The Gibson Assembly® HiFi 1 Step Kit provides a simple and effective method to seamlessly construct synthetic genes, genetic pathways, as well as entire genomes. This method allows researchers to achieve complex assembly of large DNA constructs, with multiple inserts, in 1 hour.
- No restriction digestion required
- Assembles up to 5 inserts at one time
- Compatible with fragments from 500 bp to 21 kb
- Convenient single step reaction
- >90% cloning efficiency
- >95% sequence fidelity
The Gibson Assembly® method allows the insertion of one or more linear double stranded DNA fragments into 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 to 40 bp overlapping ends. For the Gibson Assembly® HiFi 1-Step reaction, these DNA fragments are combined with a 2X Gibson Assembly® HiFi 1-Step Master Mix and incubated. During this time complementary ends are generated, annealed, and sealed. This one-step, isothermal process can be performed in less than 60 minutes and results in a transfection or transformation ready, double stranded, fully ligated DNA construct.
The Gibson Assembly® Hi-Fi 1 Step Master Mix (2X) contains a proprietary mixture of enzymes and reagents optimised to facilitate one-step assembly of double standed DNA fragments. This mastermix includes a proof-reading polymerase that mediates junction repair resulting assembled constructs with low rates of junction errors and high sequence fidelity.
The Gibson Assembly® Positive Control (2X) is sufficient for 2 reactions (5 and 10 reaction kits) or 5 reactions (50 reaction kit). This control consists of a mixture of 10 ng of a 1,5 kb insert and 30 ng of a 2,7 kb vector containing an ampicillin resistance gene. Selection for the 4,2 kb assembled construct can be performed using LB agar plates with 100 μg/ml ampicillin, 0,1 mM IPTG, and 40 μg/ml X-Gal.