The Gibson Assembly® Method is a well-established assembly reaction that can be leveraged to join multiple, mutagenised DNA fragments with overlapping ends. Following mutagenesis, DNA fragments of various lengths are uniformly assembled using complementary overlaps between fragments. The inherent flexibility of this approach lends itself to small and large constructs and encompasses both single and multiple insert assemblies.
- No restriction digestion required
- Incorporate up to 5 site-specific mutations into plasmid or BAC constructs in a single reaction
- Compatible with fragments from 500 bp to 5,5 kb
- Allows for up to 40 bp insertions and any size deletion
- Optimal for assembly of up to 5 fragments
- Achieve efficiencies of 95% for single mutation and 55% for 5-site mutations
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.
Firstly, 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 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.
Tilaustiedot: All kits contain one vial of Gibson Assembly® SDM PCR Mix (2X), one vial of Gibson Assembly® SDM Assembly Mix A (2X), one vial of Gibson Assembly® SDM Assembly Mix B (2X), one vial of Control Plasmid, one vial of Control Primer Mix A , Control Primer Mix B, and a detailed instruction manual.