Storage temp.
−20°C
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General description
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 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.
Application
Production of C-terminal FLAG-tagged fusion proteins.
Components
- 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
Legal Information
Purchase of this product includes an immunity from suit under patents specified in the product insert to use only the amount purchased for the purchaser′s own internal research. No other patent rights (such as 5′ Nuclease Process patent rights) are conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained by contacting the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA.
AccuTaq is a trademark of Sigma-Aldrich Co. LLC
Director is a trademark of Sigma-Aldrich Co. LLC
ExoClone is a trademark of Sigma-Aldrich Co. LLC
FLAG is a registered trademark of Merck KGaA, Darmstadt, Germany
JumpStart is a trademark of Sigma-Aldrich Co. LLC
REDAccuTaq is a registered trademark of Merck KGaA, Darmstadt, Germany
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