">DNP Detection</a> </li>
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                      <li><a href="https://2015.igem.org/Team:Edinburgh/Notebook/FluidDynamics">Fluid Dynamics</a> </li>
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                  <li><a href="https://2015.igem.org/Team:Edinburgh/MedalCriteria">Accomplishments</a></li> 
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       <header class="intro">
 
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                 <h1 class="brand-heading">InterLab Study</h1>
 
                 <h1 class="brand-heading">InterLab Study</h1>
 
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                 <p class="intro-text">
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                 </p>
 
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                <div align="center">
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                    <a href="#about">
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                        <span class="arrowtext">Scroll down to read more</span>
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                        <img src="https://static.igem.org/mediawiki/2014/3/3e/Aalto_Helsinki_Nuoli.png" class="arrow">
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                    </a>
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       </header>
 
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         <div class="container">
 
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                 <div class="col-lg-12 text-center">
 
                 <div class="col-lg-12 text-center">
                     <h2 class="section-heading">OVERVIEW</h2>
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                     <h2 class="section-heading">Overview</h2>
 
                     <h3 class="section-subheading text-muted">
 
                     <h3 class="section-subheading text-muted">
                    stuff
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                      The Edinburgh iGEM 2015 team is participating in the second year of the iGEM InterLab study along with 82 other teams. The 2015 InterLab study focuses on obtaining consistent fluorescence data for three genetic devices expressing GFP. The devices consist of a constitutive promoter with either high (J23101), medium (J23106) or low (J23117) promoter strengths, fused to GFP (I13504).
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The promoters and GFP from the Biobrick distribution kit were transformed into E.coli Top10 cells then digested to have complementary sticky ends and ligated resulting in the final devices. The devices were validated by restriction digest and sequence data. Each promoter fused to GFP was transformed once more grown in cultures overnight and then measurements of fluorescence were taken.
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More detailed information regarding the cloning process can be found below and on our InterLab lab notebook page.
 
                     </h3>
 
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    <div class="container">
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                <div class="col-lg-12 text-center">
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                    <h2 class="section-heading">Protocols</h2>
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                    <h3 class="section-subheading text-muted">
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                      All other protocols used in the process of the study can be found on the <a href="2015.igem.org/Team:Edinburgh/Project/Protocols">protocols</a> page.
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                    </h3>                 
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<div class="container">  
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<div class="container">
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            <a role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseOne" aria-expanded="false" aria-controls="collapseOne">
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              1% Agarose Gel
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            <div class="panel-heading">
            </a>
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          </h4>
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                <h4 class="panel-title">
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseOne">What protocol was followed for growing cells for measurement?</a>
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                    <p>The protocol supplied in the iGEM 2015 InterLab Protocol  form was followed with a few minor changes.
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                    <br>
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                    Each device and the two controls were streaked out onto LB agar plates supplemented with chloramphenicol and incubated at 37°C for 22 hours.  3 colonies from each plate were inoculated into 50ml Falcon tubes containing 10ml of LB media and chloramphenicol.  The tubes were incubated upright on a platform shaker in a room kept at 37°C for 16 hours.</p>
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         <div id="collapseOne" class="panel-collapse collapse in" role="tabpanel" aria-labelledby="headingOne">
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              <p style="color: black;">
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            <div class="panel-heading">
              <h2>Materials</h2>
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              <ul>
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                <h4 class="panel-title">
                 <li>1g Agarose
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                <li>100ml 1X TAE buffer
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwo">What controls were used?</a>
                <li>5µl GelRed stain
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              </ul>
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                 </h4>
            </p>
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              <p class="text-muted">
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              <h2>Procedure</h2>
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              <ul>
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                <div class="panel-body">
                <li>1. Mix the agarose with the 1X TAE buffer in a flask.
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                <li>2. Heat the mixture until all the agarose is dissolved.
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                    <p>The positive control used was the recommended BBa_I20270, a chloramphenicol resistant GFP expression device in pSB1C3.  
                <li>3. Swirl the flask under cold running water to cool the mixture.
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<br>
                <li> 4. Add the gel stain.
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The negative control used consisted of cells transformed with BioBrick number BBa_R0040. This part consists of the promoter pTetR and should therefore have very low fluorescence</p>
                 <li>5. Pour into an assembled gel tray and let it cool.
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              </uL>
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        <div class="panel-heading" role="tab" id="headingTwo">
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          <h4 class="panel-title">
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            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseTwo" aria-expanded="false" aria-controls="collapseTwo">
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            Agarose Gel Electrophoresis
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                <h4 class="panel-title">
            </a>
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          </h4>
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseThree">What agar was used to grow the cells?</a>
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                </h4>
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                    <p>The cells were grown on LB agar supplemented with 1000x chloramphenicol.</p>
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                </div>
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            <div class="col-md-6">
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              <p>
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            <div class="panel-heading">
              <h2>Materials</h2>
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              <ul>
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                <h4 class="panel-title">
                 <li>1% Agarose
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                <li>1X TAE buffer
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                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseFour">How were the cells prepared for measurement?</a>
                <li>5X loading dye
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                <li>DNA ladder
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                 </h4>
                <li>DNA samples
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              </ul>
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            </p>
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             </div>
            <div class="col-md-6">
 
              <p>
 
              <h2>Procedure</h2>
 
              <ul>
 
                <li>1. Place gel tray into the electrophoresis apparatus.
 
                <li>2. Pour 1X TAE so that the gel is covered by buffer.
 
                <li>3. Prepare the samples by adding the appropriate amount of loading dye.
 
                <li>4. Load samples and DNA ladder into wells on the gel.
 
                <li>5. Run the gel at roughly 100V for around an hour
 
  
              </uL>
+
            <div id="collapseFour" class="panel-collapse collapse">
            </p>
+
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>A colony of cells was inoculated into 10ml of liquid LB with 10µl of 1000x chloramphenicol. The cultures were grown in a 50ml falcon tube, upright on shaking platform at 275rpm in 37°C room overnight.
 +
<br>
 +
For each device three colonies were inoculated to act as the three biological replicates.</p>
 +
 
 +
                </div>
 +
 
 
             </div>
 
             </div>
          </div>
+
 
 
         </div>
 
         </div>
      </div>
+
       
      <div class="panel panel-default">
+
                <div class="panel panel-default">
        <div class="panel-heading" role="tab" id="headingThree">
+
 
          <h4 class="panel-title">
+
            <div class="panel-heading">
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseThree" aria-expanded="false" aria-controls="collapseThree">
+
 
              Culture
+
                <h4 class="panel-title">
            </a>
+
 
          </h4>
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseFive">How was the fluorescence of each device measured? (followed protocol)</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseFive" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>After the overnight incubation period of 16 hours the liquid cultures were removed from the 37°C room. 200µl from each culture was added to a 96-well plate. The initial optical density of each sample was measured at 600nm using a plate reader. All samples were then diluted appropriately so as to have an OD600 value within 5% of 0.5.  Two additional technical replicates for each of the biological replicates were set up at this point. The OD600 was measured again to ensure all samples had an optical density within 5% of 0.5 before the fluorescence measurements were taken.
 +
<br>
 +
To measure fluorescence, the cells were excited at a wavelength of 485nm and emitted light at 538nm as measured.
 +
<br>
 +
The values obtained for fluorescence were in relative fluorescence units.</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
        <div id="collapseThree" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingThree">
+
 
          <div class="panel-body">
+
                <div class="panel panel-default">
          <div class="col-md-6">
+
 
              <p>
+
            <div class="panel-heading">
              <h2>Materials</h2>
+
 
              <ul>
+
                <h4 class="panel-title">
                 <li>10ml Luria Broth (LB)
+
 
                <li>10µl Specific Antibiotic at 1000x (Chloramphenicol, Ampicillin or Kanamycin)
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseSix">What instrument was used to measure the fluorescence of each device?</a>
                <li>Loop (for picking colony)
+
 
                <li>Ethanol
+
                 </h4>
              </ul>
+
 
            </p>
+
 
             </div>
 
             </div>
             <div class="col-md-6">
+
 
              <p>
+
             <div id="collapseSix" class="panel-collapse collapse">
              <h2>Procedure</h2>
+
 
              <ul>
+
                <div class="panel-body">
                <li>1. Pour 10ml of LB into a 50ml Falcon tube.  
+
 
                <li>2. Pipette 10µl of antibiotic into the broth.
+
                    <p>A SpectaMax M5 plate reader was used to obtain the values of fluorescence.</p>
                <li>3. Dip loop in ethanol and flame to sterilise. Once it is cool, pick colony and transfer to a 50ml Falcon tube.
+
 
                 <li>4. Incubate at 37°C overnight in a shaking incubator.
+
                 </div>
              </uL>
+
 
            </p>
+
 
             </div>
 
             </div>
          </div>
+
 
 
         </div>
 
         </div>
      </div>
+
 
      <div class="panel panel-default">
+
    </div>
        <div class="panel-heading" role="tab" id="headingFour">
+
</div>
          <h4 class="panel-title">
+
 
             <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseFour" aria-expanded="false" aria-controls="collapseFour">
+
<section id="about">
            Gel Purification using QIAquick Gel Extraction Kit
+
    <div class="container">
            </a>
+
            <div class="row">
          </h4>
+
                <div class="col-lg-12 text-center">
 +
                    <h2 class="section-heading">Results</h2>                   
 +
                </div>
 +
            </div>
 +
  </div>
 +
</section>
 +
 
 +
<div class="container">
 +
 
 +
    <div id="accordion" class="panel-group">
 +
 
 +
        <div class="panel panel-default">
 +
 
 +
             <div class="panel-heading">
 +
 
 +
                <h4 class="panel-title">
 +
 
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseSeven">How was the raw data processed?</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseSeven" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>The background absorbance of 6 wells containing 200µl of LB was measured.  The average of these readings was then subtracted from the fluorescence values obtained for the devices.</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
         <div id="collapseFour" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingFour">
+
 
          <div class="panel-body">
+
         <div class="panel panel-default">
            <div class="col-md-6">
+
 
              <p>
+
            <div class="panel-heading">
              <h2>Materials</h2>
+
 
              <ul>
+
                <h4 class="panel-title">
                 <li>Buffer QG
+
 
                <li>10µl 3M sodium acetate
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseEight">What were the units of the results?</a>
                <li>Isopropanol
+
 
                <li>750µl Buffer PE
+
                 </h4>
                <li>25µl Buffer EB
+
 
              </ul>
+
            </p>
+
 
             </div>
 
             </div>
             <div class="col-md-6">
+
 
              <p>
+
             <div id="collapseEight" class="panel-collapse collapse">
              <h2>Procedure</h2>
+
 
              All centrifuge steps are carried out at 13,000 rpm.
+
                 <div class="panel-body">
              <ul>
+
 
                 <li>1. Excise the region of gel containing the DNA fragment using a scalpel.  Cut close to the DNA to minimise the gel volume.
+
                    <p>The data is in relative fluorescent units.</p>
                <li>2. Place the gel slice in a 1.5ml tube and weigh it. Record the volume of the gel.
+
                 </div>
                <li>3.  Add 300µl of Buffer QG for each 100mg of gel.
+
 
                <li>4. Incubate at 50°C for 10 minutes or until the gel has completely dissolved. Mix by vortexing the tube every 2 minutes during the incubation.
+
                <li>5. Once the gel is completely dissolved, the mixture should be yellow. If the mixture is orange or violet add 10 µl of 3M sodium acetate and mix until it turns yellow. Yellow colour indicates the solution is the optimum pH for DNA binding to the QIAquick membrane.
+
                <li>6. Add 1 gel volume of isopropanol to the solution and mix (1:1 volumes of isopropanol to gel slice).
+
                <li>7. Place a QIAquick spin column in a 2ml collection tube.
+
                <li>8. Pipette the sample onto the QIAquick column and centrifuge. Discard flow-through.
+
                 <li>9. Place column back in same collection tube. Add 500µl of Buffer QG to the column and centrifuge for 1 minute to remove all traces of agarose.
+
                <li>10. Wash column by adding 750µl buffer PE. Let it stand for 2-5 min and then centrifuge for 1 minute.
+
                <li>11. Discard the flow-through. Centrifuge for 1 minute to remove the residual buffer PE.
+
                <li>12. Then place the column in a clean, labelled 1.5ml Eppendorf tube.
+
                <li>13. To elute the DNA, add 25µl of Buffer EB to the centre of the column membrane, let it stand for 1 minute and then centrifuge for 1 minute.
+
                <li>14. Using a pipette, transfer the flow-through back into the centre of the column. Let it stand for 1 minute and then centrifuge for 1 minute. The DNA will now be in the flow-through.
+
              </uL>
+
            </p>
+
 
             </div>
 
             </div>
          </div>
+
 
 
         </div>
 
         </div>
      </div>
+
 
      <div class="panel panel-default">
+
        <div class="panel panel-default">
        <div class="panel-heading" role="tab" id="headingFive">
+
 
          <h4 class="panel-title">
+
            <div class="panel-heading">
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseFive" aria-expanded="false" aria-controls="collapseFive">
+
 
            Glycerol Stock
+
                <h4 class="panel-title">
            </a>
+
 
          </h4>
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseNine">Fluorescence of 3 biological replicates of positive control</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseNine" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
<p>1.) 1278.712 <br>  2.) 1216.887  <br>  3.) 1110.834</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
        <div id="collapseFive" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingFive">
+
 
          <div class="panel-body">
+
          <div class="panel panel-default">
            <div class="col-md-6">
+
 
              <p>
+
            <div class="panel-heading">
              <h2>Materials</h2>
+
 
              <ul>
+
                <h4 class="panel-title">
                 <li>500µl 50% glycerol solution
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTen">Fluorescence of 3 biological replicates of negative control</a>
                <li>500µl cultured cells
+
 
              </ul>
+
                 </h4>
            </p>
+
 
 
             </div>
 
             </div>
             <div class="col-md-6">
+
 
              <p>
+
             <div id="collapseTen" class="panel-collapse collapse">
              <h2>Procedure</h2>
+
 
              <ul>
+
                <div class="panel-body">
                <li>1. Add the 50% glycerol to a sterile Eppendorf tube.
+
 
                <li>2. Add 500µl of cells to the tube and vortex to mix.
+
                    <p>1.) -36.863 <br>   2.) -33.066 <br>   3.) -44.696</p>
                <li>3. Freeze at -80°C.
+
 
              </uL>
+
                </div>
            </p>
+
 
 
             </div>
 
             </div>
          </div>
+
 
 
         </div>
 
         </div>
      </div>
+
 
      <div class="panel panel-default">
+
 
        <div class="panel-heading" role="tab" id="headingSix">
+
          <div class="panel panel-default">
          <h4 class="panel-title">
+
 
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseSix" aria-expanded="false" aria-controls="collapseSix">
+
            <div class="panel-heading">
            Heat Shock Transformation
+
 
            </a>
+
                <h4 class="panel-title">
          </h4>
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseEleven">Fluorescence of 3 biological replicates of J23101 + I13504, mean and st.dev (device 1)</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseEleven" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>1.)5082.078 <br> 2.) 5879.239 <br> 3.) 5973.973
 +
                        <br>
 +
                        Mean = 5645.097
 +
                        <br>
 +
                        Standard Deviation = 489.884
 +
                  </p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
        <div id="collapseSix" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingSix">
+
 
          <div class="panel-body">
+
          <div class="panel panel-default">
            <div class="col-md-6">
+
 
              <p>
+
            <div class="panel-heading">
              <h2>Materials</h2>
+
 
              <ul>
+
                <h4 class="panel-title">
                 <li>2.5µl ligated DNA or 0.5µl purified plasmid
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwelve">Fluorescence of 3 biological replicates of J23106 + I13504, mean and st.dev (device 2)</a>
                <li>50µl chemically competent DH5α E.coli cells
+
 
                <li>250µl Luria Broth
+
                 </h4>
                <li>Petri dish with LB agar and specific antibiotic (chloramphenicol or ampicillin)
+
 
              </ul>
+
            </p>
+
 
             </div>
 
             </div>
             <div class="col-md-6">
+
 
              <p>
+
             <div id="collapseTwelve" class="panel-collapse collapse">
              <h2>Procedure</h2>
+
 
              <ul>
+
                <div class="panel-body">
                <li>1. Thaw the competent cells on ice.
+
 
                <li>2. Add 50µl of cells to a pre-chilled, labelled microcentrifuge tube.
+
                    <p>
                <li>3. Add 2.5µl of DNA suspension to the tube. Pipette up and down to mix.
+
                      1.) 3185.188 <br> 2.) 3119.913 <br> 3.) 2396.115
                <li>4. Incubate tube on ice for 30 min.
+
                      <br>
                <li>5. Heat shock in a water bath at 42°C for 60s.
+
                      Mean = 2900.405
                <li>6. Place tube back on ice for 3 min.
+
                      <br>
                <li>7. Add 250µl of LB to the tube.
+
                      Standard Deviation = 437.946
                <li>8. Incubate the tube at 37°C for 1 hour.
+
                  </p>
                <li>9. Prepare two plates for each transformation, one plated with 200µl of cells and another plated with 100µl of cells.
+
 
                 <li>10. Incubate at 37°C overnight (12-14 hours).
+
                 </div>
              </uL>
+
 
            </p>
+
 
             </div>
 
             </div>
          </div>
+
 
 
         </div>
 
         </div>
      </div>
+
 
      <div class="panel panel-default">
+
 
        <div class="panel-heading" role="tab" id="headingSeven">
+
          <div class="panel panel-default">
          <h4 class="panel-title">
+
 
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseSeven" aria-expanded="false" aria-controls="collapseSeven">
+
            <div class="panel-heading">
            Miniprep using QIAprep Spin Miniprep Kit
+
 
            </a>
+
                <h4 class="panel-title">
          </h4>
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseThirteen">Fluorescence of 3 biological replicates of J23117 + I13504, mean and st.dev (device3)</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseThirteen" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>
 +
                      1.) -14.003 <br> 2.) -27.149 <br> 3.) -16.631
 +
                      <br>
 +
                      Mean = -19.261
 +
                      <br>
 +
                      Standard Deviation = 6.956
 +
                  </p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
        <div id="collapseSeven" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingSeven">
+
    </div>
          <div class="panel-body">
+
</div>
            <div class="col-md-6">
+
 
              <p>
+
<section id="about">
              <h2>Materials</h2>
+
    <div class="container">
              <ul>
+
            <div class="row">
                 <li>10ml cell culture
+
                <div class="col-lg-12 text-center">
                <li>250µl Buffer P1
+
                    <h2 class="section-heading">Extra Credit</h2>                  
                <li>250µl Buffer P2
+
                 </div>
                <li>350µl Buffer N3
+
                <li>750µl Buffer PE
+
                <li>50µl Buffer EB
+
              </ul>
+
            </p>
+
 
             </div>
 
             </div>
            <div class="col-md-6">
+
  </div>
              <p>
+
</section>
              <h2>Procedure</h2>
+
 
              <ul>
+
<div class="container">
                <li>1. Centrifuge 10ml cell culture at 4,500 x g for 5 minutes and pour off the supernatant. Centrifuge at 4,500 x g for 2 minutes and remove the supernatant by pipetting.
+
 
                <li>2. Resuspend pelleted cells in 250 µl Buffer P1 and transfer the solution to a labelled Eppendorf tube.
+
    <div id="accordion" class="panel-group">
                <li>3. Add 250 µl Buffer P2 and gently invert the tube 4–6 times to mix. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
+
 
                <li>4. Add 350 µl Buffer N3 and invert the tube immediately but gently 4–6 times. The solution should become cloudy.
+
        <div class="panel panel-default">
                <li>5. Centrifuge for 10 min at 13,000 rpm in an Eppendorf tube. A compact white pellet will form.
+
 
                <li>6. Apply the supernatant to the QIAprep Spin Column by pipetting.
+
            <div class="panel-heading">
                <li>7. Centrifuge for 60s at 13,000 rpm. Discard the flow-through.
+
 
                <li>8. Wash QIAprep Spin Column by adding 750µl Buffer PE and centrifuging for 60s at 13,000 rpm.
+
                 <h4 class="panel-title">
                 <li>9. Discard the flow-through, and centrifuge for an additional 60s at 13,000 rpm to remove residual wash buffer.
+
 
                <li>10. Place the QIAprep column in a clean, labelled Eppendorf tube. To double elute DNA, add 50 µl Buffer EB to the centre of each QIAprep Spin Column, let stand for 1 min, and centrifuge for 1 min at 13,000 rpm. Re-apply flow-through to the centre of the QIAprep Spin Column, let stand for 1 min and centrifuge for 1 min at 13,000 rpm.
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseFourteen">Fluorescence of 2nd and 3rd technical replicates of J23101 + I13504 (device 1) biological replicate 1</a>
                <li>11. Discard column. Flow-through contains the DNA.
+
 
              </uL>
+
                </h4>
            </p>
+
 
 
             </div>
 
             </div>
          </div>
+
 
 +
            <div id="collapseFourteen" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>2nd Replicate = 5202.079 <br> 3rd replicate = 5187.619</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
      </div>
+
 
      <div class="panel panel-default">
+
        <div class="panel panel-default">
        <div class="panel-heading" role="tab" id="headingEight">
+
 
          <h4 class="panel-title">
+
            <div class="panel-heading">
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseEight" aria-expanded="false" aria-controls="collapseEight">
+
 
            Nanodrop
+
                <h4 class="panel-title">
            </a>
+
 
          </h4>
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseFifteen">Fluorescence of 2nd and 3rd technical replicates of J23101 + I13504 (device 1) biological replicate 2</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseFifteen" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>2nd replicate = 6008.646 <br> 3rd replicate = 6135.483</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
         <div id="collapseEight" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingEight">
+
 
          <div class="panel-body">
+
         <div class="panel panel-default">
            <div class="col-md-6">
+
 
              <p>
+
            <div class="panel-heading">
              <h2>Materials</h2>
+
 
              <ul>
+
                <h4 class="panel-title">
                 <li>Buffer EB to blank
+
 
                <li>DNA sample
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseSixteen">Fluorescence of 2nd and 3rd technical replicates of J23101 + I13504 (device 1) biological replicate 3</a>
              </ul>
+
 
            </p>
+
                 </h4>
 +
 
 
             </div>
 
             </div>
             <div class="col-md-6">
+
 
              <p>
+
             <div id="collapseSixteen" class="panel-collapse collapse">
              <h2>Procedure</h2>
+
 
              <ul>
+
                <div class="panel-body">
                <li>1. Wipe the NanoDrop 2000 clean before pipetting 1µl of buffer EB and lowering the arm. Blank the NanoDrop spectrophotometer.
+
 
                <li>2. Clean the buffer EB from the pedestal and apply 1µl of sample. Measure and record the absorbance, and repeat for remainder of samples.
+
                    <p>2nd replicate =6634.865 <br> 3rd replicate = 6254.715</p>
                <li>3. Clean the NanoDrop pedestal after use.
+
 
              </uL>
+
                </div>
            </p>
+
 
 
             </div>
 
             </div>
          </div>
+
 
 
         </div>
 
         </div>
      </div>
+
     
            <div class="panel panel-default">
+
        <div class="panel panel-default">
        <div class="panel-heading" role="tab" id="headingNine">
+
 
          <h4 class="panel-title">
+
            <div class="panel-heading">
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseNine" aria-expanded="false" aria-controls="collapseNine">
+
 
            PCR Purification using QIAquick PCR Purification Kit
+
                <h4 class="panel-title">
            </a>
+
 
          </h4>
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseSeventeen">Mean and standard deviation of technical replicates of J23101 + I13504 (device 1)</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseSeventeen" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>
 +
Mean = 5817.633
 +
<br>
 +
Standard deviation = 541.009
 +
</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
         <div id="collapseNine" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingNine">
+
          
          <div class="panel-body">
+
                <div class="panel panel-default">
            <div class="col-md-6">
+
 
              <p>
+
            <div class="panel-heading">
              <h2>Materials</h2>
+
 
              <ul>
+
                <h4 class="panel-title">
                 <li>Buffer PB
+
 
                <li>10µl of 3M sodium acetate
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseEighteen">Fluorescence of 2nd and 3rd technical replicates of J23106 + I13504 (device 2) biological replicate 1</a>
                <li>750µl buffer PE
+
 
                <li>25µl buffer EB
+
                 </h4>
              </ul>
+
 
            </p>
+
 
             </div>
 
             </div>
             <div class="col-md-6">
+
 
              <p>
+
             <div id="collapseEighteen" class="panel-collapse collapse">
              <h2>Procedure</h2>
+
 
              All centrifugation steps are carried out at 17,900 x g (13,000 rpm)
+
                 <div class="panel-body">
              <ul>
+
 
                 <li>1. Add 5 volumes of buffer PB to 1 volume of PCR sample (e.g. 500µl PB to 100µl PCR sample) and mix.
+
                    <p>2nd replicate = 3694.877 <br> 3rd replicate = 3606.107</p>
                <li>2. The pH indicator in buffer PB will cause the mixture to turn yellow. If the mixture is orange or violet add 10µl of 3M sodium acetate and mix until it turns yellow.
+
 
                <li>3. Place a QIAquick spin column into a 2ml collection tube.
+
                 </div>
                <li>4. Apply the sample to the centre of the column and centrifuge for 60s.
+
 
                <li>5. Discard the flow-through and replace the column in the same collection tube.
+
                <li>6. Add 750µl of buffer PE to the column and centrifuge for 60s.
+
                 <li>7. Discard the flow-through, replace the column in the collection tube and centrifuge for a further 60s to remove residual buffer.
+
                <li>8. Place the column in a clean 1.5ml Eppendorf tube.
+
                <li>9. Elute the DNA by adding 25µl buffer EB to the centre of the membrane in the column. Let it stand for 1 minute then centrifuge for 1 minute.
+
                <li>10. Using a pipette, transfer the flow-through back into the centre of the column. Let it stand for 1 minute and then centrifuge for 1 minute.
+
                <li>11. The purified DNA is now present in the flow-through.
+
              </uL>
+
            </p>
+
 
             </div>
 
             </div>
          </div>
+
 
 
         </div>
 
         </div>
      </div>
+
 
            <div class="panel panel-default">
+
                <div class="panel panel-default">
        <div class="panel-heading" role="tab" id="headingTen">
+
 
          <h4 class="panel-title">
+
            <div class="panel-heading">
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseTen" aria-expanded="false" aria-controls="collapseTen">
+
 
            Resuspending gBlocks
+
                <h4 class="panel-title">
            </a>
+
 
          </h4>
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseNineteen">Fluorescence of 2nd and 3rd technical replicates of J23106 + I13504 (device 2) biological replicate 2</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseNineteen" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>2nd replicate = 3036.241 <br> 3rd replicate = 3140.1</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
        <div id="collapseTen" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingTen">
+
 
          <div class="panel-body">
+
    <div class="panel panel-default">
            <div class="col-md-6">
+
 
              <p>
+
            <div class="panel-heading">
              <h2>Materials</h2>
+
 
              <ul>
+
                <h4 class="panel-title">
                 <li>gBlocks dry gene fragment
+
 
                <li>100µl distilled water
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwenty">Fluorescence of 2nd and 3rd technical replicates of J23106 + I13504 (device 2) biological replicate 3</a>
              </ul>
+
 
            </p>
+
                 </h4>
 +
 
 
             </div>
 
             </div>
             <div class="col-md-6">
+
 
              <p>
+
             <div id="collapseTwenty" class="panel-collapse collapse">
              <h2>Procedure</h2>
+
 
              <ul>
+
                <div class="panel-body">
                <li>1. Centrifuge the tube containing the gBlocks gene fragment pellet at 3000 x g for 5s.
+
 
                <li>2. Add 100µl water to the tube.
+
                    <p>2nd replicate = 2871.642 <br> 3rd replicate = 2801.428</p>
                <li>3. Vortex to mix.
+
 
                <li>5. Incubate at 50°C for 20 mins.
+
                 </div>
                 <li>6. Briefly vortex and centrifuge.
+
 
              </uL>
+
            </p>
+
 
             </div>
 
             </div>
          </div>
+
 
 
         </div>
 
         </div>
      </div>
+
 
            <div class="panel panel-default">
+
    <div class="panel panel-default">
        <div class="panel-heading" role="tab" id="headingEleven">
+
 
          <h4 class="panel-title">
+
            <div class="panel-heading">
            <a class="collapsed" role="button" data-toggle="collapse" data-parent="#accordion" href="#collapseEleven" aria-expanded="false" aria-controls="collapseEleven">
+
 
            Sequencing
+
                <h4 class="panel-title">
            </a>
+
 
          </h4>
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyOne">Mean and standard deviation of technical replicates of J23106 + I13504 (device 2)</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseTwentyOne" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>Mean = 3094.623
 +
<br>
 +
Standard deviation = 396.837</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 
         </div>
 
         </div>
        <div id="collapseEleven" class="panel-collapse collapse" role="tabpanel" aria-labelledby="headingEleven">
+
 
          <div class="panel-body">
+
    <div class="panel panel-default">
            <div class="col-md-6">
+
 
              <p>
+
            <div class="panel-heading">
              <h2>Materials</h2>
+
 
              <ul>
+
                <h4 class="panel-title">
                 <li>200-500ng DNA sample
+
 
                <li>0.32µl 10µM Vf2 and Vr primers
+
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyTwo">Fluorescence of 2nd and 3rd technical replicates of J23117 + I13504 (device 3) biological replicate 1</a>
                <li>Water
+
 
              </ul>
+
                 </h4>
            </p>
+
 
 
             </div>
 
             </div>
             <div class="col-md-6">
+
 
              <p>
+
             <div id="collapseTwentyTwo" class="panel-collapse collapse">
              <h2>Procedure</h2>
+
 
              <ul>
+
                <div class="panel-body">
                <li>1. Prepare the DNA for sequencing by adding DNA, primers and water to a final volume of 6µl.
+
 
                <li>2. Prepare two samples for each DNA sequence, one containing the forward primer, and another separate sample containing the reverse primer.
+
                    <p>2nd replicate = 7.859 <br> 3rd replicate = 0.659</p>
                <li>3. Send the samples to be Sanger sequenced by Edinburgh Genomics using an ABI 3730 DNA analyser.
+
 
                 <li>4. Edinburgh Genomics will send an email with the sequence data.
+
                 </div>
              </uL>
+
 
            </p>
+
 
             </div>
 
             </div>
          </div>
+
 
 
         </div>
 
         </div>
      </div>
+
 
 +
    <div class="panel panel-default">
 +
 
 +
            <div class="panel-heading">
 +
 
 +
                <h4 class="panel-title">
 +
 
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyThree">Fluorescence of 2nd and 3rd technical replicates of J23117 + I13504 (device 3) biological replicate 2</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseTwentyThree" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>2nd replicate = 28.499 <br> 3rd replicate = 11.4</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 +
        </div>
 +
 
 +
    <div class="panel panel-default">
 +
 
 +
            <div class="panel-heading">
 +
 
 +
                <h4 class="panel-title">
 +
 
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyFour">Fluorescence of 2nd and 3rd technical replicates of J23117 + I13504 (device 3) biological replicate 3</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseTwentyFour" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>2nd replicate = -17.823 <br> 3rd replicate = -6.295</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 +
        </div>
 +
 
 +
    <div class="panel panel-default">
 +
 
 +
            <div class="panel-heading">
 +
 
 +
                <h4 class="panel-title">
 +
 
 +
                    <a data-toggle="collapse" data-parent="#accordion" href="#collapseTwentyFive">Mean and standard deviation of technical replicates of J23117 + I13504 (device 3)</a>
 +
 
 +
                </h4>
 +
 
 +
            </div>
 +
 
 +
            <div id="collapseTwentyFive" class="panel-collapse collapse">
 +
 
 +
                <div class="panel-body">
 +
 
 +
                    <p>Mean = -3.720
 +
<br>
 +
Standard deviation = 17.489</p>
 +
 
 +
                </div>
 +
 
 +
            </div>
 +
 
 +
        </div>
 +
 
 
     </div>
 
     </div>
  </div>
+
</div>
 +
 
 +
 
  
 
       <footer>
 
       <footer>

Latest revision as of 18:58, 20 November 2015

Overview

The Edinburgh iGEM 2015 team is participating in the second year of the iGEM InterLab study along with 82 other teams. The 2015 InterLab study focuses on obtaining consistent fluorescence data for three genetic devices expressing GFP. The devices consist of a constitutive promoter with either high (J23101), medium (J23106) or low (J23117) promoter strengths, fused to GFP (I13504).

The promoters and GFP from the Biobrick distribution kit were transformed into E.coli Top10 cells then digested to have complementary sticky ends and ligated resulting in the final devices. The devices were validated by restriction digest and sequence data. Each promoter fused to GFP was transformed once more grown in cultures overnight and then measurements of fluorescence were taken.

More detailed information regarding the cloning process can be found below and on our InterLab lab notebook page.

Protocols

All other protocols used in the process of the study can be found on the protocols page.

The protocol supplied in the iGEM 2015 InterLab Protocol form was followed with a few minor changes.
Each device and the two controls were streaked out onto LB agar plates supplemented with chloramphenicol and incubated at 37°C for 22 hours. 3 colonies from each plate were inoculated into 50ml Falcon tubes containing 10ml of LB media and chloramphenicol. The tubes were incubated upright on a platform shaker in a room kept at 37°C for 16 hours.

The positive control used was the recommended BBa_I20270, a chloramphenicol resistant GFP expression device in pSB1C3.
The negative control used consisted of cells transformed with BioBrick number BBa_R0040. This part consists of the promoter pTetR and should therefore have very low fluorescence

The cells were grown on LB agar supplemented with 1000x chloramphenicol.

A colony of cells was inoculated into 10ml of liquid LB with 10µl of 1000x chloramphenicol. The cultures were grown in a 50ml falcon tube, upright on shaking platform at 275rpm in 37°C room overnight.
For each device three colonies were inoculated to act as the three biological replicates.

After the overnight incubation period of 16 hours the liquid cultures were removed from the 37°C room. 200µl from each culture was added to a 96-well plate. The initial optical density of each sample was measured at 600nm using a plate reader. All samples were then diluted appropriately so as to have an OD600 value within 5% of 0.5. Two additional technical replicates for each of the biological replicates were set up at this point. The OD600 was measured again to ensure all samples had an optical density within 5% of 0.5 before the fluorescence measurements were taken.
To measure fluorescence, the cells were excited at a wavelength of 485nm and emitted light at 538nm as measured.
The values obtained for fluorescence were in relative fluorescence units.

A SpectaMax M5 plate reader was used to obtain the values of fluorescence.

Results

The background absorbance of 6 wells containing 200µl of LB was measured. The average of these readings was then subtracted from the fluorescence values obtained for the devices.

The data is in relative fluorescent units.

1.) 1278.712
2.) 1216.887
3.) 1110.834

1.)5082.078
2.) 5879.239
3.) 5973.973
Mean = 5645.097
Standard Deviation = 489.884

1.) 3185.188
2.) 3119.913
3.) 2396.115
Mean = 2900.405
Standard Deviation = 437.946

1.) -14.003
2.) -27.149
3.) -16.631
Mean = -19.261
Standard Deviation = 6.956

Extra Credit