|
Download
protocol
278kb
pdf? |
|
Abstract for E. coli S30 Extract System for Circular DNA
The E. coli S30 Extract for Circular DNA simplifies the
transcription/translation of DNA sequences cloned in plasmid or lambda vectors,
providing a powerful tool for identifying and characterizing polypeptides. The
investigator needs only to supply the cloned DNA containing the appropriate
prokaryotic
promoter and ribosome binding sites. The S30 Extract System for Circular DNA
is prepared by modifications of the method described by Zubay from an E.
coli strain B deficient in OmpT endoproteinase and lon protease activity.
This results in a greater stability of expressed proteins that would otherwise
be degraded by proteases if expressed in vivo. The S30 in vitro system also
allows higher expression levels of proteins that are normally expressed at low
levels in vivo due to the action of host-encoded repressors.
Expression of the luciferase gene in the S30 System for Circular DNA can be
easily assayed by non-radioactive methods using the provided Luciferase Assay
Reagent. 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.
|
