Merck, 11031163001, T3 RNA Polymerase

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.
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.

Biochem/physiol Actions
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.
Features and Benefits
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.
Packaging
1 kit containing 2 components
Preparation Note
Activator: BSA/spermine
Keep container tightly closed in a dry and well-ventilated place.
Analysis Note
Absence of endonucleases, nicking activity, and RNases tested according to the current Quality Control procedures. Function tested with the SP6/T7 Transcription Kit.
Other Notes
For life science research only. Not for use in diagnostic procedures.
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