Description
General description
With this system, homogeneously labeled single-stranded RNA can be generated. 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.
Contents:
SP6 RNA Polymerase, ≥20 U/μl, in buffer, pH 7.9
Transcription Buffer, 10x concentrated
Application
SP6 RNA Polymerase can transcribe RNA from cloned DNA templates that are downstream from an SP6 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.
The synthesis of antisense RNA probes
Biochem/physiol Actions
Promoter specifity
SP6 RNA polymerase is extremely promoter-specific and only transcribes bacteriophage SP6 DNA or DNA cloned downstream of a SP6 promoter (e.g., pSPTBM20; pSPTBM21).
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
Synthesis of hybridization probes: SP6 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 SP6 RNA Polymerase.
Preparation Note
Activator: BSA/spermine
Keep container tightly closed in a dry and well-ventilated place
Analysis Note
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 SP6 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 > 30 U.
2. 1 μg Eco RI/Hind III fragments of lambda DNA is incubated with SP6 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 > 30 U.
Absence of Nicking Activity
1 μg pBR322 DNA is incubated with SP6 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 > 30.
Absence of RNases
4 μg MS2 RNA are incubated with SP6 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 > 30.
Performance in Transcription Assay
SP6 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 pST18 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.
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
