RPOLT3-RO

T3 RNA Polymerase

from Escherichia coli HB101

• Synonym(s): mRNA, polymerase
• Enzyme Commission number: 2.7.7.6 (BRENDA, IUBMB)
• UNSPSC Code: 12352204

Properties

• biological source: Escherichia coli (HB101)
• Quality Level: 100
• Assay: 100% (SDS-PAGE)
• form: solution
• specific activity: ≥20 U/μL
• mol wt: 100 kDa (single polypeptide chain)
• packaging: pkg of 1,000 U (11031163001); pkg of 5,000 U (11031171001)
• manufacturer/tradename: Roche
• concentration: <0.1 % (w/w)
• technique(s): DNA sequencing: suitable; Northern blotting: suitable; Southern blotting: suitable; hybridization: suitable

Description

General description

With this enzyme, homogeneously labeled single-stranded RNA can be generated. Transcripts can be nonradioactively labeled with biotin or DIG-11-UTP or radioactively labeled to high specific activity with [α-³²P]- or [α-³⁵S]-labeled nucleotides. T3 RNA polymerase is a DNA-dependent RNA polymerase that is highly specific for its promoter. Only DNA downstream from the T3 promoter will be transcribed.

Specificity

Promotor specifity
T3 RNA polymerase is extremely promoter-specific and only transcribes bacteriophage T3 DNA or DNA cloned downstream of a T3 promoter.
Heat inactivation: Stop the reaction by adding 2 μl 0.2 M EDTA (pH 8.0) and/or heat to 65 °C.

Application

T3 RNA Polymerase can transcribe RNA from cloned DNA templates that are downstream from a T3 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.

Packaging

1 kit containing 2 components

Quality

Absence of endonucleases, nicking activity, and RNases tested according to the current Quality Control procedures. Function tested with the SP6/T7 Transcription Kit.

Specifications

Volume Activity: =20U/μl
Synthesis of hybridization probes: T3 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, DIG-11-UTP, or fluorescein-12-UTP. They may also be radioactively labeled to high specific activity with [a-32P]- or [a-35S]-labeled nucleotides.

Note: Roche has 10x concentrated RNA Labeling Mixes that are specially designed for DIG-, biotin-, and fluorescein-labeling. These mixes work well with T3 RNA Polymerase.

Unit Definition

One unit is the enzyme activity which incorporates 1 nMol CMP in acid-precipitable RNA products within 60 minutes at +37 °C.

Volume Activity: ≥20 U/μl

Preparation Note

Activator: BSA/spermine

Storage and Stability

Keep container tightly closed in a dry and well-ventilated place.

Other Notes

For life science research only. Not for use in diagnostic procedures.

Kit Components Only

• T3 RNA Polymerase, in buffer, pH 7.9 ≥20 U/μl
• Transcription Buffer 10x concentrated

Safety Information

• Pictograms: GHS07
• Signal Word: Warning
• Hazard Statements: H319
• Precautionary Statements: P264 - P280 - P305 + P351 + P338 - P337 + P313
• Hazard Classifications: Eye Irrit. 2
• Storage Class Code: 12 - Non Combustible Liquids
• WGK: WGK 2
• Flash Point(F): does not flash
• Flash Point(C): does not flash