Team:British Columbia/Protocols/GibsonAssembly

From 2012.igem.org

(Difference between revisions)
(Created page with "{{Template:Team:British Columbia Header}}")
 
(7 intermediate revisions not shown)
Line 1: Line 1:
{{Template:Team:British Columbia Header}}
{{Template:Team:British Columbia Header}}
 +
<html>
 +
<style>
 +
#sponsormap {width:320px;float:left; background-color: white; margin-left: 8px;  margin-top:10px;}
 +
#protocol {width:600px;float:left; background-color: white; margin-left: 8px;  margin-top:10px;}
 +
</style>
 +
<map name="sponsormap">
 +
<area shape="rect" coords="40,40,255,100" href="https://2012.igem.org/Team:British_Columbia/Protocols/Competent_Cell_Production" />
 +
<area shape="rect" coords="40,120,255,180" href="http://partsregistry.org/Help:Protocols/Competent_Cells" />
 +
<area shape="rect" coords="40,200,255,260" href="http://www.neb.com/nebecomm/products/protocol631.asp" />
 +
<area shape="rect" coords="40,280,255,340" href="https://2012.igem.org/Team:British_Columbia/Protocols/Restriction_Digests" />
 +
<area shape="rect" coords="40,360,255,420" href="https://2012.igem.org/Team:British_Columbia/Protocols/Site_Directed_Mutagenesis" />
 +
<area shape="rect" coords="40,440,255,500" href="https://2012.igem.org/Team:British_Columbia/Protocols/GibsonAssembly" />
 +
<area shape="rect" coords="40,520,255,580" href="https://2012.igem.org/Team:British_Columbia/Protocols/DNAPrepBacteria" />
 +
 +
<area shape="rect" coords="40,640,255,700" href="https://2012.igem.org/Team:British_Columbia/Protocols/ConsortiaFluor" />
 +
<area shape="rect" coords="40,720,255,780" href="https://2012.igem.org/Team:British_Columbia/Protocols/AArate" />
 +
<area shape="rect" coords="40,800,255,860" href="https://2012.igem.org/Team:British_Columbia/Protocols/Monod" />
 +
<area shape="rect" coords="40,880,255,940" href="https://2012.igem.org/Team:British_Columbia/Protocols/Biodesulfurization" />
 +
</map>
 +
 +
 +
 +
 +
<div id="sponsormap"><img align="left" src="https://static.igem.org/mediawiki/2012/d/db/Ubcigemnotebookmenu2.jpg" usemap="#sponsormap" alt="UBC iGEM 2012 protocols"> </div><div id=protocol></html>
 +
 +
<h1>Gibson Assembly Protocol</h1>
 +
This protocol closely follows the suggested protocol in the NEB Gibson Assembly guidebook.
 +
 +
#Using purified DNA, add 50-100ng plasmid and 2-3X molar concentration of the inserts, or 5X molar concentration if using inserts of 200 bp or less. Add dH2O up to 10 uL. Set up a positive control using 10uL Gibson Assembly Positive Control.
 +
#Add 10 uL Gibson Assembly Master Mix to each product.
 +
#Incubate at 50°C for 1 hour.
 +
#Using 2uL product, transform into competent cells. Since the Gibson Assembly Master Mix contains PEG, avoid using PEG sensitive competent cells. The provided positive control results in an amp-resistant plasmid.

Latest revision as of 01:23, 4 October 2012

British Columbia - 2012.igem.org
UBC iGEM 2012 protocols

Gibson Assembly Protocol

This protocol closely follows the suggested protocol in the NEB Gibson Assembly guidebook.

  1. Using purified DNA, add 50-100ng plasmid and 2-3X molar concentration of the inserts, or 5X molar concentration if using inserts of 200 bp or less. Add dH2O up to 10 uL. Set up a positive control using 10uL Gibson Assembly Positive Control.
  2. Add 10 uL Gibson Assembly Master Mix to each product.
  3. Incubate at 50°C for 1 hour.
  4. Using 2uL product, transform into competent cells. Since the Gibson Assembly Master Mix contains PEG, avoid using PEG sensitive competent cells. The provided positive control results in an amp-resistant plasmid.
Retrieved from "http://2012.igem.org/Team:British_Columbia/Protocols/GibsonAssembly"