RNA Polymerase T7

Application

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
 

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.

Other Notes

Test Buffer
40 mM Tris-HCl, pH 8.0 (+20°C), 6 mM MgCl2, 10 mM dithiothreitol, 2 mM spermidine, pH approximately 8.0 (+20°C).
Absence of Endonucleases
1. 1 μg lambda DNA is incubated with T7 RNA polymerase for 4 hours at +37°C in 25 μl test buffer. The number of enzyme units which show no degradation of lambda DNA is >100 U.
2. 1 μg Eco RI/Hind III fragments of lambda DNA is incubated with T7 RNA polymerase for 4 hours at +37°C in 25 μl test buffer. The number of enzyme units which show no alteration of the banding pattern is >100 U.
Absence of Nicking Activity
1 μg pBR322 DNA is incubated with T7 RNA polymerase for 4 hours at +37°C in 25 μl test buffer. The number of enzyme units which show no relaxing of supercoiled structure is >100 U.
Absence of RNases
4 μg MS2 RNA are incubated with T7 RNA polymerase for 4 hours at +37°C in 50 μl test buffer. The number of enzyme units which show no degradation of MS2 RNA is >100 U.
Performance in Transcription Assay
T7 RNA polymerase is function tested in the SP6/T7 Transcription Kit (Cat. No. 10 999 644 001). The incorporation rate in the standard assay with 0.5 μg pSPT19 neo DNA linearized with Eco RI and 50 mCi [alpha-32P] CTP, [400 Ci/mmol (15 TBq/mmol)] gives >50% of the input radioactivity in 20 minutes.

For life science research only. Not for use in diagnostic procedures.
Roche has 10x concentrated RNA Labeling Mixes that are specially designed for DIG- or biotin-labeling. These mixes work well with T7 RNA Polymerase.

包装

1 kit containing 2 components

Preparation Note

Activator: The T7 RNA polymerases are strongly stimulated by BSA or spermidine.

Specificity

Promotor specifity
T7 RNA polymerase is extremely promoter-specific and only transcribes bacteriophage T7 DNA or DNA cloned downstream of a T7 promoter. Although the T7 and T3 promoter sequences differ only by 3 bp, T7 RNA polymerase only transcribes DNA cloned downstream of its promoter.
Heat inactivation: Stop the reaction by adding 2 μl 0.2 M EDTA (pH 8.0) and/or heat to 65 °C.

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