In vitro transcription with T7 RNA polymerase
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In vitro T7 transcription is the synthesis of RNAs using a T7 promoter and purified enzyme. It is the standard method of making up to several mg of RNAs longer than about 20nts with relatively high quality as compared to solid phase synthesis.
In vitro transcribed RNAs like those from T7 or similar viral promoters like T3 and SP6 are important components for many molecular biology experiments. They can be used to generate (antisense) RNA probes for blot hybridisation and nuclease protection assays that are more sensitive than randomly primed DNA probes. Modified nucleotides containing isotopes like 32P or detectable epitopes like DIG can be integrated into the RNA via T7 transcription. Synthesis can be scaled up for microinjection, viral RNA infection studies, in vitro translation, and binding experiments.
The Sauer lab has an excellent, detailed protocol: Sauer:In vitro transcription with T7 RNA polymerase.
For a detailed description of PCR-based attachment of T7 promoters see Making RNA probes with T7 transcription.
Related OWW pages
- Rebuilding T7
- Endy:Dedicated systems/Transcription
- vectors with an N-terminal T7 sequence: pET11a-d, pET-3a-d
- vector for blue/white screening and T7 transcription pETBlue