Purification and characterization of transcribed RNAs using gel filtration chromatography

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Abstract

RNA synthesis using in vitro transcription by phage T7 RNA polymerase allows preparation of milligram quantities of RNA for biochemical, biophysical and structural investigations. Previous purification approaches relied on gel electrophoretic or gravity-flow chromatography methods. We present here a protocol for the in vitro transcription of RNAs and subsequent purification using fast-performance liquid chromatography. This protocol greatly facilitates production of RNA in a single day from transcription to purification.

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Figure 1: Schematic outline of RNA sample preparation.
Figure 2: Sample elution profiles obtained from the gel filtration chromatography step, demonstrating the separation of in vitro-transcribed RNA (10-ml reaction) from plasmid and nucleotide triphosphates (NTPs).
Figure 3: Example of a monomer/dimer equilibrium after an in vitro transcription (10-ml reaction).
Figure 4: RNA sample purity.

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Correspondence to Joseph D Puglisi.

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