t4 dna ligase  (New England Biolabs)


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    Name:
    T4 DNA Ligase
    Description:
    T4 DNA Ligase 100 000 units
    Catalog Number:
    m0202l
    Price:
    256
    Category:
    DNA Ligases
    Size:
    100 000 units
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    New England Biolabs t4 dna ligase
    T4 DNA Ligase
    T4 DNA Ligase 100 000 units
    https://www.bioz.com/result/t4 dna ligase/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "The longevity SNP rs2802292 uncovered: HSF1 activates stress-dependent expression of FOXO3 through an intronic enhancer"

    Article Title: The longevity SNP rs2802292 uncovered: HSF1 activates stress-dependent expression of FOXO3 through an intronic enhancer

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky331

    HSF1 mediates the occurrence of a promoter–enhancer interaction at FOXO3 locus involving the 5′UTR and the rs2802292 region. ( A ) Top: physical map of the human FOXO3 gene. The scheme shows the Csp6I restriction enzyme sites flanking the baits (red for the 5′UTR and blue for the rs2802292 region). Bottom: schematic representation of the 3C and ChIP-loop assay. Crosslinked chromatin was digested with Csp6I and immunoprecipitated with anti-HSF1. The immunoprecipitated samples were diluted in a ligation buffer and ligated with the T4 DNA Ligase. After reversing the crosslinks, the ligated DNA was purified and amplified by PCR with various combinations of primers as indicated in (B). ( B ) This strategy allows the amplification of sequences ligated to the bait in circular DNA. The arrows indicate the positions of the primers within the bait sequence. Five different couples of primers were designed to analyze the five possible ligation products. Purified DNA was analyzed by PCR with primers specific for the various possible combinations of chromatin fragments. The values are the results of the densitometric analysis and are expressed as fold induction. HEK-293 cells and primary human fibroblasts (GG, n = 3; TT, n = 3) were collected after induction of oxidative stress (1 h H 2 O 2 , 100 μM). The presented results are representative of three independent experiments. P -values were derived from t -tests: * P ≤ 0.05.
    Figure Legend Snippet: HSF1 mediates the occurrence of a promoter–enhancer interaction at FOXO3 locus involving the 5′UTR and the rs2802292 region. ( A ) Top: physical map of the human FOXO3 gene. The scheme shows the Csp6I restriction enzyme sites flanking the baits (red for the 5′UTR and blue for the rs2802292 region). Bottom: schematic representation of the 3C and ChIP-loop assay. Crosslinked chromatin was digested with Csp6I and immunoprecipitated with anti-HSF1. The immunoprecipitated samples were diluted in a ligation buffer and ligated with the T4 DNA Ligase. After reversing the crosslinks, the ligated DNA was purified and amplified by PCR with various combinations of primers as indicated in (B). ( B ) This strategy allows the amplification of sequences ligated to the bait in circular DNA. The arrows indicate the positions of the primers within the bait sequence. Five different couples of primers were designed to analyze the five possible ligation products. Purified DNA was analyzed by PCR with primers specific for the various possible combinations of chromatin fragments. The values are the results of the densitometric analysis and are expressed as fold induction. HEK-293 cells and primary human fibroblasts (GG, n = 3; TT, n = 3) were collected after induction of oxidative stress (1 h H 2 O 2 , 100 μM). The presented results are representative of three independent experiments. P -values were derived from t -tests: * P ≤ 0.05.

    Techniques Used: Chromatin Immunoprecipitation, Immunoprecipitation, Ligation, Purification, Amplification, Polymerase Chain Reaction, Sequencing, Derivative Assay

    Related Articles

    Blocking Assay:

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries
    Article Snippet: .. Because our approach relies on T4 DNA Ligase, which is sensitive to base-pair mismatches and gaps , these variations can adversely affect the efficacy of the blocking (Supplementary Figure S4). .. Although variation at the 5′ end has similar effects on the 5′ blocking approach (Supplementary Figure S5), 5′ end variants generally represent a smaller fraction of the total.

    Ligation:

    Article Title: The longevity SNP rs2802292 uncovered: HSF1 activates stress-dependent expression of FOXO3 through an intronic enhancer
    Article Snippet: To further confirm this observation, we performed a ChIP-loop analysis of HEK-293 cells and primary human fibroblasts (GG and TT). .. H2 O2 -stressed cells were treated with formaldehyde to obtain cross-linked chromatin, after which DNA/protein complexes were first digested with the Csp6I restriction enzyme and then selected with anti-HSF1 antibodies; at last, ligation of chromatin fragments was carried out with the T4 DNA Ligase (Figure ). .. Purified DNA was analyzed by PCR with primers specific for the various possible combinations of ligated chromatin fragments (Figure ).

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
    Article Snippet: We focused only on T4 DNA ligase, since DNA ligase III activity was undetectable even with wild type RNase H1 as discussed above (Figure ). .. While wild type RNase H1 together with T4 DNA ligase produced a ligated product, negligible ligation was seen with the mutant proteins (Figure ). .. Longer extension products were formed with the V142I mutant compared to A185V; this can be explained by V142I being able to remove some ribonucleotides (Figure , compare lanes 7–9 with lanes 12–14) and thus making a longer gap for POLγ to fill.

    Produced:

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
    Article Snippet: We focused only on T4 DNA ligase, since DNA ligase III activity was undetectable even with wild type RNase H1 as discussed above (Figure ). .. While wild type RNase H1 together with T4 DNA ligase produced a ligated product, negligible ligation was seen with the mutant proteins (Figure ). .. Longer extension products were formed with the V142I mutant compared to A185V; this can be explained by V142I being able to remove some ribonucleotides (Figure , compare lanes 7–9 with lanes 12–14) and thus making a longer gap for POLγ to fill.

    Mutagenesis:

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
    Article Snippet: We focused only on T4 DNA ligase, since DNA ligase III activity was undetectable even with wild type RNase H1 as discussed above (Figure ). .. While wild type RNase H1 together with T4 DNA ligase produced a ligated product, negligible ligation was seen with the mutant proteins (Figure ). .. Longer extension products were formed with the V142I mutant compared to A185V; this can be explained by V142I being able to remove some ribonucleotides (Figure , compare lanes 7–9 with lanes 12–14) and thus making a longer gap for POLγ to fill.

    other:

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL
    Article Snippet: For this, we performed the POLRMT-primed minicircle assay with T4 DNA ligase, and then tested whether the closed circular products could be subsequently converted to nicked circles by KOH treatment ( ).

    Article Title: Ligation with Nucleic Acid Sequence-Based Amplification
    Article Snippet: Only when P3, P5, Target, T4 DNA Ligase, and the two primers were present was a 72-nt band observed.

    Incubation:

    Article Title: Preparation of Mammalian Expression Vectors Incorporating Site-Specifically Platinated-DNA Lesions
    Article Snippet: The gapped plasmid (40 µg) was added at 1:10 to 1:100 molar ratio (vs. the insertion strands) and the mixture was annealed in a thermocycler (80 °C for 10 min, then cooling to 4 °C at −1 °C/min). .. The reaction was supplemented with T4 DNA ligase (80 U) in buffer (50 mM Tris·HCl pH 7.6, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) and incubated at 16 °C for 12 h. The mixture was supplemented with 6X loading buffer (NEB), heated at 75 °C for 15 min and separated using preparative 0.8% w/v agarose gel electrophoresis containing 0.5 µg/mL EtdBr. ..

    Agarose Gel Electrophoresis:

    Article Title: Preparation of Mammalian Expression Vectors Incorporating Site-Specifically Platinated-DNA Lesions
    Article Snippet: The gapped plasmid (40 µg) was added at 1:10 to 1:100 molar ratio (vs. the insertion strands) and the mixture was annealed in a thermocycler (80 °C for 10 min, then cooling to 4 °C at −1 °C/min). .. The reaction was supplemented with T4 DNA ligase (80 U) in buffer (50 mM Tris·HCl pH 7.6, 10 mM MgCl2 , 10 mM DTT, 1 mM ATP) and incubated at 16 °C for 12 h. The mixture was supplemented with 6X loading buffer (NEB), heated at 75 °C for 15 min and separated using preparative 0.8% w/v agarose gel electrophoresis containing 0.5 µg/mL EtdBr. ..

    DNA Ligation:

    Article Title: Mechanical properties of DNA-like polymers
    Article Snippet: .. DNA ligase-catalyzed cyclization reactions were performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl2 , 1 mM ATP, 10 mM dithiothreitol; NEB) and a final concentration of 100 U/ml T4 DNA ligase (NEB). .. Aliquots (10 µl) were removed at 5, 10, 15 and 20 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels (29:1 acrylamide:bisacylamide, BioRad) in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3), followed by drying and storage phosphor imaging.

    Concentration Assay:

    Article Title: Mechanical properties of DNA-like polymers
    Article Snippet: .. DNA ligase-catalyzed cyclization reactions were performed at ∼22°C with 1 nM DNA restriction fragment, T4 DNA ligation buffer (50 mM Tris–HCl, pH 7.5, 10 mM MgCl2 , 1 mM ATP, 10 mM dithiothreitol; NEB) and a final concentration of 100 U/ml T4 DNA ligase (NEB). .. Aliquots (10 µl) were removed at 5, 10, 15 and 20 min time points, quenched by addition of EDTA to 20 mM and then analyzed by electrophoresis through 5% native polyacrylamide gels (29:1 acrylamide:bisacylamide, BioRad) in 0.5× TBE buffer (50 mM Tris base, 55 mM boric acid and 1 mM EDTA, pH 8.3), followed by drying and storage phosphor imaging.

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  • 99
    New England Biolabs t4 dna ligase
    HSF1 mediates the occurrence of a promoter–enhancer interaction at FOXO3 locus involving the 5′UTR and the rs2802292 region. ( A ) Top: physical map of the human FOXO3 gene. The scheme shows the Csp6I restriction enzyme sites flanking the baits (red for the 5′UTR and blue for the rs2802292 region). Bottom: schematic representation of the 3C and ChIP-loop assay. Crosslinked chromatin was digested with Csp6I and immunoprecipitated with anti-HSF1. The immunoprecipitated samples were diluted in a ligation buffer and ligated with the <t>T4</t> DNA Ligase. After reversing the crosslinks, the ligated DNA was purified and amplified by PCR with various combinations of primers as indicated in (B). ( B ) This strategy allows the amplification of sequences ligated to the bait in circular DNA. The arrows indicate the positions of the primers within the bait sequence. Five different couples of primers were designed to analyze the five possible ligation products. Purified DNA was analyzed by PCR with primers specific for the various possible combinations of chromatin fragments. The values are the results of the densitometric analysis and are expressed as fold induction. HEK-293 cells and primary human fibroblasts (GG, n = 3; TT, n = 3) were collected after induction of oxidative stress (1 h H 2 O 2 , 100 μM). The presented results are representative of three independent experiments. P -values were derived from t -tests: * P ≤ 0.05.
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 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 1 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase - by Bioz Stars, 2021-03
    99/100 stars
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    HSF1 mediates the occurrence of a promoter–enhancer interaction at FOXO3 locus involving the 5′UTR and the rs2802292 region. ( A ) Top: physical map of the human FOXO3 gene. The scheme shows the Csp6I restriction enzyme sites flanking the baits (red for the 5′UTR and blue for the rs2802292 region). Bottom: schematic representation of the 3C and ChIP-loop assay. Crosslinked chromatin was digested with Csp6I and immunoprecipitated with anti-HSF1. The immunoprecipitated samples were diluted in a ligation buffer and ligated with the T4 DNA Ligase. After reversing the crosslinks, the ligated DNA was purified and amplified by PCR with various combinations of primers as indicated in (B). ( B ) This strategy allows the amplification of sequences ligated to the bait in circular DNA. The arrows indicate the positions of the primers within the bait sequence. Five different couples of primers were designed to analyze the five possible ligation products. Purified DNA was analyzed by PCR with primers specific for the various possible combinations of chromatin fragments. The values are the results of the densitometric analysis and are expressed as fold induction. HEK-293 cells and primary human fibroblasts (GG, n = 3; TT, n = 3) were collected after induction of oxidative stress (1 h H 2 O 2 , 100 μM). The presented results are representative of three independent experiments. P -values were derived from t -tests: * P ≤ 0.05.

    Journal: Nucleic Acids Research

    Article Title: The longevity SNP rs2802292 uncovered: HSF1 activates stress-dependent expression of FOXO3 through an intronic enhancer

    doi: 10.1093/nar/gky331

    Figure Lengend Snippet: HSF1 mediates the occurrence of a promoter–enhancer interaction at FOXO3 locus involving the 5′UTR and the rs2802292 region. ( A ) Top: physical map of the human FOXO3 gene. The scheme shows the Csp6I restriction enzyme sites flanking the baits (red for the 5′UTR and blue for the rs2802292 region). Bottom: schematic representation of the 3C and ChIP-loop assay. Crosslinked chromatin was digested with Csp6I and immunoprecipitated with anti-HSF1. The immunoprecipitated samples were diluted in a ligation buffer and ligated with the T4 DNA Ligase. After reversing the crosslinks, the ligated DNA was purified and amplified by PCR with various combinations of primers as indicated in (B). ( B ) This strategy allows the amplification of sequences ligated to the bait in circular DNA. The arrows indicate the positions of the primers within the bait sequence. Five different couples of primers were designed to analyze the five possible ligation products. Purified DNA was analyzed by PCR with primers specific for the various possible combinations of chromatin fragments. The values are the results of the densitometric analysis and are expressed as fold induction. HEK-293 cells and primary human fibroblasts (GG, n = 3; TT, n = 3) were collected after induction of oxidative stress (1 h H 2 O 2 , 100 μM). The presented results are representative of three independent experiments. P -values were derived from t -tests: * P ≤ 0.05.

    Article Snippet: H2 O2 -stressed cells were treated with formaldehyde to obtain cross-linked chromatin, after which DNA/protein complexes were first digested with the Csp6I restriction enzyme and then selected with anti-HSF1 antibodies; at last, ligation of chromatin fragments was carried out with the T4 DNA Ligase (Figure ).

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Ligation, Purification, Amplification, Polymerase Chain Reaction, Sequencing, Derivative Assay

    RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.

    Journal: Nucleic Acids Research

    Article Title: A two-nuclease pathway involving RNase H1 is required for primer removal at human mitochondrial OriL

    doi: 10.1093/nar/gky708

    Figure Lengend Snippet: RNase H1 processing coupled to POLγ dependent DNA synthesis does not produce ligatable nicks. ( A ) Schematic of the coupled nuclease gap-filling ligation assay performed on a gapped OriL substrate (a). The upstream oligonucleotide was radioactively labelled at the 5′-end. The possible products are illustrated (b–d). (–) Coupled nuclease gap-filling ligation assay as shown in A. POLγ filled the gap (lane 2, marked b) and had limited strand displacement activity (SD). Note though that POLγ completely displaces the downstream oligonucleotide in a small fraction of templates (80 nt band, lanes 3–6,), RNase H1 cleaved the RNA in the substrate (lane 3), enabling further gap-filling (marked c). Only very low levels of ligated products were formed in the presence of 80–320 fmol DNA ligase III (lanes 4–6). A prominent 80 nt ligated product was formed with T4 DNA ligase (lane 7, marked d). The letters a-d correspond to the illustrations in panel A. ( C ) Ligation assay on a nicked substrate containing RNA tracts of varying length downstream of the nick in the presence of 80–320 fmol DNA ligase III. DNA ligase III discriminates against nicked substrates that contain increasing stretches of ribonucleotides. Two, but not five or more ribonucleotides, can be ligated. ( D ) As in C, except performed with T4 ligase (1–8 U). T4 ligase can ligate five but not 10 ribonucleotides. ( E ) T4 ligase-mediated ligation is abolished in the presence of the mutant RNase H1 proteins. The letters a-d correspond to the illustrations in panel A.

    Article Snippet: While wild type RNase H1 together with T4 DNA ligase produced a ligated product, negligible ligation was seen with the mutant proteins (Figure ).

    Techniques: DNA Synthesis, Ligation, Activity Assay, Mutagenesis

    Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.

    Journal: Nucleic Acids Research

    Article Title: Blocking of targeted microRNAs from next-generation sequencing libraries

    doi: 10.1093/nar/gkv724

    Figure Lengend Snippet: Modification of miRNA sequencing library generation protocol to allow for blocking of targeted species. ( A ) In the standard protocol, a pre-adenylated adaptor is ligated to the 3′ end of a small RNA pool using T4 RNA Ligase 2, truncated. Subsequently, a second adaptor is added to the 5′ end of the miRNA with T4 RNA Ligase 1, followed by reverse transcription and PCR. ( B ) In our modification, a hairpin oligonucleotide with an overhang complementary to the 5′ end of the targeted miRNA is attached via ligation with T4 DNA Ligase to the 5′ end of the miRNA subsequent to the ligation of the adaptor to the 3′ end. This prevents the ligation of the second adaptor to the 5′ end of the miRNA, resulting in a product that does not amplify during PCR. ( C ) Sequencing libraries were generated from human heart total RNA using a titration of a blocking oligonucleotide targeting hsa-miR-16–5p. The fraction of hsa-miR-16–5p present in the blocked library compared to the unblocked library is shown on the y-axis.

    Article Snippet: Because our approach relies on T4 DNA Ligase, which is sensitive to base-pair mismatches and gaps , these variations can adversely affect the efficacy of the blocking (Supplementary Figure S4).

    Techniques: Modification, Sequencing, Blocking Assay, Polymerase Chain Reaction, Ligation, Generated, Titration