Difference between revisions of "Team:UiOslo Norway/Experiments/SDS-Page"

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<ul>
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<p><h3>Reagent</h3> </br>
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<li><p>Resuspend cell pellet in 600 ul PBS buffer (pH 7.4) </p></li>
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<ol>
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<li><p>Add glass beads and disrupt cells by vortexing</p></li>
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  <li><p><b>Stacking Gel:</b></br>
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<li><p>Centrifuge at 16.000 x g for 5 minutes</p></li>
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</br>
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<li><p>Transfer soluble fraction into a new tube and resuspend insoluble fraction in 600 ul PBS buffer (pH 7.4)</p></li>
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10 µl 5X Phusion HF Buffer </br>
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</p>
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5 µl dNTPS [2 mM]</br>
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2.5 µl Forward Primer [10 µM]</br>
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2.5 µl Reverse Primer  [10 µM]</br>
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1 µl Template DNA  </br>
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0.5 µl Phusion HF DNA Polymerase</br>
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28.5 µl MilliQ Water</br>
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<hr style="height:2px;border:none;color:#472400;background-color:#333;" />
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<p>50 µl Total Reaction </p></li>
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</br>
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  <li><p><b>Separating gel:</b></br>
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<table id="t01">
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  <tr>
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    <th>Step</th>
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    <th>Temperature</th>
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    <th>Time</th>
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  </tr>
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  <tr>
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    <td>Initial Denaturation</td>
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    <td>98 °C</td>
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    <td>30 seconds</td>
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  </tr>
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  <tr>
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    <td rowspan="3"></br></br>35 Cycles</td>
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    <td>98 °C</td>
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    <td>10 seconds</td>
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    </tr>
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    <tr>
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    <td>45 °C – 72 °C</td>
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    <td>30 seconds</td>
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  </tr>
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    </tr>
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    <tr>
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    <td>72 °C</td>
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    <td>30 seconds / 1 kb</td>
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  </tr>
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  <tr>
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    <td>Final Extension</td>
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    <td>72 °C</td>
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    <td>5 minutes</td>
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  </tr>
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  <tr>
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    <td>Hold</td>
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    <td>4 °C</td>
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    <td>forever</td>
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  </tr>
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</p></table>
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</li>
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Revision as of 11:44, 14 August 2015

SDS-Page:

Back to Protocols


Reagent


  1. Stacking Gel:

    10 µl 5X Phusion HF Buffer
    5 µl dNTPS [2 mM]
    2.5 µl Forward Primer [10 µM]
    2.5 µl Reverse Primer [10 µM]
    1 µl Template DNA
    0.5 µl Phusion HF DNA Polymerase
    28.5 µl MilliQ Water

    50 µl Total Reaction


  2. Separating gel:

    Step Temperature Time
    Initial Denaturation 98 °C 30 seconds


    35 Cycles    		 98 °C 10 seconds
    45 °C – 72 °C 30 seconds
    72 °C 30 seconds / 1 kb
    Final Extension 72 °C 5 minutes
    Hold 4 °C forever