Difference between revisions of "Team:Hong Kong HKU/Experiments"

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{{Hong_Kong_HKU}}
 
 
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{{Hong_Kong_HKU/Header}}
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<p>Describe the experiments, research and protocols you used in your iGEM project.</p>
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<body>
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</div>
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<div class="container" align="center">
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    <h2>Notebook</h2>
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<div class="column half_size">
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    <ul class="nav nav-pills">
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<h5>What should this page contain?</h5>
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      <li class="active"><a href="">Working Log</a></li>
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<ul>
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      <li><a href="https://2016.igem.org/Team:Hong_Kong_HKU/Experiments">Experiments and Protocol</a></li>
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<li> Protocols </li>
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      <li><a href="https://2016.igem.org/Team:Hong_Kong_HKU/Safety">Safety</a></li>
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<li> Experiments </li>
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    </ul>
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<li>Documentation of the development of your project </li>
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    <div class="tab-content">
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</ul>
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      <div id="Protocol" class="tab-pane fade in active">
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        <h3>Protocol</h3>
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        <div class="panel-group" id="ProtocolContent" role="tablist" aria-multiselectable="true">
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          <div class="panel panel-transparent">
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<div class="column half_size">
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            <div class="panel-heading" role="tab">
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<h5>Inspiration</h5>
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              <h4 class="panel-title"><a data-toggle="collapse" data-parent="#ProtocolContent" href="#PAGE"><h3>Native Polyacrylamide gel electrophoresis</h3></a></h4>
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<ul>
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            </div>
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<li><a href="https://2014.igem.org/Team:Colombia/Protocols">2014 Colombia </a></li>
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            <div id="PAGE" class="panel-collapse collapse in">
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<li><a href="https://2014.igem.org/Team:Imperial/Protocols">2014 Imperial </a></li>
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              <div class="panel-body">
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<li><a href="https://2014.igem.org/Team:Caltech/Project/Experiments">2014 Caltech </a></li>
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              <h4>General preparation of Native Polyacrylamide gel (Native PAGE gel)</h4>
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</ul>
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              <p align="left"><font size="3">
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</div>
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              <br>
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              <b>Specifications</b><br>
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              A piece of PAGE Gel (~12mL in volume)<br>
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<div class="clear"></div>
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              <b>Requiring Materials</b><br>
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              <table class="table" border="0" >
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                  <thead>
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<div class="column half_size">
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                      <tr>
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                          <th>Materials (12% PAGE gel)</th>
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                          <th>Quantity</th>
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                      </tr>
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                  </thead>
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                  <tbody>
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                      <tr>
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                          <td>Gel kit</td>
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                          <td>1 Set</td>
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                      </tr>
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                      <tr>
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                          <td>Distilled water</td>
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                          <td>6.0 mL</td>
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                      </tr>
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                      <tr>
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                          <td>10X TBE</td>
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                          <td>1.2 mL</td>
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                      </tr>
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                      <tr>
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                          <td>30% Acrylamide (29:1)</td>
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                          <td>4.8 mL</td>
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                      </tr>
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                      <tr>
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                          <td>10% APS</td>
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                          <td>200 μL</td>
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                      </tr>
 +
                      <tr>
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                          <td>TEMED</td>
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                          <td>200 μL</td>
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                      </tr>
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                  </tbody>
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              </table>
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              <b>Storage</b><br>
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              4°C, keep wet in 1x TBE to prevent drying<br>
 +
              <b>Steps</b><br>
 +
              1. Clean the glass plates and spacers thoroughly. Rinse the plates withdeionized water and ethanol and set them aside to dry.<br>
 +
              2. Assemble the glass plates with spacers in gel caster. Make sure there is no water leakage.<br>
 +
              3. Prepare the gel solution according to the desired polyacrylamide percentage. Note the addition of TEMED will immediately trigger the gel to polymerize.<br>
 +
              4. Vortex the solution roughly for 5 seconds.<br>
 +
              5. Quickly piette the gel solution into the caster. Insert the appropriate comb into the gel, prevent to trap air bubbles under the teeth.<br>
 +
              6. Let the polymerization go for 30 minutes at room temperature.<br>
 +
              7. Surround the comb and the top of the gel with paper towels that have been soaked in 1x TBE.
 +
              Then seal the entire gel in  plastic bag and store it at 4°C until needed.<br>
 +
              <b>Notes</b><br>
 +
              Different polyacrylamide percentage results in different speed and discrimating ability for sample running down in electrophoresis.
 +
              The quantity of the solutions varies with the percentage.
 +
              The required quantity of native PAGE gel in common polyacrylamide percentage is listed below. <br>
 +
              <table class="table" border="0">
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                  <thead>
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                      <tr>
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                          <th> Polyacrylamide percentage </th>
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                          <th> Distilled water </th>
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                          <th> 30% Acrylamide (29:1) </th>
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                          <th> 10X TBE </th>
 +
                          <th> 10% APS </th>
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                          <th> TEMED  </th>
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                      </tr>
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                  </thead>
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                  <tbody>
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                      <tr>
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                          <td style="text-align:center" >8%</td>
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                          <td style="text-align:center" >7.6 mL</td>
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                          <td style="text-align:center" >3.2 mL</td>
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                          <td style="text-align:center" >1.2 mL</td>
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                          <td style="text-align:center" >200 μL</td>
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                          <td style="text-align:center" >10 μL</td>
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                      </tr>
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                      <tr>
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                          <td style="text-align:center" >10%</td>
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                          <td style="text-align:center" >6.8 mL</td>
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                          <td style="text-align:center" >4.0mL</td>
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                          <td style="text-align:center" >1.2 mL</td>
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                          <td style="text-align:center" >200 μL</td>
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                          <td style="text-align:center" >10 μL</td>
 +
                      </tr>
 +
                      <tr>
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                          <td style="text-align:center" >12%</td>
 +
                          <td style="text-align:center" >6.0 mL</td>
 +
                          <td style="text-align:center" >4.8 mL</td>
 +
                          <td style="text-align:center" >1.2 mL</td>
 +
                          <td style="text-align:center" >200 μL</td>
 +
                          <td style="text-align:center" >10 μL</td>
 +
                      </tr>
 +
                      <tr>
 +
                          <td style="text-align:center" >15%</td>
 +
                          <td style="text-align:center" >5.2 mL</td>
 +
                          <td style="text-align:center" >5.6 mL</td>
 +
                          <td style="text-align:center" >1.2 mL</td>
 +
                          <td style="text-align:center" >200 μL</td>
 +
                          <td style="text-align:center" >10 μL</td>
 +
                      </tr>
 +
                  </tbody>
 +
              </table>
 +
              </font></p>
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              </div>
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            </div>
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          </div>
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          <div class="panel panel-transparent">
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            <div class="panel-heading">
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              <h4 class="panel-title"><a data-toggle="collapse" data-parent="#ProtocolContent" href="#ABTS"><h3>ABTS Assay</h3></a></h4>
 +
            </div>
 +
            <div id="ABTS" class="panel-collapse collapse">
 +
              <div class="panel-body">
 +
                <p class="text-justify" align="left"><font size="3">
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 +
                </font></p>
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              </div>
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            </div>
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          </div>
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        </div>
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      </div>
 +
    </div>
 
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Revision as of 11:53, 16 October 2016

Notebook

Protocol

General preparation of Native Polyacrylamide gel (Native PAGE gel)


Specifications
A piece of PAGE Gel (~12mL in volume)
Requiring Materials

Materials (12% PAGE gel) Quantity
Gel kit 1 Set
Distilled water 6.0 mL
10X TBE 1.2 mL
30% Acrylamide (29:1) 4.8 mL
10% APS 200 μL
TEMED 200 μL
Storage
4°C, keep wet in 1x TBE to prevent drying
Steps
1. Clean the glass plates and spacers thoroughly. Rinse the plates withdeionized water and ethanol and set them aside to dry.
2. Assemble the glass plates with spacers in gel caster. Make sure there is no water leakage.
3. Prepare the gel solution according to the desired polyacrylamide percentage. Note the addition of TEMED will immediately trigger the gel to polymerize.
4. Vortex the solution roughly for 5 seconds.
5. Quickly piette the gel solution into the caster. Insert the appropriate comb into the gel, prevent to trap air bubbles under the teeth.
6. Let the polymerization go for 30 minutes at room temperature.
7. Surround the comb and the top of the gel with paper towels that have been soaked in 1x TBE. Then seal the entire gel in plastic bag and store it at 4°C until needed.
Notes
Different polyacrylamide percentage results in different speed and discrimating ability for sample running down in electrophoresis. The quantity of the solutions varies with the percentage. The required quantity of native PAGE gel in common polyacrylamide percentage is listed below.
Polyacrylamide percentage Distilled water 30% Acrylamide (29:1) 10X TBE 10% APS TEMED
8% 7.6 mL 3.2 mL 1.2 mL 200 μL 10 μL
10% 6.8 mL 4.0mL 1.2 mL 200 μL 10 μL
12% 6.0 mL 4.8 mL 1.2 mL 200 μL 10 μL
15% 5.2 mL 5.6 mL 1.2 mL 200 μL 10 μL

ABTS Assay


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