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

{background-color: rgba(255,255,255,0.4);

       <li class="active"><a href="#">Experiments and Protocol</a></li>

               <h4 class="panel-title"><a data-toggle="collapse" data-parent="#ProtocolContent" href="#Tetra"><h3>Assembly of tetrahedron</h3></a></h4>

                <p class="text-justify" align="left"><font size="3">

                    1. Wipe the bench and gloves with 70% ethanol.<br>

                                <th style="text-align:center">Volume of 1X TM buffer</th>

                            <td style="text-align:center">O1 (5μM)</td>

 

                    TM Buffer is a Tris buffer containing Magnesium ion. Here, we prepare TM Buffer of 40 mM Tris, 100mM MgCl₂ and is maintained at pH 8.0 condition.<br>

                    For the steps, it is noted that the pH of the buffer is adjusted to 8 by adding HCl with the aid of a pH meter.

                    The above quantity is in 2X concentarion, therefore the solution is further diluted to 1X by mixing 115μL 2X TM buffer with 115μL distilled water to an eppendorf.<br>

              <h4 class="panel-title"><a data-toggle="collapse" data-parent="#ProtocolContent" href="#PAGE"><h3>Native Polyacrylamide gel electrophoresis</h3></a></h4>

              <h4>General preparation of Native Polyacrylamide gel (Native PAGE gel)</h4>

              <table class="table" border="0" >

                          <td>6.0 mL</td>

                          <td>30% Acrylamide (29:1)</td>

              4°C, keep wet in 1x TBE to prevent drying<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>

              The quantity of the solutions varies with the percentage.

              The required quantity of native PAGE gel in common polyacrylamide percentage is listed below. <br>

              For running the electrophoresis, the buffer is further diluted for 10 times to 1X concentration. <br>

              <table class="table">

              Load the followings. (DNA ladder: 2μL, DNA sample: 8μL, 1X loading dye: 8μL)

              Run the gel at constant voltage at 100V for until the bands of dye reach ¾ of the length of the gel.

          <div class="panel panel-transparent">

              <table class="table" border="0">

<td>100mL</td>

                     </tr>

                  <thead>

                    <td></td>

                         <td></td>

                    </tr>

                          <th style="text-align:center">Lane 1</th>

                          <th style="text-align:center">Lane 2</th>

                      <td style="text-align:center">DNA ladder (2 log)</td>

                5. Run the gel at a constant voltage of 100V for 1 hour.<br>

                <p class="text-justify" align="left"><font size="3">

                <br><b>Specification</b><br>

                        <th>Matrials</th>

                            <td>95 mL</td>

                        <td>1M Tris</td>

                             <td>5 mL</td>

                        <td>NH₄Cl</td>

                             <td>0.8023 g</td>

                        <td>KCl</td>

                             <td>0.1491 g</td>

                        <td>HCl</td>

                             <td>variable amount</td>

                        <td>Trition X-100</td>

                             <td>30 μL</td>

                    </tbody>

                </table>

                 <br>

                1. Dilute 1M Tris buffer into 50mM by adding 5mL 1M Tris buffer to 95mL of distilled water.

                2. Dissolve 0.8g NH₄Cl and 0.15g KCl into the solution.

                3. Adjust pH of the solution to 7.5 by adding 1M HCl drop by drop with the aid of a pH meter.

                4. Add 30μL of Triton X-100 to the solution and shake vigorously.

                 </font></p>

               <h4 class="panel-title"><a data-toggle="collapse" data-parent="#ProtocolContent" href="#Cell"><h3>Cell culture</h3></a></h4>

             <div id="Cell" class="panel-collapse collapse">

                 <br><b>Steps</b><br>

                1. Mix thoroughly the above (except the antibodic) with 1L of distilled water.<br>