The simple three step procedure (PCR, Exonuclease III digestion and rapid ligation/transformation) can be completed in one day. The typical cloning efficiency using this method is greater than 80%.
Features and Benefits
- Universal - PCR amplicon can be cloned into any expression vector
- High Cloning Efficiency - Typically >80%
- High Expression Efficiency - Typically >66%
- High Fidelity - Long and accurate, hotstart enzyme generates amplicons up to 20 kb with fidelity up to 6.5× greater than standard Taq DNA polymerase
- Fast - Simple three-step procedure allows completion in less than one day
The Director Universal Cloning System provides a simple, rapid and universal method to directionally clone PCR products into a vector cleaved with 5′ overhang-producing restriction endonucleases.
Directionality is achieved by pairing directionally designed PCR primers (e.g., containing restriction sites) with any appropriately digested plasmid. The kit contains an optimized nucleotide triphosphate mix, containing dATPαS and dGTPαS, that is used for the PCR step. After PCR, the cohesive 5′ termini of the amplicon are generated by Exonuclease III digestion instead of being generated by traditional restriction enzyme digestions. The dA/GTPαS that was incorporated into the amplicon during PCR protects it from over-digestion by Exonuclease III. The nucleotide mix in the kit is specially formulated so that the amplicon terminates at a statistically determined array of 3′ dA/GαS sites. PCR primers are designed such that the 5′ termini complement the 5′ overhangs of the predigested plasmid.
Director is a trademark of Sigma-Aldrich Co. LLC