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Team:Calgary/Notebook/Protocols/Gibson Assembly
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| - | <p>In 2012, the iGEM Calgary team tried its first attempt at Gibson Assembly. We inteded to use this method to assemble parts from plasmids into larger constructs and save assembly time. For our first construction, we attempted to build the rhamnose promoter with GFP and its control protein | + | <p>In 2012, the iGEM Calgary team tried its first attempt at Gibson Assembly. We inteded to use this method to assemble parts from plasmids into larger constructs and save assembly time. For our first construction, we attempted to build the <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902065">rhamnose promoter</a> with <a href="http://partsregistry.org/Part:BBa_K082003">GFP</a> and its <a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K902068">control protein</a>. Primers were designed to be fully compatible with the Biobrick Standard—scar sites and cloning sites were introduced where appropriate.</p> |
<p>We custom designed primers for and used <a href="http://www.thermoscientificbio.com/pcr-enzymes-master-mixes-and-reagents/pfu-dna-polymerase/">Pfu polymerase</a> to PCR the genes out from plasmid vectors. Also, we purchased commercial <a href="http://www.neb.com/nebecomm/products/productE2611.asp">Gibson master mix</a> from New England Biolabs; primers were designed to meet parameters in their protocol.</p> | <p>We custom designed primers for and used <a href="http://www.thermoscientificbio.com/pcr-enzymes-master-mixes-and-reagents/pfu-dna-polymerase/">Pfu polymerase</a> to PCR the genes out from plasmid vectors. Also, we purchased commercial <a href="http://www.neb.com/nebecomm/products/productE2611.asp">Gibson master mix</a> from New England Biolabs; primers were designed to meet parameters in their protocol.</p> | ||
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Latest revision as of 23:01, 26 October 2012
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Gibson Assembly
In 2012, the iGEM Calgary team tried its first attempt at Gibson Assembly. We inteded to use this method to assemble parts from plasmids into larger constructs and save assembly time. For our first construction, we attempted to build the rhamnose promoter with GFP and its control protein. Primers were designed to be fully compatible with the Biobrick Standard—scar sites and cloning sites were introduced where appropriate.
We custom designed primers for and used Pfu polymerase to PCR the genes out from plasmid vectors. Also, we purchased commercial Gibson master mix from New England Biolabs; primers were designed to meet parameters in their protocol.
