Team:Exeter/lab book/proto/10

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 ...")
 
Line 89: Line 89:
  </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 23:50, 26 September 2012

Protocol 10

Linearized Plasmid Backbone Digestion and Ligation Protocol
  • For digestion of the linearized plasmid backbone, make an enzyme master-mix of:
    • 5 µL NEB Buffer 2
    • 0.5 µL BSA
    • 0.5 µL EcoRI-HF
    • 0.5 µL PstI
    • 18.5 µL dH20

  • Then add 4µL of linearized plasmid backbone (25ng/µL for 100ng total) into 4µL of enzyme master mix.

  • Digest for 37C/30 min and then heat kill at 80C/20 min.

  • Once heat killed, a ligation mix is made-up containing:
    • 2µl of digested plasmid backbone (25ng)
    • Equimolar amount of EcoRI-HF SpeI digested fragment (< 3 µL)
    • Equimolar amount of XbaI PstI digested fragment (< 3 µL)
    • 1 µL T4 DNA ligase buffer.
    • 0.5 µL T4 DNA ligase
    • Top up solution to 10µL with water

  • Ligate at 16C/30 min and then heat kill at 80C for 20 min.

  • Transform with 1-2µL of product.
    •

    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/10"