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

Line 18: Line 18:
 
   <li><p><b>Stacking Gel:</b></br>
 
   <li><p><b>Stacking Gel:</b></br>
 
</br>
 
</br>
−
10 µl 5X Phusion HF Buffer </br>
+
0.7 ml 30 % Acrylamide (37.5:1)</br>
−
5 µl dNTPS [2 mM]</br>
+
1.25 ml Stacking gel buffer (1 M Tris pH 6.8)</br>
−
2.5 µl Forward Primer [10 µM]</br>
+
3 ml Water</br>
−
2.5 µl Reverse Primer  [10 µM]</br>
+
50 ul 10 % (w/v) SDS</br>
−
1 µl Template DNA  </br>
+
5 ul TEMED</br>
−
0.5 µl Phusion HF DNA Polymerase</br>
+
50 ul APS</br>
−
28.5 µl MilliQ Water</br>
+
 
−
<hr style="height:2px;border:none;color:#472400;background-color:#333;" />
+
</li>
−
<p>50 µl Total Reaction </p></li>
+
  
 
</br>
 
</br>
  
 
   <li><p><b>Separating gel:</b></br>
 
   <li><p><b>Separating gel:</b></br>
−
 
+
</br>
−
<table id="t01">
+
12 % 18 %</br>
−
  <tr>
+
<hr style="height:2px;border:none;color:#472400;background-color:#333;" />
−
    <th>Step</th>
+
<p>2 ml 2 ml Seperating buffer (1.5 M Tris pH 8.8)</br>
−
    <th>Temperature</th>
+
3.2 ml 4.8 ml    30 % Acrylamide (37.5:1)</br>
−
    <th>Time</th>
+
2.7 ml 1.1 ml Water</br>
−
  </tr>
+
80 ul 80 ul 10% (w/v) SDS</br>
−
  <tr>
+
8 ul 8 ul TEMED</br>
−
    <td>Initial Denaturation</td>
+
80 ul 80 ul APS</br>
−
    <td>98 °C</td>
+
</br>
−
    <td>30 seconds</td>
+
Run SDS-Page for 1 hour at 30 mA per gel
−
  </tr>
+
−
  <tr>
+
−
    <td rowspan="3"></br></br>35 Cycles</td>
+
−
    <td>98 °C</td>
+
−
    <td>10 seconds</td>
+
−
    </tr>
+
−
    <tr>
+
−
    <td>45 °C – 72 °C</td>
+
−
    <td>30 seconds</td>
+
−
  </tr>
+
−
    </tr>
+
−
    <tr>
+
−
    <td>72 °C</td>
+
−
    <td>30 seconds / 1 kb</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>Final Extension</td>
+
−
    <td>72 °C</td>
+
−
    <td>5 minutes</td>
+
−
  </tr>
+
−
  <tr>
+
−
    <td>Hold</td>
+
−
    <td>4 °C</td>
+
−
    <td>forever</td>
+
−
  </tr>
+
−
</p></table>
+
−
 
+
 
</li>
 
</li>
 +
<p>
  
  

Revision as of 11:47, 14 August 2015

SDS-Page:

Back to Protocols


Reagent


  1. Stacking Gel:

    0.7 ml 30 % Acrylamide (37.5:1)
    1.25 ml Stacking gel buffer (1 M Tris pH 6.8)
    3 ml Water
    50 ul 10 % (w/v) SDS
    5 ul TEMED
    50 ul APS


  2. Separating gel:

    12 % 18 %

    2 ml 2 ml Seperating buffer (1.5 M Tris pH 8.8)
    3.2 ml 4.8 ml 30 % Acrylamide (37.5:1)
    2.7 ml 1.1 ml Water
    80 ul 80 ul 10% (w/v) SDS
    8 ul 8 ul TEMED
    80 ul 80 ul APS

    Run SDS-Page for 1 hour at 30 mA per gel