For general questions please contact our Customer Service:
Merck KGaA
Frankfurter Str. 250
64293 Darmstadt
Germany
Phone: +49 6151 72-0
Fax: +49 6151 72 2000
15 February 2012
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Any DNA coding sequence inserted downstream from a T7 or SP6 promoter can be quickly expressed as protein simply by adding it to the linked reaction. Suitable templates can be supercoiled or linearized plasmid DNA or PCR products. Compared with standard methods for translation of synthetic RNA templates, several tedious manipulations are avoided, including restriction enzyme digestion, linearized plasmid DNA purification, and RNA purification, which normally require four to six hours to perform. In addition, the STP3 method efficiently produces protein directly from PCR amplification products generated using appropriately designed primers. Performance can be further enhanced using the Novagen pCITE® or pT7Blue-2 vectors containing eukaryotic translation enhancer sequences.
In the standard STP3 reaction, the DNA template (typically 0.5 µg plasmid or 2 µl chloroform-extracted PCR amplification reaction) is transcribed at 30°C for 15 minutes, followed by the addition of translation mix and continued incubation for 60 minutes. All components are premixed such that the only reagents to be added are template, water, and a choice of unlabeled methionine (included) or 35S-methionine (not included).
The kit has enough reagents to perform 50 standard 50-µl reactions or 100 small-scale (25-µl) reactions. A positive control DNA containing the E. coli β-galactosidase gene is included with the kits to monitor performance. The translation product of the STP3 control reaction can be measured by standard incorporation assays, nonradioactive S•Tag™ Rapid Assay or FRETWorks™ S•Tag Assay, S•Tag™ Western Blot, or direct measurement of β-galactosidase activity with the BetaFluor™ β-Gal Assay Kit. Careful quality control ensures that the Single Tube Protein System 3 provides the highest activity, lowest background, and most consistent performance available.
| Related products | |
|---|---|
| 70192 | STP3®, T7 |
| 70205 | Introductory STP3®, T7 |
| Related products for STP3®, SP6 | |
| 70192 | STP3®, T7 |
| 70205 | Introductory STP3®, T7 |
| Product information | ||||||||||||||||||
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| Avoid freeze/thaw | Yes | |||||||||||||||||
| Components |
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| Features and benefits |
Strategies for STP3® PCR Template Preparation Many approaches are possible for rapid protein analysis with the Single Tube Protein® System 3. By using appropriately designed primers, STP3 templates can be synthesized by PCR directly from bacterial colonies, plasmid preparations, baculovirus lysates, or from ligation reactions with various vectors. In addition, by incorporating a T7 or SP6 promoter, upstream spacer, and translation signals (if necessary) into a 5′-primer (e.g., T7/polh primer), templates can be prepared from nonexpressing vectors, vectors lacking T7 or SP6 promoters, genomic DNA exons, or via RT-PCR from cellular mRNA. |
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| Store and ship information | |||
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| Storage | ≤ -70°C | ||
| Ship |
Dry Ice Only
Standard Handling |
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