Team:Exeter/lab book/proto/13

From 2012.igem.org

(Difference between revisions)
(Created page with "{{Template:Team:Exeter/e-candi_banner}} <html xmlns="http://www.w3.org/1999/xhtml"> <head> <meta http-equiv="Content-Type" content="text/html; charset=utf-8" /> <title>Protocol ...")
 
(One intermediate revision not shown)
Line 59: Line 59:
   <font color="#1b1b1d" size="2" face="Verdana">
   <font color="#1b1b1d" size="2" face="Verdana">
   <p>SDS-PAGE was run using 10% NuPAGE<sup>®</sup> Novel<sup>®</sup> Bis-Tris Mini Gels.</p>
   <p>SDS-PAGE was run using 10% NuPAGE<sup>®</sup> Novel<sup>®</sup> Bis-Tris Mini Gels.</p>
-
   <li type="1">Prepare 10µL total volume samples containing:</li>
+
   <li type="1">Prepare a master-mix containing (procedure for 1 sample only):</li>
     <ul type="square">
     <ul type="square">
-
     <li>XµL sample</li>
+
     <li>2x NuPAGE<sup>®</sup> LDS Sample Buffer (4x provided)</li>
-
    <li>2.5µL NuPAGE<sup>®</sup> LDS Sample Buffer (4 x)</li>
+
    <li>2x Sample Reducing Buffer (10x provided)</li>
-
     <li>Up to 7.5µL MilliQ H<sub>2</sub>O</li>
+
     <li>Top up to 50µL using MilliQ H<sub>2</sub>O</li>
     </ul>
     </ul>
     <br>
     <br>
Line 83: Line 83:
  </tr>
  </tr>
 +
</table>
 +
 +
<table width="980" align="center" cellspacing="20">
 +
<tr align="center">
 +
  <td>
 +
  <font color="#57B947" size="1" face="Verdana">
 +
    <p><u>Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley</u> &nbsp;&nbsp;|&nbsp;&nbsp;
 +
    <a href="https://igem.org/Team.cgi?id=764" style="color:#57B947" target="_blank"><u>Contact Us</u></a>  &nbsp;&nbsp;|&nbsp;&nbsp;
 +
    <a href="https://2012.igem.org/Team:Exeter/site_map" style="color:#57B947"><u>Site Map</u></a></p>
 +
  </font>
 +
  </td>
 +
</tr>
</table>
</table>
</body>
</body>
</html>
</html>

Latest revision as of 23:51, 26 September 2012

Protocol 13

SDS-PAGE Protocol

SDS-PAGE was run using 10% NuPAGE® Novel® Bis-Tris Mini Gels.

  • Prepare a master-mix containing (procedure for 1 sample only):
    • 2x NuPAGE® LDS Sample Buffer (4x provided)
    • 2x Sample Reducing Buffer (10x provided)
    • Top up to 50µL using MilliQ H2O

  • Heat samples at 70oC for 10 minutes.

  • Prepare 1 x SDS Running Buffer by adding 50mL 20 x NuPAGE® MES or MOPS SDS Running Buffer to 950mL of deionised water.

  • Load the appropriate concentration of your protein sample on the gel.

  • To load the buffer, fill the Upper (inner) Buffer Chamber with 200mL 1 x NuPAGE® SDS Running Buffer. Fill the Lower (outer) Buffer Chamber with 600mL 1 x NuPAGE® SDS Running Buffer.

  • Then run the SDS-PAGE gel at 200V constant for 35 minutes (for MES Buffer) or 50 minutes (for MOPS Buffer).

  • Once finished, stain using SimplyBlue™ SafeStain, leave for approximately an hour, and then wash using MilliQ H2O for direct visualisation.

    • Website Designed and Built by: Ryan Edginton, James Lynch & Alex Clowsley   |   Contact Us   |   Site Map

    Retrieved from "http://2012.igem.org/Team:Exeter/lab_book/proto/13"