t4 ligase  (New England Biolabs)


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    Structured Review

    New England Biolabs t4 ligase
    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, <t>T4</t> ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).
    T4 Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 273 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 ligase/product/New England Biolabs
    Average 99 stars, based on 273 article reviews
    Price from $9.99 to $1999.99
    t4 ligase - by Bioz Stars, 2020-05
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    Images

    1) Product Images from "Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro"

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro

    Journal: Nucleic Acids Research

    doi:

    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).
    Figure Legend Snippet: Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).

    Techniques Used: Plasmid Preparation, Ligation, Generated

    Related Articles

    Sequencing:

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression
    Article Snippet: .. To determine the terminal sequence of viral negative-strand genomic RNA and sgmRNA, total cellular RNA was treated with tobacco acid pyrophosphatase (Epicentre), ligated with T4 RNA ligase I (New England Biolabs) and primer 1: BCV3′UTR1(−) was used for RT; for PCR, primers BCV3′UTR(−) and BCV107(+), and primers BCV3′UTR(−) and RYN(+) were used for determining terminal sequence of negative-strand genomic RNA and subgenomic mRNA, respectively. .. The resulting 50-µl PCR mixture was heated to 94°C for 2 min and subjected to 50 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C.

    Polymerase Chain Reaction:

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression
    Article Snippet: .. To determine the terminal sequence of viral negative-strand genomic RNA and sgmRNA, total cellular RNA was treated with tobacco acid pyrophosphatase (Epicentre), ligated with T4 RNA ligase I (New England Biolabs) and primer 1: BCV3′UTR1(−) was used for RT; for PCR, primers BCV3′UTR(−) and BCV107(+), and primers BCV3′UTR(−) and RYN(+) were used for determining terminal sequence of negative-strand genomic RNA and subgenomic mRNA, respectively. .. The resulting 50-µl PCR mixture was heated to 94°C for 2 min and subjected to 50 cycles of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C.

    Incubation:

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression
    Article Snippet: .. A 3-µl aliquot of 10X ligase buffer and 2 U (in 2 µl) of T4 RNA ligase I (New England Biolabs) were added, and the mixture was incubated for 16 h at 16°C. .. After ligation, RNA was phenol-chloroform-extracted and quantitated, and 1 µg of ligated RNA was used for an RT reaction to synthesize cDNA with SuperScript III reverse transcriptase (Invitrogen).

    Article Title: A Leaderless Genome Identified during Persistent Bovine Coronavirus Infection Is Associated with Attenuation of Gene Expression
    Article Snippet: .. The extracted RNA in 25 µl of water, 3 µl of 10X ligase buffer, and 2 U (in 2 µl) of T4 RNA ligase I (New England Biolabs) were combined, and the mixture was incubated for 16 h at 16°C. ..

    Article Title: High-throughput determination of RNA structure by proximity ligation
    Article Snippet: .. Following end-repair, complexes were immediately transferred to 450 uL ligation reaction mix (50 uL 10X T4 DNA Ligase Buffer w/ 10 mM ATP (NEB); 5 uL SuperASE-In (Ambion), 12.5 uL T4 RNA Ligase I (NEB), 382.5 uL 1X PBS w/ 0.2% IGEPAL), and incubated overnight in a 16 °C water bath, after which complexes were added to 1.5 mL TriZOL (Ambion). .. Samples were then purified using Direct-ZOL spin columns (Zymo) according to manufacturer's protocols.

    other:

    Article Title: Detecting RNA-RNA interactions in E. coli using a modified CLASH method
    Article Snippet: Free RNA overhangs adjacent to duplexes were ligated using T4 RNA ligase 1.

    Ligation:

    Article Title: High-throughput determination of RNA structure by proximity ligation
    Article Snippet: .. Following end-repair, complexes were immediately transferred to 450 uL ligation reaction mix (50 uL 10X T4 DNA Ligase Buffer w/ 10 mM ATP (NEB); 5 uL SuperASE-In (Ambion), 12.5 uL T4 RNA Ligase I (NEB), 382.5 uL 1X PBS w/ 0.2% IGEPAL), and incubated overnight in a 16 °C water bath, after which complexes were added to 1.5 mL TriZOL (Ambion). .. Samples were then purified using Direct-ZOL spin columns (Zymo) according to manufacturer's protocols.

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    New England Biolabs t4 rna ligase buffer
    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using <t>T4</t> RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.
    T4 Rna Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    New England Biolabs t4 rna ligase synthesized mir173 rna standard
    Effect of various modifications on the 3′ terminal nucleotide of a small <t>RNA</t> on <t>T4</t> RNA ligase- and yeast PAP-catalyzed reactions. ( a ) T4 RNA ligase-mediated ligation of various <t>miR173</t> forms to an RNA linker. ( b ) Activity of yeast PAP on various forms of miR173 in the presence of 2 pmol [α- 32 P]-ATP. The ladders or smears represent products of PAP-catalyzed reaction. ( c ) Activity of yeast PAP on various forms of miR173 in the presence of 10 pmol [α- 32 P]-ATP.
    T4 Rna Ligase Synthesized Mir173 Rna Standard, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 rna ligase synthesized mir173 rna standard/product/New England Biolabs
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs t4 dna ligase
    Schematic illustration of the multiple patch cloning procedure. DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and <t>T4</t> DNA ligase.
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3478 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna ligase/product/New England Biolabs
    Average 99 stars, based on 3478 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2020-05
    99/100 stars
      Buy from Supplier

    Image Search Results


    DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Journal: Genome Research

    Article Title: Strand-specific deep sequencing of the transcriptome

    doi: 10.1101/gr.094318.109

    Figure Lengend Snippet: DSSS protocol workflow. ( A ) Fragmentation. RNA is fragmented to sizes in the range of 60–200 nt. ( B ) Dephosphorylation. 5′ phosphates are removed from RNA by treatment with alkaline phosphatase. ( C ) 3′ adapter ligation. Dephosphorylated 200-nt-long RNA fragments are selected by urea-PAGE. The 3′ adapter is ligated to the 3′ ends using T4 RNA ligase I. ( D ) Rephosphorylation. Fragments are rephosphorylated by treatment with T4 polynucleotide kinase as preparation for the next ligation step. ( E ) 5′ adapter ligation, preceded by removal of the nonligated 3′adapter by urea-PAGE size selection. ( F ) Reverse transcription (RT) and amplification of library. Molecules with 5′ and 3′ adapters were selected by urea-PAGE. First strand cDNA synthesis and PCR amplification were carried out with the indicated primers. ( G ) Sequencing.

    Article Snippet: We incubated the following reaction mixture for 30 min at 37°C: 10 μL of sample, 1 μL of 10× T4 RNA ligase buffer (as fresh ATP supply), 10 U of polynucleotide kinase (New England BioLabs), 3 μL of RNase free water.

    Techniques: De-Phosphorylation Assay, Ligation, Polyacrylamide Gel Electrophoresis, Selection, Amplification, Polymerase Chain Reaction, Sequencing

    Effect of various modifications on the 3′ terminal nucleotide of a small RNA on T4 RNA ligase- and yeast PAP-catalyzed reactions. ( a ) T4 RNA ligase-mediated ligation of various miR173 forms to an RNA linker. ( b ) Activity of yeast PAP on various forms of miR173 in the presence of 2 pmol [α- 32 P]-ATP. The ladders or smears represent products of PAP-catalyzed reaction. ( c ) Activity of yeast PAP on various forms of miR173 in the presence of 10 pmol [α- 32 P]-ATP.

    Journal: Nucleic Acids Research

    Article Title: HEN1 recognizes 21-24 nt small RNA duplexes and deposits a methyl group onto the 2? OH of the 3? terminal nucleotide

    doi: 10.1093/nar/gkj474

    Figure Lengend Snippet: Effect of various modifications on the 3′ terminal nucleotide of a small RNA on T4 RNA ligase- and yeast PAP-catalyzed reactions. ( a ) T4 RNA ligase-mediated ligation of various miR173 forms to an RNA linker. ( b ) Activity of yeast PAP on various forms of miR173 in the presence of 2 pmol [α- 32 P]-ATP. The ladders or smears represent products of PAP-catalyzed reaction. ( c ) Activity of yeast PAP on various forms of miR173 in the presence of 10 pmol [α- 32 P]-ATP.

    Article Snippet: Ligation with T4 RNA ligase Synthesized miR173 RNA standard and miR173 with a methyl group on either the 2′ or 3′ OH (miR173-2′OMe or miR173-3′OMe) were treated with calf intestine alkaline phosphatase (New England Biolabs) to remove the 5′P to prevent self-ligation.

    Techniques: Ligation, Activity Assay

    Schematic illustration of the multiple patch cloning procedure. DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.

    Journal: BMC Biotechnology

    Article Title: Patch cloning method for multiple site-directed and saturation mutagenesis

    doi: 10.1186/1472-6750-13-91

    Figure Lengend Snippet: Schematic illustration of the multiple patch cloning procedure. DNA fragments are amplified by polymerase chain reaction using two sets of oligo-DNA primers (shown in red and blue). The star on the primer indicates the site of mismatch. The resultant DNA fragments and digested vector DNA containing 16 bp homologous regions (shown in yellow) were assembled at 37°C by T5 exonuclease, Klenow fragment and T4 DNA ligase.

    Article Snippet: The MUPAC enzyme stock solution was prepared by mixing 10 μL of T5 exonuclease (10 U/μL; New England Biolabs), 1 μL of Klenow fragment exo– (5 U/μL; New England Biolabs), 2.5 μL of T4 DNA ligase (400 U/μL; New England Biolabs), and 11.5 μL of the storage buffer (50 mM Tris–HCl pH 7.5, 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% Glycerol, and 0.1% Triton X-100).

    Techniques: Clone Assay, Amplification, Polymerase Chain Reaction, Plasmid Preparation

    In vitro reconstitution of the BER pathway using 8oxoA•T duplex DNA substrate. 5 nM 40 mer 8oxoA•T duplex was incubated in the presence of 20 nM hTDG, 5 nM APE1, 2 nM FEN1, 0.1 U POL-β and 5 nM T4 DNA ligase in buffer containing 20 µCi of [α- 32 P]dATP, 50 µM dNTPs, 50 mM HEPES–KOH (pH 7.6), 30 mM NaCl, 0.1 mg/ml BSA, 2 mM DTT, 2 mM ATP and 3 mM MgCl 2 for 5 and 30 min at 37°C. Lane 1, 30 min in the absence of hTDG and T4 DNA ligase; lane 2, 5 min in the absence of T4 DNA ligase; lane 3, same as 2, but 30 min; lane 4, 30 min in the presence of all proteins. For details see ‘Materials and Methods’ section.

    Journal: Nucleic Acids Research

    Article Title: 7,8-dihydro-8-oxoadenine, a highly mutagenic adduct, is repaired by Escherichia coli and human mismatch-specific uracil/thymine-DNA glycosylases

    doi: 10.1093/nar/gks1149

    Figure Lengend Snippet: In vitro reconstitution of the BER pathway using 8oxoA•T duplex DNA substrate. 5 nM 40 mer 8oxoA•T duplex was incubated in the presence of 20 nM hTDG, 5 nM APE1, 2 nM FEN1, 0.1 U POL-β and 5 nM T4 DNA ligase in buffer containing 20 µCi of [α- 32 P]dATP, 50 µM dNTPs, 50 mM HEPES–KOH (pH 7.6), 30 mM NaCl, 0.1 mg/ml BSA, 2 mM DTT, 2 mM ATP and 3 mM MgCl 2 for 5 and 30 min at 37°C. Lane 1, 30 min in the absence of hTDG and T4 DNA ligase; lane 2, 5 min in the absence of T4 DNA ligase; lane 3, same as 2, but 30 min; lane 4, 30 min in the presence of all proteins. For details see ‘Materials and Methods’ section.

    Article Snippet: Restriction enzymes and T4 DNA ligase were from New England Biolabs France (Evry, France).

    Techniques: In Vitro, Incubation