Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides.
We present a fast and simple protocol for large-scale preparation and purification of RNA oligonucleotides. RNA oligonucleotides are prepared by in vitro transcription with T7 RNA polymerase from linearized plasmid DNA templates constructed by PCR. In place of denaturing polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography is employed to purify the RNA oligonucleotide from the transcription mixture yielding >99% pure RNA product. In contrast to PAGE-based purification, the gel filtration method does not require denaturation of the RNA oligonucleotide, which is desirable for larger RNAs, and the product is free of low-molecular-weight acrylamide contaminants, which greatly benefits NMR, crystallographic, and other biophysical studies of large RNAs and RNA-protein complexes.
Lukavsky
Peter J
PJ
Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305-5126, USA.
Puglisi
Joseph D
JD
D000180:Acrylic Resins / Q000302:isolation & purification; D001483:Base Sequence; D002850:Chromatography, Gel / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000096:biosynthesis / Q000302:isolation & purification*; D010957:Plasmids / Q000378:metabolism; D014158:Transcription, Genetic; D000099166:Polyacrylamides
D000180:Acrylic Resins; D009838:Oligodeoxyribonucleotides; D000099166:Polyacrylamides