E. coli S30 Extract System for Linear Templates

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

1. Lesley, S.A. et al. (1991) J. Biol. Chem. 266, 2632–8.