ID0010
Sigma
In Vitro Director™ PCR System
3′ FLAG®
Description
| 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 |
| Application | Production of C-terminal FLAG-tagged fusion proteins. |
| 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 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. | |
| 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. 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. |
| ExoClone is a trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co. | |
| Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,079,352, 5,789,224, 5,618,711, 6,127,155 and claims outside the US corresponding to US Patent No. 4,889,818. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product for the purchaser’s own internal research. No right under any other patent claim (such as the patented 5′ Nuclease Process claims in US Patents Nos. 5,210,015 and 5,487,972), no right to perform any patented method, and no right to perform commercial services of any kind, including without limitation reporting the results of purchaser’s activities for a fee or other commercial consideration, is conveyed expressly, by implication, or by estoppel. This product is for research use only. Diagnostic uses under Roche patents require a separate license from Roche. 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. | |
| JumpStart is a trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co. | |
| AccuTaq is a trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co. | |
| REDAccuTaq is a registered trademark of Sigma-Aldrich Biotechnology LP and Sigma-Aldrich Co. | |
| Director 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. |
Properties
| storage temp. | −20°C |
Safety
| Hazard Codes | Xi |
| Risk Statements | 36/37/38-52 |
| Safety Statements | 26-36 |
