Director^™ Universal PCR System 

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%.

• 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 Biotechnology LP and Sigma-Aldrich Co.

Purchase of this product is accompanied by a limited license for use in the Polymerase Chain Reaction (PCR) process for research purposes only and in conjunction with a thermal cycler whose use in the automated performance of the PCR process is covered by an up-front license fee, either by payment to Applied Biosystems or as purchased, i.e., and authorized thermal cycler. Antibody licensed for in vitro research use under U.S. Patent No. 5,338,671 and 5,587,287, and corresponding patents in other countries. Licensed under U.S. Patent Number 5,436,149 owned by Takara Shuzo Co., Ltd.

sufficient for 25 PCR reactions

wet ice

−20°C

10x AccuTaq™ LA DNA Polymerase Buffer 500 μL

Control PCR Template, 1ng/μl 10 μL

Control RDC primer-R (with 5′ phosphorylation) 25 μL

Control RDC primer-F (with 5′ phosphorylation) 25 μL

ExoNuclease III, 100 units/μl 25 μL

ExoClone^™ 20x DNTP Mix 1 vl

JumpStart™ REDAccuTaq™ LA DNA Polymerase, 1 unit/μl 62.5 μL

Molecular biology grade water 500 μL