Incorporate up to 5 different site-specific mutations into plasmid or BAC constructs in a single reaction and assemble your constructs without the need for compatible restriction digestion sites

The Gibson Assembly Site Directed Mutagenesis (SDM) process begins by creating PCR primers for substitution, insertion, or deletion mutagenesis. PCR is used to introduce the mutations and create overlapping ends using the 2X GA SDM PCR Mix. To generate the site directed mutant construct, a two-step, Gibson Assembly process is performed. First, the mutant DNA fragments and vector 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 this highly efficient and robust approach results in double stranded, fully ligated DNA construct ready for downstream analysis.

Gibson Assembly was developed by Dr. Dan Gibson and his team at the J. Craig Venter™ Institute in 2009 and is backed by over 600 peer reviewed publications. Clones produced by Gibson Assembly can be used for a variety of applications including transfection, transformation, PCR, rolling circle amplification, etc. Both small and simple as well as large and complex constructs have been generated by Gibson Assembly. Using this method and multi-stage reactions the Gibson method has been used to create synthetic DNA constructs up to 100 kb in length.

This flexible site directed mutagenesis kit is based on the Gibson Assembly® Method and allows laboratories to easily incorporate base changes, insertions of up to 40 bp, and deletions of any size. Suitable for reactions requiring from one to five fragments with sizes of 500 bp to 5.5 kb you can achieve efficiencies of 95% for a single site mutation and 55% for five site mutations.