t4 rna ligase  (Thermo Fisher)


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    Structured Review

    Thermo Fisher t4 rna ligase
    Formation of natural and unnatural lariat RNA isomers in the <t>T4</t> RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which
    T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    t4 rna ligase - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "A general two-step strategy to synthesize lariat RNAs"

    Article Title: A general two-step strategy to synthesize lariat RNAs

    Journal: RNA

    doi: 10.1261/rna.2259406

    Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which
    Figure Legend Snippet: Formation of natural and unnatural lariat RNA isomers in the T4 RNA ligase loop-closure reaction, and the blocking and capping approaches to control which isomer is formed. On each structure is marked the cleavage site for debranching enzyme Dbr1p, which

    Techniques Used: Blocking Assay

    2) Product Images from "Synthesis and Labeling of RNA In Vitro"

    Article Title: Synthesis and Labeling of RNA In Vitro

    Journal: Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]

    doi: 10.1002/0471142727.mb0415s102

    Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule
    Figure Legend Snippet: Schematic representation of radiolabeling of RNA at its 3′ end. T4 RNA ligase catalyzes the ligation reaction where 5′[ 32 P]pCp is covalently attached to the 3′ end of the single-stranded RNA substrate. The radiolabeled RNA molecule

    Techniques Used: Radioactivity, Ligation

    Related Articles

    Clone Assay:

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: Clones for the terminal sequences of the dsRNA were generated by T4 RNA ligase oligonucleotide-mediated amplification as described by Lambden et al. ( ). .. The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    Amplification:

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: Clones for the terminal sequences of the dsRNA were generated by T4 RNA ligase oligonucleotide-mediated amplification as described by Lambden et al. ( ). .. The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    Molecular Cloning:

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: Paragraph title: cDNA synthesis, molecular cloning, and sequencing. ... The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    Autoradiography:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase. ..

    Electrophoresis:

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: The resulting PCR product was fractionated by electrophoresis on 1% agarose gel and purified using a gel extraction kit (Axygen). .. The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    Microarray:

    Article Title: MicroRNA expression in the adult mouse central nervous system
    Article Snippet: Paragraph title: Microarray printing, labeling, and hybridization ... Total RNA (2–4 μg) was 3′-end-labeled using T4 RNA ligase and a Cy3-labeled RNA linker ( ; ) by the following procedure: RNA in 4.5 μL of water was combined with 0.8 μL of T4 RNA ligase buffer (10×) (Ambion), 1.1 μL of polyethyleneglycol (50% [w/v]), 0.8 μL of RNA-linker (250 μM; DNA Technology), and 0.8 μL of T4 RNA ligase (Ambion).

    Incubation:

    Article Title: A general two-step strategy to synthesize lariat RNAs
    Article Snippet: The sample was incubated at 37°C for 1.5 h and quenched with 60 μL of stop solution. .. Then, loop closure catalyzed by T4 RNA ligase was performed as follows: Branched RNA (5 pmol) was annealed in 7.5 μL of 5 mM HEPES (pH 7.5), 15 mM NaCl, and 0.1 mM EDTA by heating at 95°C for 3 min and cooling on ice for 5 min. Portions of 5× ligation buffer and T4 RNA ligase (Fermentas) were added, bringing the final conditions to 50 mM HEPES (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50μM ATP with 1 U/μL of T4 RNA ligase in 10 μL total volume.

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: Denatured dsRNA was reverse transcribed using RevertAid Moloney murine leukemia virus (M-MuLV) reverse transcriptase (Fermentas) and a sequence-specific reverse primer and incubated for 60 min at 45°C. .. The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    Article Title: Long Noncoding RNA NEAT1 Suppresses Proliferation and Promotes Apoptosis of Glioma Cells Via Downregulating MiR-92b
    Article Snippet: A total of 50 pmol NEAT1 RNA was labeled using biotin and T4 RNA ligase via a PierceTM RNA 3′End Desthiobiotinylation Kit (Thermo Fisher Scientific). .. Briefly, the cells were lysed with Pierce IP Lysis Buffer and incubated on ice for 5 minutes.

    Modification:

    Article Title: A general two-step strategy to synthesize lariat RNAs
    Article Snippet: For loop closure catalyzed by T4 RNA ligase (step 2), the branched product from step 1 was prepared by a modification of the above procedure. .. Then, loop closure catalyzed by T4 RNA ligase was performed as follows: Branched RNA (5 pmol) was annealed in 7.5 μL of 5 mM HEPES (pH 7.5), 15 mM NaCl, and 0.1 mM EDTA by heating at 95°C for 3 min and cooling on ice for 5 min. Portions of 5× ligation buffer and T4 RNA ligase (Fermentas) were added, bringing the final conditions to 50 mM HEPES (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50μM ATP with 1 U/μL of T4 RNA ligase in 10 μL total volume.

    Article Title: Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27
    Article Snippet: CLIP-seq We used a modified version of eCLIP. .. 3′ RNA linker was added by T4 RNA ligase (Thermo Fisher Scientific) overnight and [γ-32 P]ATP (PerkinElmer) was used to label the 5′ end of RNA by T4 PNK (NEB).

    Derivative Assay:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase. ..

    Hybridization:

    Article Title: MicroRNA expression in the adult mouse central nervous system
    Article Snippet: Paragraph title: Microarray printing, labeling, and hybridization ... Total RNA (2–4 μg) was 3′-end-labeled using T4 RNA ligase and a Cy3-labeled RNA linker ( ; ) by the following procedure: RNA in 4.5 μL of water was combined with 0.8 μL of T4 RNA ligase buffer (10×) (Ambion), 1.1 μL of polyethyleneglycol (50% [w/v]), 0.8 μL of RNA-linker (250 μM; DNA Technology), and 0.8 μL of T4 RNA ligase (Ambion).

    Transfection:

    Article Title: Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27
    Article Snippet: Briefly, infected and transfected HeLa cells were exposed to 254 nM UV with 400 mJ/cm2 , and then scrapped and re-suspended in lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitor). .. 3′ RNA linker was added by T4 RNA ligase (Thermo Fisher Scientific) overnight and [γ-32 P]ATP (PerkinElmer) was used to label the 5′ end of RNA by T4 PNK (NEB).

    Ligation:

    Article Title: Genomic characterization, phylogenetic position and in situ localization of a novel putative mononegavirus in Lepeophtheirus salmonis
    Article Snippet: To increase the efficiency of RNA ligation, the 5’ triphosphate residues of the RNA were removed by incubating 5 μg of total RNA with 5 units of 5’ RNA pyrophosphohydrolase (Rpph; New England Biolabs) in 40 μl of 1 × NEBuffer 2 for 30 min at 37 °C [ ]. .. Purified dephosphorylated RNA (1 μg) was then ligated with 10 U of T4 RNA ligase (ThermoScientific) in 50 μl of 1 × reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNAseOUT (Invitrogen) for 1 h at 37 °C.

    Article Title: A general two-step strategy to synthesize lariat RNAs
    Article Snippet: .. Then, loop closure catalyzed by T4 RNA ligase was performed as follows: Branched RNA (5 pmol) was annealed in 7.5 μL of 5 mM HEPES (pH 7.5), 15 mM NaCl, and 0.1 mM EDTA by heating at 95°C for 3 min and cooling on ice for 5 min. Portions of 5× ligation buffer and T4 RNA ligase (Fermentas) were added, bringing the final conditions to 50 mM HEPES (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50μM ATP with 1 U/μL of T4 RNA ligase in 10 μL total volume. .. To prepare the lariat RNA samples for the assays of Figure 4 , the entire sample was quenched after 15 min with 20 μL of stop solution, and the lariat RNA product was purified by 12% PAGE.

    Article Title: The complete genome sequence of CrRV-Ch01, a new member of the family Rhabdoviridae in the parasitic copepod Caligus rogercresseyi present on farmed Atlantic salmon (Salmo salar) in Chile
    Article Snippet: To increase the efficiency of RNA ligation, the 5’-triphosphate residues of the RNA were removed by incubating 5 μg of total RNA with 5 units of 5’ RNA pyrophosphohydrolase (Rpph; New England Biolabs) in 40 μl of 1X NEBuffer 2 for 30 min at 37 °C [ ], followed by RNA clean-up using an RNeasy Mini Kit (QIAGEN). .. Four hundred ng of purified dephosphorylated RNA was then ligated for 1 h at 37 °C using 10 U of T4 RNA ligase (Thermo Scientific) in 20-μl of 1x reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNaseOUT™ (Invitrogen).

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas). .. The oligonucleotide-ligated dsRNA was denatured and used for the RT reaction with RevertAid M-MuLV reverse transcriptase and 10 pmol of a primer with a sequence complementary to that of the oligonucleotide used for the RNA ligation (oligoREV, 5′-GGCAATTAACCCTCACTAAAG-3′).

    Article Title: Regulation of gene expression by the action of a fungal lncRNA on a transactivator
    Article Snippet: .. CY5-labelling of HAX1 CY5-coupled cytidine-5ʹ-phosphate-3ʹ-(aminohexyl)-phosphate (pCp-CY5, from Jena Bioscience, Jena, Germany) was linked to the 3ʹ end of in vitro synthesized HAX1 299 RNA using T4 RNA Ligase (Thermo Scientific) based on the instructions provided but scaled up for labelling 294.67 pmol (30 µg) HAX1 RNA with equimolar amounts of pCp-CY5 in a 30 µl ligation mixture. .. Subsequently, the CY5-labelled HAX1 RNA was purified using Sephadex G-25 Quick Spin Columns for radiolabelled RNA purification (Roche, Basel, Switzerland) according to the manufacturer’s instructions.

    Protease Inhibitor:

    Article Title: Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27
    Article Snippet: Briefly, infected and transfected HeLa cells were exposed to 254 nM UV with 400 mJ/cm2 , and then scrapped and re-suspended in lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitor). .. 3′ RNA linker was added by T4 RNA ligase (Thermo Fisher Scientific) overnight and [γ-32 P]ATP (PerkinElmer) was used to label the 5′ end of RNA by T4 PNK (NEB).

    Synthesized:

    Article Title: Regulation of gene expression by the action of a fungal lncRNA on a transactivator
    Article Snippet: .. CY5-labelling of HAX1 CY5-coupled cytidine-5ʹ-phosphate-3ʹ-(aminohexyl)-phosphate (pCp-CY5, from Jena Bioscience, Jena, Germany) was linked to the 3ʹ end of in vitro synthesized HAX1 299 RNA using T4 RNA Ligase (Thermo Scientific) based on the instructions provided but scaled up for labelling 294.67 pmol (30 µg) HAX1 RNA with equimolar amounts of pCp-CY5 in a 30 µl ligation mixture. .. Subsequently, the CY5-labelled HAX1 RNA was purified using Sephadex G-25 Quick Spin Columns for radiolabelled RNA purification (Roche, Basel, Switzerland) according to the manufacturer’s instructions.

    Infection:

    Article Title: Genomic characterization, phylogenetic position and in situ localization of a novel putative mononegavirus in Lepeophtheirus salmonis
    Article Snippet: Determination of the 5′ and 3′ terminal sequences RNA from infected lice was ligated to allow circularization and sequencing of the LsNSRV-1 genome termini. .. Purified dephosphorylated RNA (1 μg) was then ligated with 10 U of T4 RNA ligase (ThermoScientific) in 50 μl of 1 × reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNAseOUT (Invitrogen) for 1 h at 37 °C.

    Article Title: Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27
    Article Snippet: Briefly, infected and transfected HeLa cells were exposed to 254 nM UV with 400 mJ/cm2 , and then scrapped and re-suspended in lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitor). .. 3′ RNA linker was added by T4 RNA ligase (Thermo Fisher Scientific) overnight and [γ-32 P]ATP (PerkinElmer) was used to label the 5′ end of RNA by T4 PNK (NEB).

    Generated:

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: Clones for the terminal sequences of the dsRNA were generated by T4 RNA ligase oligonucleotide-mediated amplification as described by Lambden et al. ( ). .. The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    Article Title: Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27
    Article Snippet: Extracted RNA was added with 5′ RNA linker by T4 RNA ligase and reverse-transcribed with Superscript III (Invitrogen). .. Libraries for CLIP-seq were generated by PCR using Illumina universal primer and index primers.

    Polymerase Chain Reaction:

    Article Title: Genomic characterization, phylogenetic position and in situ localization of a novel putative mononegavirus in Lepeophtheirus salmonis
    Article Snippet: Purified dephosphorylated RNA (1 μg) was then ligated with 10 U of T4 RNA ligase (ThermoScientific) in 50 μl of 1 × reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNAseOUT (Invitrogen) for 1 h at 37 °C. .. The cDNA was subjected to nested PCR with forward primers located within the 3′ end of the putative L gene and reverse primers located within the 5′ end of ORFI, using the Expand High Fidelity PCR system (Roche).

    Article Title: The complete genome sequence of CrRV-Ch01, a new member of the family Rhabdoviridae in the parasitic copepod Caligus rogercresseyi present on farmed Atlantic salmon (Salmo salar) in Chile
    Article Snippet: Four hundred ng of purified dephosphorylated RNA was then ligated for 1 h at 37 °C using 10 U of T4 RNA ligase (Thermo Scientific) in 20-μl of 1x reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNaseOUT™ (Invitrogen). .. The cDNA was subjected to nested PCR with forward primers located within the 3’ end of the L gene and reverse primers located within the 5’ end of the N gene (Table ), using the Expand™ High Fidelity PCR System (Roche).

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: The PCR product was cloned into the pMD18-T vector. .. The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    Article Title: Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27
    Article Snippet: Extracted RNA was added with 5′ RNA linker by T4 RNA ligase and reverse-transcribed with Superscript III (Invitrogen). .. Libraries for CLIP-seq were generated by PCR using Illumina universal primer and index primers.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Genomic characterization, phylogenetic position and in situ localization of a novel putative mononegavirus in Lepeophtheirus salmonis
    Article Snippet: Purified dephosphorylated RNA (1 μg) was then ligated with 10 U of T4 RNA ligase (ThermoScientific) in 50 μl of 1 × reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNAseOUT (Invitrogen) for 1 h at 37 °C. .. For cDNA synthesis, 2.5 μl of ligated RNA was used directly as template for SuperScript III reverse transcriptase (SuperScript III First-Strand Synthesis System for RT-PCR, Invitrogen), with gene-specific primers annealing to the putative L gene in the genomic RNA.

    Article Title: The complete genome sequence of CrRV-Ch01, a new member of the family Rhabdoviridae in the parasitic copepod Caligus rogercresseyi present on farmed Atlantic salmon (Salmo salar) in Chile
    Article Snippet: Four hundred ng of purified dephosphorylated RNA was then ligated for 1 h at 37 °C using 10 U of T4 RNA ligase (Thermo Scientific) in 20-μl of 1x reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNaseOUT™ (Invitrogen). .. For cDNA synthesis, 2.5 μl of ligated RNA was used directly as template for SuperScript™ III reverse transcriptase (SuperScript™ III First-Strand Synthesis System for RT-PCR, Invitrogen), with gene-specific primers annealing to the L gene in the genomic RNA.

    Nucleic Acid Electrophoresis:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase. .. If high purity is required for the subsequent experiment, further purify 3′ end-radiolabeled RNA by 8 M urea-PAGE gel electrophoresis followed by autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution and ethanol precipitation as described in Basic Protocol 1, steps 10 to 13.

    Pull Down Assay:

    Article Title: Long Noncoding RNA NEAT1 Suppresses Proliferation and Promotes Apoptosis of Glioma Cells Via Downregulating MiR-92b
    Article Snippet: A total of 50 pmol NEAT1 RNA was labeled using biotin and T4 RNA ligase via a PierceTM RNA 3′End Desthiobiotinylation Kit (Thermo Fisher Scientific). .. The RNA pull-down assay was performed according to the PierceTM Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific).

    Labeling:

    Article Title: MicroRNA expression in the adult mouse central nervous system
    Article Snippet: Paragraph title: Microarray printing, labeling, and hybridization ... Total RNA (2–4 μg) was 3′-end-labeled using T4 RNA ligase and a Cy3-labeled RNA linker ( ; ) by the following procedure: RNA in 4.5 μL of water was combined with 0.8 μL of T4 RNA ligase buffer (10×) (Ambion), 1.1 μL of polyethyleneglycol (50% [w/v]), 0.8 μL of RNA-linker (250 μM; DNA Technology), and 0.8 μL of T4 RNA ligase (Ambion).

    Article Title: Long Noncoding RNA NEAT1 Suppresses Proliferation and Promotes Apoptosis of Glioma Cells Via Downregulating MiR-92b
    Article Snippet: .. A total of 50 pmol NEAT1 RNA was labeled using biotin and T4 RNA ligase via a PierceTM RNA 3′End Desthiobiotinylation Kit (Thermo Fisher Scientific). .. The RNA pull-down assay was performed according to the PierceTM Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific).

    Purification:

    Article Title: Genomic characterization, phylogenetic position and in situ localization of a novel putative mononegavirus in Lepeophtheirus salmonis
    Article Snippet: .. Purified dephosphorylated RNA (1 μg) was then ligated with 10 U of T4 RNA ligase (ThermoScientific) in 50 μl of 1 × reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNAseOUT (Invitrogen) for 1 h at 37 °C. .. For cDNA synthesis, 2.5 μl of ligated RNA was used directly as template for SuperScript III reverse transcriptase (SuperScript III First-Strand Synthesis System for RT-PCR, Invitrogen), with gene-specific primers annealing to the putative L gene in the genomic RNA.

    Article Title: A general two-step strategy to synthesize lariat RNAs
    Article Snippet: The branched RNA product was purified by 12% PAGE. .. Then, loop closure catalyzed by T4 RNA ligase was performed as follows: Branched RNA (5 pmol) was annealed in 7.5 μL of 5 mM HEPES (pH 7.5), 15 mM NaCl, and 0.1 mM EDTA by heating at 95°C for 3 min and cooling on ice for 5 min. Portions of 5× ligation buffer and T4 RNA ligase (Fermentas) were added, bringing the final conditions to 50 mM HEPES (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50μM ATP with 1 U/μL of T4 RNA ligase in 10 μL total volume.

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase. ..

    Article Title: The complete genome sequence of CrRV-Ch01, a new member of the family Rhabdoviridae in the parasitic copepod Caligus rogercresseyi present on farmed Atlantic salmon (Salmo salar) in Chile
    Article Snippet: .. Four hundred ng of purified dephosphorylated RNA was then ligated for 1 h at 37 °C using 10 U of T4 RNA ligase (Thermo Scientific) in 20-μl of 1x reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNaseOUT™ (Invitrogen). .. For cDNA synthesis, 2.5 μl of ligated RNA was used directly as template for SuperScript™ III reverse transcriptase (SuperScript™ III First-Strand Synthesis System for RT-PCR, Invitrogen), with gene-specific primers annealing to the L gene in the genomic RNA.

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: The resulting PCR product was fractionated by electrophoresis on 1% agarose gel and purified using a gel extraction kit (Axygen). .. The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    Article Title: Regulation of gene expression by the action of a fungal lncRNA on a transactivator
    Article Snippet: CY5-labelling of HAX1 CY5-coupled cytidine-5ʹ-phosphate-3ʹ-(aminohexyl)-phosphate (pCp-CY5, from Jena Bioscience, Jena, Germany) was linked to the 3ʹ end of in vitro synthesized HAX1 299 RNA using T4 RNA Ligase (Thermo Scientific) based on the instructions provided but scaled up for labelling 294.67 pmol (30 µg) HAX1 RNA with equimolar amounts of pCp-CY5 in a 30 µl ligation mixture. .. Subsequently, the CY5-labelled HAX1 RNA was purified using Sephadex G-25 Quick Spin Columns for radiolabelled RNA purification (Roche, Basel, Switzerland) according to the manufacturer’s instructions.

    Sequencing:

    Article Title: Genomic characterization, phylogenetic position and in situ localization of a novel putative mononegavirus in Lepeophtheirus salmonis
    Article Snippet: Determination of the 5′ and 3′ terminal sequences RNA from infected lice was ligated to allow circularization and sequencing of the LsNSRV-1 genome termini. .. Purified dephosphorylated RNA (1 μg) was then ligated with 10 U of T4 RNA ligase (ThermoScientific) in 50 μl of 1 × reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNAseOUT (Invitrogen) for 1 h at 37 °C.

    Article Title: The complete genome sequence of CrRV-Ch01, a new member of the family Rhabdoviridae in the parasitic copepod Caligus rogercresseyi present on farmed Atlantic salmon (Salmo salar) in Chile
    Article Snippet: RNA from 4-5 pooled lice from one location was ligated to allow circularization and sequencing of the genome termini of CrRV-Ch01. .. Four hundred ng of purified dephosphorylated RNA was then ligated for 1 h at 37 °C using 10 U of T4 RNA ligase (Thermo Scientific) in 20-μl of 1x reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNaseOUT™ (Invitrogen).

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: Paragraph title: cDNA synthesis, molecular cloning, and sequencing. ... The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    Immunoprecipitation:

    Article Title: Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27
    Article Snippet: After RNase I digestion, 80 μl of protein G Dynabeads beads and 5 μg of ICP27 antibody per IP were used for immunoprecipitation overnight. .. 3′ RNA linker was added by T4 RNA ligase (Thermo Fisher Scientific) overnight and [γ-32 P]ATP (PerkinElmer) was used to label the 5′ end of RNA by T4 PNK (NEB).

    Polyacrylamide Gel Electrophoresis:

    Article Title: A general two-step strategy to synthesize lariat RNAs
    Article Snippet: The branched RNA product was purified by 12% PAGE. .. Then, loop closure catalyzed by T4 RNA ligase was performed as follows: Branched RNA (5 pmol) was annealed in 7.5 μL of 5 mM HEPES (pH 7.5), 15 mM NaCl, and 0.1 mM EDTA by heating at 95°C for 3 min and cooling on ice for 5 min. Portions of 5× ligation buffer and T4 RNA ligase (Fermentas) were added, bringing the final conditions to 50 mM HEPES (pH 7.5), 10 mM MgCl2 , 10 mM DTT, and 50μM ATP with 1 U/μL of T4 RNA ligase in 10 μL total volume.

    Article Title: Regulation of gene expression by the action of a fungal lncRNA on a transactivator
    Article Snippet: CY5-labelling of HAX1 CY5-coupled cytidine-5ʹ-phosphate-3ʹ-(aminohexyl)-phosphate (pCp-CY5, from Jena Bioscience, Jena, Germany) was linked to the 3ʹ end of in vitro synthesized HAX1 299 RNA using T4 RNA Ligase (Thermo Scientific) based on the instructions provided but scaled up for labelling 294.67 pmol (30 µg) HAX1 RNA with equimolar amounts of pCp-CY5 in a 30 µl ligation mixture. .. Before application for the EMSAs, the sample eluted from the column was again analysed by denaturing PAGE as described in the prior section.

    Lysis:

    Article Title: Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27
    Article Snippet: Briefly, infected and transfected HeLa cells were exposed to 254 nM UV with 400 mJ/cm2 , and then scrapped and re-suspended in lysis buffer (50 mM Tris-HCl pH 7.4, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and protease inhibitor). .. 3′ RNA linker was added by T4 RNA ligase (Thermo Fisher Scientific) overnight and [γ-32 P]ATP (PerkinElmer) was used to label the 5′ end of RNA by T4 PNK (NEB).

    Article Title: Long Noncoding RNA NEAT1 Suppresses Proliferation and Promotes Apoptosis of Glioma Cells Via Downregulating MiR-92b
    Article Snippet: A total of 50 pmol NEAT1 RNA was labeled using biotin and T4 RNA ligase via a PierceTM RNA 3′End Desthiobiotinylation Kit (Thermo Fisher Scientific). .. Briefly, the cells were lysed with Pierce IP Lysis Buffer and incubated on ice for 5 minutes.

    Nested PCR:

    Article Title: Genomic characterization, phylogenetic position and in situ localization of a novel putative mononegavirus in Lepeophtheirus salmonis
    Article Snippet: Purified dephosphorylated RNA (1 μg) was then ligated with 10 U of T4 RNA ligase (ThermoScientific) in 50 μl of 1 × reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNAseOUT (Invitrogen) for 1 h at 37 °C. .. The cDNA was subjected to nested PCR with forward primers located within the 3′ end of the putative L gene and reverse primers located within the 5′ end of ORFI, using the Expand High Fidelity PCR system (Roche).

    Article Title: The complete genome sequence of CrRV-Ch01, a new member of the family Rhabdoviridae in the parasitic copepod Caligus rogercresseyi present on farmed Atlantic salmon (Salmo salar) in Chile
    Article Snippet: Four hundred ng of purified dephosphorylated RNA was then ligated for 1 h at 37 °C using 10 U of T4 RNA ligase (Thermo Scientific) in 20-μl of 1x reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNaseOUT™ (Invitrogen). .. The cDNA was subjected to nested PCR with forward primers located within the 3’ end of the L gene and reverse primers located within the 5’ end of the N gene (Table ), using the Expand™ High Fidelity PCR System (Roche).

    Plasmid Preparation:

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: The PCR product was cloned into the pMD18-T vector. .. The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    Agarose Gel Electrophoresis:

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: The resulting PCR product was fractionated by electrophoresis on 1% agarose gel and purified using a gel extraction kit (Axygen). .. The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    In Vitro:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase. ..

    Article Title: Regulation of gene expression by the action of a fungal lncRNA on a transactivator
    Article Snippet: .. CY5-labelling of HAX1 CY5-coupled cytidine-5ʹ-phosphate-3ʹ-(aminohexyl)-phosphate (pCp-CY5, from Jena Bioscience, Jena, Germany) was linked to the 3ʹ end of in vitro synthesized HAX1 299 RNA using T4 RNA Ligase (Thermo Scientific) based on the instructions provided but scaled up for labelling 294.67 pmol (30 µg) HAX1 RNA with equimolar amounts of pCp-CY5 in a 30 µl ligation mixture. .. Subsequently, the CY5-labelled HAX1 RNA was purified using Sephadex G-25 Quick Spin Columns for radiolabelled RNA purification (Roche, Basel, Switzerland) according to the manufacturer’s instructions.

    Ethanol Precipitation:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 10 × buffer for T4 RNA ligase (see recipe) 10 mM ATP (Thermo Scientific) RNA substrate with 3′ hydroxyl end derived from in vitro transcription (Basic Protocol 1) or purified directly from cells (endogenous RNA; ) 5′ 10 µCi/µl [32 P]pCp (3000 Ci/mmol; PerkinElmer) 10 U/µl T4 RNA ligase (Thermo Scientific) G50 buffer (see recipe) Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) Prepare the following reaction mixture at room temperature in a microcentrifuge tube by combining the reagents in the indicated order (total reaction volume, 20 µl): 2 µl 10× buffer for T4 RNA ligase 1 µl distilled, deionized H2 O 1 µl 10 mM ATP 5 µl RNA substrate with a 3′-hydroxyl end (30 pmol) 10 µl 10 µCi/µl 5′ [32 P]pCp (3000 Ci/mmol) 1 µl 10 U/µl T4 RNA ligase. ..

    Concentration Assay:

    Article Title: MicroRNA expression in the adult mouse central nervous system
    Article Snippet: Probes were diluted to a final concentration of 10 μM in printing buffer (150 mM sodium phosphate at pH 8.5) and printed onto Codelink slides (GE Healthcare) using a MicroGrid TAS II arrayer (Biorobotics). .. Total RNA (2–4 μg) was 3′-end-labeled using T4 RNA ligase and a Cy3-labeled RNA linker ( ; ) by the following procedure: RNA in 4.5 μL of water was combined with 0.8 μL of T4 RNA ligase buffer (10×) (Ambion), 1.1 μL of polyethyleneglycol (50% [w/v]), 0.8 μL of RNA-linker (250 μM; DNA Technology), and 0.8 μL of T4 RNA ligase (Ambion).

    Gel Extraction:

    Article Title: Genomic characterization, phylogenetic position and in situ localization of a novel putative mononegavirus in Lepeophtheirus salmonis
    Article Snippet: Purified dephosphorylated RNA (1 μg) was then ligated with 10 U of T4 RNA ligase (ThermoScientific) in 50 μl of 1 × reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNAseOUT (Invitrogen) for 1 h at 37 °C. .. Finally, the nested PCR products were gel purified (QIAquick Gel Extraction Kit, QIAGEN) and sequenced by the Sanger method using the same primers that were used for the nested PCR.

    Article Title: The complete genome sequence of CrRV-Ch01, a new member of the family Rhabdoviridae in the parasitic copepod Caligus rogercresseyi present on farmed Atlantic salmon (Salmo salar) in Chile
    Article Snippet: Four hundred ng of purified dephosphorylated RNA was then ligated for 1 h at 37 °C using 10 U of T4 RNA ligase (Thermo Scientific) in 20-μl of 1x reaction buffer for T4 RNA ligase supplemented with 0.1 mg of BSA per ml and 40 units of RNaseOUT™ (Invitrogen). .. Finally, the nested PCR products were gel purified (QIAquick® Gel Extraction Kit, QIAGEN) and sequenced by the Sanger method.

    Article Title: A Novel Mycovirus That Is Related to the Human Pathogen Hepatitis E Virus and Rubi-Like Viruses ▿ and Rubi-Like Viruses ▿ †
    Article Snippet: The resulting PCR product was fractionated by electrophoresis on 1% agarose gel and purified using a gel extraction kit (Axygen). .. The 3′ terminus of each strand of dsRNA was ligated at 5 to 15°C for 16 to 18 h with the 5′-end phosphorylated oligonucleotide 5′-GCATTGCATCATGATCGATCGAATTCTTTAGTGAGGGTTAATTGCC-(NH2 )-3′ using T4 RNA ligase (Fermentas).

    Cross-linking Immunoprecipitation:

    Article Title: Herpes simplex virus blocks host transcription termination via the bimodal activities of ICP27
    Article Snippet: Paragraph title: CLIP-seq ... 3′ RNA linker was added by T4 RNA ligase (Thermo Fisher Scientific) overnight and [γ-32 P]ATP (PerkinElmer) was used to label the 5′ end of RNA by T4 PNK (NEB).

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  • 99
    Thermo Fisher t4 rna ligase
    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using <t>T4</t> RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).
    T4 Rna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t4 dna ligase
    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using <t>T4</t> DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.
    T4 Dna Ligase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/Thermo Fisher
    Average 99 stars, based on 382 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-04
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    Image Search Results


    Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Journal: RNA Biology

    Article Title: Identification of host RNAs that interact with EBV noncoding RNA EBER2

    doi: 10.1080/15476286.2018.1518854

    Figure Lengend Snippet: Identifying EBER2-interacting RNAs by combining psoralen crosslinking, ASO-mediated selection, and RNase V1 treatment. (A) The psoralen derivative AMT is used to crosslink RNA duplexes in intact cells to preserve in vivo RNA-RNA interactions. An EBER2-targeting ASO is then used to select EBER2 together with crosslinked interacting RNAs. These duplexes are eluted from the ASO beads using TEACl-containing buffer and are subjected to RNase V1 digestion. Following cleavage of double-stranded regions, a linker is ligated to the newly-generated 5′ phosphate group at the cut site using T4 RNA ligase (inset). Only one possible cleavage event is depicted for simplicity. After deep sequencing, not only can the interacting RNAs be identified, but also the site of RNA-RNA interactions can be deduced, which are specified by the junction of the linker and interacting RNA. (B) Cobra venom fractions were examined for activity towards doubled-stranded and single-stranded substrates. The double-stranded substrate consists of a shRNA with a pyrimidine-rich loop, which can be digested by single-strand specific RNases, such as RNase A. The trimmed RNA duplex with no loop region migrates faster in a native polyacrylamide gel. Digestion within the stem region by a double-strand specific RNase results in the disappearance of radioactive signal, as observed after digestion with all input material as well as hydroxyapatite (HAP) fraction 15; note that the weak activity of the MonoS input sample is due to the great dilution of protein concentration following size exclusion chromatography. Indicated fractions were also used in a ligation assay (outlined in D) to verify the compatibility of RNase V1 digest with T4 RNA ligase reaction. A silver-stained gel of the purified fractions is shown in the bottom panel, revealing the partial purification only of RNase V1; many other proteins are present in our sample preparation, which, importantly, do not interfere with RNase V1 activity. (C) Purification scheme of RNase V1 from Naja oxiana venom. (D) Outline of ligation reaction after RNase V1 digest. An oligonucleotide blocked at the 3′ end with puromycin was 5′ end-labeled (arrow in B, third panel from top) and annealed to a partially complementary oligonucleotide with a 3′ amino modifier. A free 3′ OH group is created only after RNase V1 digest, to which a 5′ phosphorylated linker blocked at the 3′ end with puromycin can be ligated using T4 RNA ligase. This ligation product is the only one that can be visualized by autoradiography as shown in B (arrowhead, third panel from top).

    Article Snippet: RNA was resuspended in 14.5 µl H2 O and subjected to T4 RNA Ligase reaction by adding 1 µl of 20 µM 5′-phosporylated RL3 (5′-P -GUGUCAGUCACUUCCAGCGG-Puromycin-3′), 2 µl 10× T4 Ligase Buffer, 2 µl BSA, 0.5 µl T4 RNA Ligase (ThermoFisher), and incubated overnight at 16°C.

    Techniques: Allele-specific Oligonucleotide, Selection, In Vivo, Generated, Sequencing, Combined Bisulfite Restriction Analysis Assay, Activity Assay, shRNA, Protein Concentration, Size-exclusion Chromatography, Ligation, Staining, Purification, Sample Prep, Labeling, Autoradiography

    Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Journal: Chembiochem

    Article Title: Practical Synthesis of Cap‐4 RNA

    doi: 10.1002/cbic.201900590

    Figure Lengend Snippet: Enzymatic ligation of T. cruzi cap‐4 spliced leader RNA using T4 DNA ligase. A) RNA sequences and sequence of the 20‐nt DNA splint; B) HPLC analysis of a typical ligation reaction after 3 h reaction time; reaction conditions: 10 μ m RNA 10 , 12.5 μ m RNA 11 , 12.5 μ m splint; 0.5 m m ATP, 40 m m Tris ⋅ HCl (pH 7.8), 10 m m MgCl 2 , 10 m m DTT, 5 % ( w / v ) PEG 4000, 0.5 U μL −1 T4 DNA ligase; C) LC–ESI mass spectrum of the purified 39‐nt cap‐4 RNA ligation product.

    Article Snippet: The 38‐nt T. cruzi cap‐4 RNA was prepared by splinted enzymatic ligation of an 11‐nt cap‐4 RNA and a chemically synthesized 5′‐phosphorylated 27‐nt RNA by using T4 DNA ligase (Thermo Fisher) in analogy to ref. .

    Techniques: Ligation, Sequencing, High Performance Liquid Chromatography, Purification

    DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Journal: Biophysical Journal

    Article Title: Multiplex Single-Molecule DNA Barcoding Using an Oligonucleotide Ligation Assay

    doi: 10.1016/j.bpj.2018.08.013

    Figure Lengend Snippet: DNA barcoding experimental scheme. Target DNA strands are immobilized on a microscope slide, and dye-labeled barcodes are introduced together with T4 DNA ligase in the microfluidic chamber (1). Complementary barcodes bind transiently to the target site (2), whereas mismatched barcodes bind on an even shorter timescale (2′). Successful ligation is observed for the complementary barcodes (3) but not for the mismatched barcodes (3′). Ligation product shows stable binding to the target DNA (4), whereas mismatched barcodes dissociate and are washed away before imaging. To see this figure in color, go online.

    Article Snippet: For ligation, T4 DNA ligase was used in standard conditions (25°C, 10 mM MgCl2 ), and the GC content of the target site was ∼50%.

    Techniques: Microscopy, Labeling, Ligation, Binding Assay, Imaging

    Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Single-stranded DNA ligation with T4 DNA ligase and CircLigase. A pool of 60 nt acceptor oligonucleotides (‘60N’) were ligated to 10 pmol of a 3΄ biotinylated donor oligonucleotide (CL78) using either T4 DNA ligase in the presence of a splinter oligonucleotide (TL38) or CircLigase. Ligation products were visualized on a 10% denaturing polyacrylamide gel stained with SybrGold. Band shifts from 60 nt to 80 nt indicate successful ligation. Schematic overviews of the reaction schemes are shown on top. The scheme developed by Kwok et al . ( 19 ) is shown for comparison. M: Single-stranded DNA size marker.

    Article Snippet: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

    Techniques: DNA Ligation, Ligation, Staining, Marker

    Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Library preparation methods for highly degraded DNA. ( A ) In the single-stranded library preparation method described here (ssDNA2.0), DNA fragments (black) are 5΄ and 3΄ dephosphorylated and separated into single strands by heat denaturation. 3΄ biotinylated adapter molecules (red) are attached to the 3΄ ends of the DNA fragments via hybridization to a stretch of six random nucleotides (marked as ‘N’) belonging to a splinter oligonucleotide complementary to the adapter and nick closure with T4 DNA ligase. Following the immobilization of the ligation products on streptavidin-coated beads, the splinter oligonucleotide is removed by bead wash at an elevated temperature. Synthesis of the second strand is carried out using the Klenow fragment of Escherichia coli DNA polymerase I and a primer with phosphorothioate backbone modifications (red stars) to prevent exonucleolytic degradation. Unincorporated primers are removed through a bead wash at an elevated temperature, preventing the formation of adapter dimers in the subsequent blunt-end ligation reaction, which is again catalyzed by T4 DNA ligase. Adapter self-ligation is prevented through a 3΄ dideoxy modification in the adapter. The final library strand is released from the beads by heat denaturation. ( B ) In the single-stranded library preparation method originally described in Gansauge and Meyer, ( 4 ), the first adapter was attached through true single-stranded DNA ligation using CircLigase. The large fragment of Bst DNA polymerase was used to copy the template strand, leaving overhanging 3΄ nucleotides, which had to be removed in a blunt-end repair reaction using T4 DNA polymerase. ( C ) The ‘454’ method of double-stranded library preparation in the implementation of Meyer and Kircher, ( 23 ), is based on non-directional blunt-end ligation of a mixture of two adapters to blunt-end repaired DNA fragments using T4 DNA ligase. To prevent adapter self-ligation, no phosphate groups are present at the 5΄ ends of the adapters, resulting in the ligation of the adapter strands only and necessitating subsequent nick fill-in with a strand-displacing polymerase. Intermittent DNA purification steps are required in-between enzymatic reactions. ( D ) The ‘Illumina’ method of double-stranded library preparation, shown here as implemented in New England Biolabs’ NEBNext Ultra II kit, requires the addition of A-overhangs (marked as ‘A’) to blunt-end repaired DNA fragments using a 3΄-5΄ exonuclease deletion mutant of the Klenow fragment of E. coli DNA polymerase I. Both adapter sequences are combined into one bell-shaped structure, which carries a 3΄ T overhang to allow sticky end ligation with T4 DNA ligase. Following ligation, adapter strands are separated by excision of uracil. Excess adapters and adapter dimers are removed through size-selective purification.

    Article Snippet: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

    Techniques: Hybridization, Ligation, Modification, DNA Ligation, DNA Purification, Mutagenesis, Purification

    Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Journal: Nucleic Acids Research

    Article Title: Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase

    doi: 10.1093/nar/gkx033

    Figure Lengend Snippet: Effects of single-stranded ligation schemes on library characteristics. ( A ) Informative sequence content of libraries prepared with CircLigase and T4 DNA ligase as a function of the input volume of ancient DNA extract used for library preparation. ( B ) Average GC content of the sequences obtained with the two ligation schemes. Note that the average GC content exceeds that of a typical mammalian genome because most sequences derive from microbial DNA, which is the dominant source of DNA in most ancient bones. ( C ) Fragment size distribution in the libraries as inferred from overlap-merged paired-end reads. Short artifacts in the library prepared from extremely little input DNA (corresponding to ∼1 mg bone) are mainly due to the incorporation of splinter fragments. ( D ) Frequencies of damage-induced C to T substitutions near the 5΄ and 3΄ ends of sequences.

    Article Snippet: Splinted end-to-end ligation of single-stranded DNA using T4 DNA ligase To explore the efficiency of splinted end-to-end ligation of single stranded DNA with T4 DNA ligase in the absence of hair-pin structures, we designed a ligation scheme where the splinter oligonucleotide is hybridized to a biotinylated adapter oligonucleotide (the donor), allowing for subsequent immobilization of ligation products on beads and removal of the splinter by mild heat treatment.

    Techniques: Ligation, Sequencing, Ancient DNA Assay