Protocol for E. coli RNA Polymerase Core Enzyme

E. coli RNA Polymerase Core Enzyme
Cat. Nos. C90100 and C90500
E. coli Core RNA Polymerase is free of the sigma subunit and thus will not initiate specific
transcription at promoter sequences on bacterial or bacteriophage DNA.* Nevertheless,
the Core Enzyme will direct low level synthesis of transcripts from the ends of DNA
molecules or from promoter sequences. Addition of sigma factor(s) renders RNA
Polymerase Core Enzyme competent to transcribe double-stranded DNA from specific
promoters.
The enzyme is supplied at a unit concentration of 1 U/μl (0.70 μg/μl, 1.4 x 10 3 U/mg) and
is purified from the rifampicin-sensitive strain BL21.
Product Specifications
Storage: Store only at –70°C.
Storage Buffer: E. coli Core RNA Polymerase is supplied in a 50% glycerol solution
containing 50 mM Tris-HCl (pH 7.5), 250 mM NaCl, 0.1 mM EDTA, and 1 mM dithiothreitol
(DTT).
Unit Definition: One unit catalyzes the incorporation of 1 nmol of ribonucleoside
triphosphates (NTPs) into RNA in 10 min. at 37°C.
Quality Control: E. coli Core RNA Polymerase is function-tested in a reaction containing:
1X E. coli RNA Polymerase Transcription Buffer, 2 mM DTT, 0.25 mM NTPs, and 1 μg T7
D111 DNA as template. The enzyme is activity assayed in a reaction containing: 1X E. coli
RNA Polymerase Transcription Buffer, 10 mM DTT, 0.5 mM ATP, 0.5 mM UTP, and 5 μg of
poly(dA-dT) template.
5X E. coli RNA Polymerase Transcription Buffer: 0.2 M Tris-HCl (pH 7.5), 0.75 M KCl,
50 mM MgCl 2
, and 0.05% Triton® X-100. Optimum buffer composition, including the
concentration of DTT, will vary based on experimental application.
Contaminating Activity Assays: E. coli Core RNA Polymerase is free of detectable DNA
exo- and endonuclease, and RNase activities.
*Core enzyme refers to E. coli RNA Polymerase without the sigma 70 (σ 70 ) subunit as determined by SDS-PAGE, and the lack of initiation of
transcription from the host promoter on bacteriophage T7 DNA. The presence of other minor sigma factors (e.g., σ 35 ), transcription factors
(e.g., GreA or GreB), or transcription accessory proteins are not excluded.
www.epicentre.com Lit. # 015 • 6/2011 1

E. coli RNA Polymerase Core Enzyme
Related Products: The following products are also available:
– E. coli RNA Polymerase Holoenzyme
– NTP Solutions
– Tagetin RNA Polymerase Inhibitor
– E. coli DNA Polymerase I
Tagetin is a trademark of Epicentre, Madison, Wisconsin.
Triton is a registered trademark of Rohm & Haas, Philadelphia, Pennsylvania.
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Notes
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