Bacteriophage T7 RNA Polymerase (RNAP) is a highly processive DNA-dependent RNA polymerase that catalyzes the transcription of T7 genes1. It acts by unwinding the double-stranded DNA to provide access to the coding strand for transcription initiation (Figure 1). The 99 kDa enzyme catalyzes in vitro RNA synthesis from a cloned DNA sequence under T7 promoters. RNA produced using the T7 RNAP is suitable for many applications in research and biotechnology.
Intact Genomics IG® T7 RNAP is a purified recombinant T7 bacteriophage enzyme that is expressed and purified from E. coli. This enzyme is designed to perform in vitro transcription of a gene under a phage T7 promoter. The physical purity of this enzyme is ≥85% as assessed by SDS-PAGE with Coomassie® blue staining and gel densitometry (Figure 2).
Fig. 1: The T7 RNAP enzyme is a protein that binds to and transcribes RNA from double-stranded DNA. A molecular surface representation of T7 RNAP in complex with DNA (PDB ID# 3e2e) is shown in the early stages of transcription. Protein residues-green, DNA-orange and blue strands, Initiating RNA strand-black.
Applications
- In vitro RNA synthesis from T7 promoter-containing DNA templates
- mRNA, sgRNA, and other functional RNA synthesis
- Radiolabeled and nonradioactive RNA probe preparation
- RNA standards and controls production
- Aptamer, ribozyme, and antisense RNA synthesis
- RNA preparation for structural and functional studies
- mRNA therapeutic research and development
- General molecular biology and in vitro translation research
Product Source
E. coli BL21 (DE3) strain expressing a recombinant T7 RNA polymerase gene (T7 gene 1) with an N-terminal 6xHis tag.
Components and Storage
The T7 RNA Polymerase product contains the items below. Store all components at -20°C.
- T7 RNA Polymerase
- 10x T7 RNA Polymerase Reaction Buffer
- NTPs mix (10 mM)
Quality Control Assays
T7 RNA Polymerase is free from detectable RNase, Endonuclease (nicking), and DNase activities.
Functional Testing
IG® T7 RNA Polymerase functional testing was done by performing in vitro transcription with the IG® sgRNA synthesis kits (Figure 3, also see Intact Genomics Catalog numbers 3203 and 3303.)
Figure 3: Synthesized RNA product analyzed on a Urea/TBE-PAGE RNA gel.
Lane 1: RNA ladder
Lane 2: IG synthesized guide RNA (60 bases)
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