The E. coli S30 Extract System for Linear Templates is prepared using minor modifications of the protocol described by Lesley and colleagues and allows successful transcription/translation of linear DNA templates. The investigator need only provide linear DNA containing a prokaryotic E. coli-like promoter (such as lacUV5, tac, λPL (con) and λ-PR). A ribosome binding site is required to direct the synthesis of proteins in vitro. In vitro-generated RNA from DNA templates lacking an E. coli promoter may also be used in this system, but protein yields will be decreased to 1–10% of that produced from linear DNA templates.
The E. coli S30 Extract System for Linear Templates allows the use of many different templates, including DNA fragments, PCR-synthesized DNA, ligated overlapping oligonucleotides, in vitro-generated RNA and prokaryotic RNA. The use of this system reduces the chance of expressed proteins degrading.
For more information, see the Protocols & Applications Guide.
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Applications
- Identification of cloned or synthesized genes from linear or closed circular DNA.
- Rapid structure mapping of genes without additional cloning steps.
- Rapid verification of in vitro-generated mutations.
- Synthesis of truncated gene products for functional domain mapping and epitope mapping studies.
- Expression from in vitro-generated RNA for genes lacking an appropriate E. coli promoter.
Reference
- Lesley, S.A. et al. (1991) J. Biol. Chem. 266, 2632–8.