The RNA pull-down assay was performed using RiboTrap Kit (MBL), according to the manufacturer’s instructions. Briefly, the cDNA fragments corresponding to the coding region or 3′-UTR of mouse Inka2 cDNA were subcloned into a pGEM-3Z plasmid vector (Promega) for in vitro transcription. The BrU–labeled RNA was prepared using a CUGA 7 in vitro Transcription Kit (Nippon Gene, Japan). Purified BrU-labeled RNA (50 pmol each) was bound on the Protein G Plus-Agarose beads conjugated to an anti-BrdU monoclonal antibody. Cytoplasmic extract prepared from N2a cells (approximately 1 × 108 cells) according to the manufacturer’s instruction (MBL) was precleared by the treatment with Protein G beads and then incubated with the BrU-labeled RNAs on antibody conjugated beads for 2 h at 4°C. After washing the sample beads four times with the low-ionic strength wash buffer I (+) supplemented with 1.5 mM DTT, BrU-RNA/protein complexes were eluted with 50 μL PBS containing BrdU by immunoblotting.
Pgem 3z plasmid vector
Manufactured by Promega
The pGEM-3Z plasmid vector is a commonly used cloning vector. It contains a multiple cloning site with recognition sequences for various restriction enzymes, allowing for the insertion and manipulation of DNA fragments. The vector also includes an ampicillin resistance gene for selection and a lac operon for blue-white screening of recombinant colonies.
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2 protocols using pgem 3z plasmid vector
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RNA-Protein Interaction Profiling
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RNA-protein Interactions Profiling
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The RNA pull-down assay was performed using RiboTrap Kit (MBL), according to the manufacturer's instructions. Briefly, the cDNA fragments corresponding to the coding region or 3′-UTR of mouse Inka2 cDNA were subcloned into a pGEM-3Z plasmid vector (Promega) for in vitro transcription. The BrU-labeled RNA was prepared using a CUGA 7 in vitro Transcription Kit (Nippon Gene, Japan). Purified BrU-labeled RNA (50 pmol each) was bound on the Protein G Plus-Agarose beads conjugated to an anti-BrdU monoclonal antibody. Cytoplasmic extract prepared from N2a cells (approximately 1 × 10 8 cells) according to the manufacturer's instruction (MBL) was precleared by the treatment with Protein G beads and then incubated with the BrUlabeled RNAs on antibody conjugated beads for 2 h at 4°C. After washing the sample beads four times with the low-ionic strength wash buffer I (+) supplemented with 1.5 mM DTT, BrU-RNA/protein complexes were eluted with 50 μL PBS containing BrdU by immunoblotting.
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