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Abstract for 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 only needs to provide linear DNA containing a
prokaryotic E. coli-like promoter and sequence for a ribosomal binding
site. 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 S30 Extract for Linear Templates is prepared from an E. coli B
strain (SL119), which is deficient in OmpT endoproteinase, lon protease and
exonuclease V (recBCD). The absence of protease activity results in
greater stability of expressed proteins. The recD mutation of the SL119
strain produces a more active S30 Extract for Linear DNA than the previously
described nuclease-deficient, recBC-derived S30 extracts. However, the
S30 Extract for Linear Templates is less active than the S30
Extract System for Circular DNA (Cat.# L1020).
An easy-to-perform, non-radioactive positive control reaction using the Luciferase Assay Reagent
provided allows detection of luciferase gene expression in the S30 System for
Linear Templates. The assay reaction produces high light output for several
minutes, allowing the researcher to choose from several methods of detection,
including simple visual observation of luminescence.
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