In Vitro Director^™ PCR System 

• JumpStart^™ REDAccuTaq^® LA DNA Polymerase
• 10X AccuTaq^™ LA DNA Polymerase Buffer
• ExoClone^™ dNTP Mix (20X)
• Control PCR Template
• Control RDC Primer-F
• Control RDC Primer-R
• Exonuclease III
• dNTP Mix, 10 mM
• Water Molecular Biology Reagent
• 5' Anchor
• 3' FLAG Anchor
• Anchor primer- Forward
• Anchor primer-Reverse

Production of C-terminal FLAG-tagged fusion proteins.

Sigma's In Vitro Director™ PCR System provides all reagents needed for the rapid generation of high quality, expression-ready DNA templates that can be used with in vitro transcription/translation systems.In vitro synthesized fusion proteins are very useful for rapid analysis of gene functions, such as enzymatic activity, protein-protein interaction, function domain mapping, and structure analysis.

The PCR System uses the Director method, a novel method that facilitates the directional ligation of transcription/translation regulatory sequences (Anchors) to the PCR products. It differs from conventional PCR kits in that it uses a specially formulated dNTP mix containing dATPaS and dGTPaS. After PCR, the cohesive 5' termini of the amplicon are produced by Exonuclease III digestion instead of being generated by traditional restriction enzyme digests. Incorporation of dA/GTPaS into the amplicon protects the amplicon from overdigestion by Exonuclease III.

The universal process of rapidly generating expression-ready DNA templates involves five steps: a first round PCR using gene-specific primers, Exonuclease III digestion, PCR clean-up, ligation of the PCR amplicon to the 5' and 3' Anchors, and a second round of PCR using Anchor primers.

The kit contains paired 5' and 3' Anchors as well as universal primers complementary to the Anchor sequences. The 5' Anchor is a double-stranded DNA containing the T7 promoter, Kozak sequence, and a Hind III overhang. The 3' FLAG Anchor is also a double-stranded DNA containing a Bgl II overhang, FLAG sequence and terminator sequence.

AccuTaq is a trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.

Director is a trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.

ExoClone is a trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.

FLAG is a registered trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.

JumpStart 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.

REDAccuTaq is a registered trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co.

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