Engineered by Daniel Gibson and his team in their pursuit to
synthesize a bacterial genome, the Ultra kit has roots in large-scale synthetic
biology. What Daniel Gibson and other leading scientists have also discovered,
is that the Ultra kit performs exceptionally well in enabling small-scale
routine cloning applications quickly and efficiently as well. The Ultra kit
mediates the seamless cloning of up to 15 DNA fragments in a two-step reaction
in a single tube and can be used with any plasmid or BAC vector. The fragments
for assembly can vary widely in size. Standard assembly of a 100 bp fragment
with a pUC19 vector yields a circular construct less than 3 kb in size. On
the other extreme, multi-stage assembly with the Ultra kit can be used to
engineer large constructs 1,000,000 bp in length (1 Mbp).
Gibson Assembly Ultra Protocol
Completed in an 80-minute reaction with less than 6 minutes
of hands-on time, constructs assembled with the Ultra kit are double-stranded,
fully sealed, and can be used immediately in downstream molecular biology
applications. To perform the Gibson Assembly® Ultra cloning method, prepare DNA
fragments and your vector of choice with overlapping ends. Combine 10–300
ng (in equimolar amounts*) of each DNA fragment, the vector, and Ultra Master
Mix A. After incubating the reaction components through a temperature cycle
enabling chew back and annealing, add Ultra Master Mix B to the reaction.
Following a 15-minute incubation at 45°C, the fully ligated constructs are
ready to transform into competent cells.
*We recommend a 5-fold molar excess
of the insert for DNA fragments.
Like the Gibson Assembly® HiFi 1-Step kit, the Ultra kit is available
as a 5-reaction starter kit. Larger kit sizes, including bulk pricing, are also
available. Whether you are planning a big or a small cloning project, our team
of experts can help. Email us at info@sgidna.com
for assistance.
Visit SGI-DNA's web page to learn more about Gibson Assembly® kits and download an electronic version of SGI-DNA's Gibson Assembly® Guide to learn about all the ways you can get #DNAMYWAY.
Gibson Assembly® is a registered trademark of Synthetic Genomics Inc.
Gibson Assembly US Patent Nos. 7,776,532, 8,435,736, and 8,968,999.
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