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T4 rna ligase 1 buffer

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T4 RNA ligase I buffer is a solution provided for use with T4 RNA ligase I, an enzyme that catalyzes the formation of a phosphodiester bond between the 5' phosphate and 3' hydroxyl termini of RNA molecules. The buffer composition optimizes the enzymatic activity of T4 RNA ligase I.

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Lab products found in correlation

Pnk buffer, by New England Biolabs (2 mentions) T4 rna ligase 1 ssrna ligase, by New England Biolabs (2 mentions) Agilent tapestation, by Agilent Technologies (1 mentions) Nanodrop one spectrophotometer, by Agilent Technologies (1 mentions) T4 rna ligase 1, by New England Biolabs (1 mentions)

Close spelling variants of this product

T4 ligase (519 citations) T4 RNA ligase 1 (175 citations) T4 RNA ligase (118 citations) T4 ligase buffer (43 citations) T4 RNA ligase buffer (25 citations) RNA ligase 1 (18 citations) RNA ligase (7 citations) 10× ligase buffer (6 citations) Ligases (5 citations) RNA ligase buffer (3 citations)

4 protocols using t4 rna ligase 1 buffer

1

Synthesis and Purification of GLuc APIE CVB3 RNA

Cited 1 time
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Construction of GLuc APIE CVB3 pAC has been described previously (Wesselhoeft et al., 2019 (link)) and was used as template for circular precursor and linear control PCRs. RNA was synthesized using in vitro transcription (IVT) kits (HiScribe T7 High Yield RNA Synthesis Kit). IVT templates were PCR amplified (Q5 Hot Start High-Fidelity 2x Master Mix) as described above and silica column purified prior to RNA synthesis (DNA Clean & Concentrator-100), or digested from plasmid and silica column purified (Purelink, ThermoFisher). One microgram of IVT template was used per reaction size, and reactions were incubated for two hours at 37°C with shaking at 1000rpm. The IVT template was degraded with DNaseI at two units per ten micrograms of expected RNA yield for 20 minutes at 37°C with shaking at 1000rpm. RNA was silica column purified prior to further enzymatic reactions, quantified using a Nanodrop One spectrophotometer, and verified using an Agilent TapeStation according to manufacturer’s recommendations. In some cases, uncircularized IVT product was subjected to a separate circularization step: T4 RNA ligase I buffer (New England Biolabs) was added to silica column-purified RNA at a final concentration of 1x with GTP to a final concentration of 2mM. Samples were heated at 55°C for 8m, and then silica column purified.
Abe B.T., Wesselhoeft R.A., Chen R., Anderson D.G, & Chang H.Y. (2022). Circular RNA migration in agarose gel electrophoresis. Molecular cell, 82(9), 1768-1777.e3.
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2

Splint-Mediated Circularization of Linear RNA

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Linear precursors for splint-mediated ligation were designed to have all of the same sequence features as PIE-circularized circRNA except for the addition of short adapter sequences onto the 5’ and 3’ ends of the precursor RNA. These adapter sequences shared homology with the splints used for circularization (Optimized splint: 5’-GTTTGTGGTTCGTGCGTCTCCGTGCTGTTCTGTTGGTGTGGG-3’). Splint ligation precursor RNA was synthesized as described previously, except a 10-fold excess of GMP was added to in vitro transcription reactions. 25ug of purified precursor RNA was heated to 70°C for 5 minutes in the presence of DNA splint at a concentration of 5uM in a 90uL reaction. The reaction was allowed to cool to room temperature, and then T4 RNA Ligase I Buffer (NEB) was added to a final concentration of 1X. ATP was added to a final concentration of 1mM. 50U of T4 RNA Ligase I (NEB) was added. Reactions were incubated at 37°C for 30 minutes and then column purified.
Wesselhoeft R.A., Kowalski P.S., Parker-Hale F.C., Huang Y., Bisaria N, & Anderson D.G. (2019). RNA circularization diminishes immunogenicity and can extend translation duration in vivo. Molecular cell, 74(3), 508-520.e4.
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3

RNA 5' End Phosphorylation and Adaptor Removal

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5′ end phosphorylation of RNA and removal of adaptor B’s phosphate blocking were simultaneously performed. The supernatant was removed and 20 μl of PNK mix (1 μl T4 PNK, 2 μl 10mM ATP, 2 μl 10× PNK buffer (NEB), 0.25 μl RNasin, 0.25 μl SUPERaseIn, 14.5 μl water) was added. The reaction was incubated at 37°C for 30 min at 1100 rpm shaking. The beads were washed once with 1 ml PNK buffer, twice with 1 ml PGB Cell Lysis Buffer (the wash was rotated for at least 5 min in cold room), and once with 1 ml PNK buffer.
The supernatant was removed and 20 μl of ligation mix (10 μl 10mM ATP, 72.5 μl water, 10 μl 10× T4 RNA ligase 1 buffer (NEB, B0216S that does not contain ATP), 5 μl T4 RNA Ligase 1 (ssRNA Ligase) (NEB, M0204), 2 μl RNasin, 0.5 μl SUPERaseIn) was added. The reaction was incubated overnight at 16°C at 1100 rpm shaking. After incubation, 800 μl of ice-cold PNK buffer was immediately added, the supernatant was removed, and the tube was transferred to ice. The beads were washed once with High Salt Wash and twice with PNK Buffer.
Sugimoto Y., Vigilante A., Darbo E., Zirra A., Militti C., D’Ambrogio A., Luscombe N.M, & Ule J. (2015). hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1. Nature, 519(7544), 491-494.
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4

RNA 5' End Phosphorylation and Adaptor Removal

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5′ end phosphorylation of RNA and removal of adaptor B’s phosphate blocking were simultaneously performed. The supernatant was removed and 20 μl of PNK mix (1 μl T4 PNK, 2 μl 10mM ATP, 2 μl 10× PNK buffer (NEB), 0.25 μl RNasin, 0.25 μl SUPERaseIn, 14.5 μl water) was added. The reaction was incubated at 37°C for 30 min at 1100 rpm shaking. The beads were washed once with 1 ml PNK buffer, twice with 1 ml PGB Cell Lysis Buffer (the wash was rotated for at least 5 min in cold room), and once with 1 ml PNK buffer.
The supernatant was removed and 20 μl of ligation mix (10 μl 10mM ATP, 72.5 μl water, 10 μl 10× T4 RNA ligase 1 buffer (NEB, B0216S that does not contain ATP), 5 μl T4 RNA Ligase 1 (ssRNA Ligase) (NEB, M0204), 2 μl RNasin, 0.5 μl SUPERaseIn) was added. The reaction was incubated overnight at 16°C at 1100 rpm shaking. After incubation, 800 μl of ice-cold PNK buffer was immediately added, the supernatant was removed, and the tube was transferred to ice. The beads were washed once with High Salt Wash and twice with PNK Buffer.
Sugimoto Y., Vigilante A., Darbo E., Zirra A., Militti C., D’Ambrogio A., Luscombe N.M, & Ule J. (2015). hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1. Nature, 519(7544), 491-494.
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