The Gibson Assembly® Ultra Kit provides a robust and efficient method to seamlessly construct synthetic genes, genetic pathways, as well as entire genomes. This method is designed for complex assembly of 2 to 15 large DNA constructs using only small amounts (nanograms) of DNA.

• No restriction digestion required
• Assembles up to 15 inserts at one time
• Compatible with fragments from 100 bp to 100 kb
• Robust two step reaction
• >95% cloning efficiency

The Gibson Assembly® method allows the insertion of one or more linear double stranded DNA fragments into a virtually any vector without the need to rely on compatible restriction sites. Gibson Assembly® is significantly faster than traditional restriction enzyme digest-based cloning and proven for the cloning of both small and large double stranded DNA fragments.

The Gibson Assembly® process begins by designing dsDNA fragments with 20 – 40 bp overlapping ends. For the Gibson Assembly® Ultra reaction, a two-step process is used. Firstly, DNA fragments are combined with a 2X Gibson Assembly® Ultra Master Mix A and incubated to generate overlapping free ends. After enzyme inactivation the reaction is slowly cooled to anneal overlapping ends. Next the 2X Gibson Assembly® Ultra Master Mix B is added to allow the generation of a fully sealed construct. This highly efficient and robust cloning method takes about 80 minutes and results in a transfection or transformation ready, double stranded, fully ligated DNA construct.

Gibson Assembly® Ultra reaction occurs in two steps using the addition of two master mixes in two sequential steps. In the first step, the Gibson Assembly® Ultra Master Mix A (2X) creates single-strand DNA 5’ overhangs by chewing back DNA from the 3’ end. This reaction is cooled under conditions that allow for annealing of complementary overlap regions. Next, the Gibson Assembly® Ultra Master Mix B (2X) is added. During this step, nucleotides are incorporated into the construct to fill in the gaps in the annealed DNA fragments and nicks are sealed to create a contiguous DNA construct.