Team:British Columbia/Protocols/GibsonAssembly

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This protocol closely follows the suggested protocol in the NEB Gibson Assembly guidebook.
 
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1. Using purified DNA, add 50-100ng plasmid and 2-3X molar concentration of the inserts, or 5X 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.
 
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2. Add 10 uL Gibson Assembly Master Mix to each product.
 
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<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>
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3. Incubate at 50°C for 1 hour.
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<h1>Gibson Assembly Protocol</h1>
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This protocol closely follows the suggested protocol in the NEB Gibson Assembly guidebook.
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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.
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#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.
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#Add 10 uL Gibson Assembly Master Mix to each product.
-
 
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#Incubate at 50°C for 1 hour.
-
 
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#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.
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<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>
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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.
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