T7 RNA POLYMERASE

characteristic

T7 RNA Polymerase is a DNA-dependent RNA polymerase that exhibits extremely high specificity for its cognate promoter sequences. Only T7 DNA or DNA cloned downstream from a T7 promoter can serve as a template for T7 RNA Polymerase-directed RNA synthesis. T7 RNA Polymerase is provided with 100mM DTT and Transcription Optimized 5X Buffer: 200mM Tris-HCl (pH 7.9 at 25°C), 30mM MgCl2, 10mM spermidine, 50mM NaCl. 

Description

T7 RNA Polymerase is a DNA-dependent RNA polymerase that is highly specific for the T7 phage promoters. The 99 KD enzyme catalyzes in vitro RNA synthesis from a cloned DNA sequence under the T7 promoters. RNA produced using the T7 RNA Polymerase is suitable for many applications in research and biotechnology.

Uses

• T7 RNA Polymerase can transcribe RNA from cloned DNA templates that are downstream from a T7 promoter. The synthesis can be performed with labeled NTPs to generate highly labeled RNA. Synthesized RNA can be used in many applications, including: RNA or DNA blotting techniques
• In situ hybridization
• RNase protection studies: Transcripts synthesized by the enzyme are used as precursor RNA for studies on RNA splicing and processing.
• Synthesis of capped RNA in vitro with addition of m7GpppG or m7GpppA in excess over GTP or ATP during the transcription reaction. The generated antisense RNA can be introduced into cells to suppress the expression of the corresponding genes.
• Microarray target synthesis

Application

Radiolabeled RNA probe preparation
RNA generation for studies of RNA structure, processing and catalysis
Expression control via anti-sense RNA
mRNA, sgRNA synthesis

General Description

T7 RNA polymerase is commonly used to transcribe DNA which has been cloned into vectors which have two phage promoters in opposite orientation. RNA can be selectively synthesized from either strand of the insert DNA with different polymerases. Homogeneously labeled single-stranded RNA can be generated with this system. Transcripts can be non-radioactively labeled with biotin or DIG-11-UTP or radioactively labeled to high specific activity with [α-³²P] or [α-³⁵S]-labeled nucleotides.

Synthesis of hybridization probes: T7 RNA polymerase allows highly efficient production of homogeneously labeled RNA. This labeled RNA may be used as hybridization probes in Southern, northern, and dot blots, as well as in situ hybridizations.
Suitable labels:Transcripts can be nonradioactively labeled with biotin-16-UTP or DIG-11-UTP. They may also be radioactively labeled to high specific activity with [α-32P]- or [α-35S]-labeled nucleotides.