t4 ligase buffer  (New England Biolabs)


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    Name:
    T4 DNA Ligase
    Description:
    T4 DNA Ligase 100 000 units
    Catalog Number:
    m0202l
    Price:
    256
    Size:
    100 000 units
    Category:
    DNA Ligases
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    Structured Review

    New England Biolabs t4 ligase buffer
    T4 DNA Ligase
    T4 DNA Ligase 100 000 units
    https://www.bioz.com/result/t4 ligase buffer/product/New England Biolabs
    Average 90 stars, based on 70 article reviews
    Price from $9.99 to $1999.99
    t4 ligase buffer - by Bioz Stars, 2020-01
    90/100 stars

    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

    Transduction:

    Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation
    Article Snippet: This lentiviral vector does not have a selection marker, but as lentiviruses were used to transduce cells the efficiencies should be high. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and T4 DNA ligase (NEB), followed by transformation into chemically competent DH5α cells.

    Clone Assay:

    Article Title: A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide
    Article Snippet: Plasmids pCR®2.1-TOPO® (Invitrogen), pQE40 (Qiagen) and pET28a+ (Novagen) were used for cloning and production of the proteins linked to a 6His-tag. .. Restriction enzymes, Taq polymerase, factor Xa and T4 DNA ligase were purchased from New England Biolabs.

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C. .. Screening for positive clones was performed by colony PCR and control restriction digests.

    Amplification:

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol. .. The pWPT-ΔGFP-X plasmids encoding the native form of the X proteins were obtained after digestion of the pWPT-GFP-X vectors using MluI and NotI and amplification of each X using Mlu1-pWPT-X-F primer (5′-GCT TAC GCG TTC TGC AGT CGA CGA ATT CAC CAT G-3′) and pWPT-R primer (5′-GTC AGC AAA CAC AGT GCA CAC CA-3′).

    Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation
    Article Snippet: For generating SBP constructs, SBP was amplified from the pTrAP vector [ ] and subcloned via BamHI and NotI sites into the pEGFP-N2 vector, replacing GFP with SBP. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and T4 DNA ligase (NEB), followed by transformation into chemically competent DH5α cells.

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: After digestion of the three DNA fragments and the target vector (pGL3-basic, Promega) using the respective restriction enzymes (gBlock1: Xho I and Pst I, gBlock2: Pst I and Pvu I, PCR amplicon 3: Pvu I and Hind III, pGL3-basic: Xho I and Hind III, NEB, 90 min 37°C) the DNA fragments and the vector were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) according manufacturer's instructions. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C.

    DNA Ligation:

    Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression
    Article Snippet: 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of T4 DNA ligase (200 U, New England Biolabs). .. DNA ligation products were recovered by extraction with phenol:chloroform (1:1 v/v) and ethanol precipitation and separated by 1% agarose gel electrophoresis.

    Synthesized:

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X coding regions (synthesized by Genewiz) from hepadnaviruses infecting the New World wooly monkey ( Lagothrix ) (WMHBx) and three distant bat species, including the roundleaf bat ( Hipposideros cf. ruber ), the horseshoe bat ( Rhinolophus Alcyone ), and the tent-making bat ( Uroderma bilobatum ) (RBHBx, HBHBx, TBHBx, respectively) , were expressed from the same vector. .. The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol.

    Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection
    Article Snippet: Restriction enzymes, T4 DNA ligase, and Taq or Phusion high-fidelity polymerase (New England Biolabs, France) were used in accordance with the manufacturer’s recommendations. .. Oligonucleotides (see in the supplemental material) were synthesized by Sigma-Proligo (Paris, France) or Eurofins-MWG (Paris, France).

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: Two fragments were synthesized (gBlock fragments-IDT), reaching from position chr15: 99589250-99589987 (gBlock1) and chr15: 99589970-99590606 (gBlock2) of the GRCm38/mm10 assembly and the third was obtained by PCR of region Chr15:99590527-99591271. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C.

    Construct:

    Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection
    Article Snippet: Restriction enzymes, T4 DNA ligase, and Taq or Phusion high-fidelity polymerase (New England Biolabs, France) were used in accordance with the manufacturer’s recommendations. .. Nucleotide sequences of all constructs were determined by Beckman Coulter Genomics (Takeley, United Kingdom).

    Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation
    Article Snippet: For generating SBP constructs, SBP was amplified from the pTrAP vector [ ] and subcloned via BamHI and NotI sites into the pEGFP-N2 vector, replacing GFP with SBP. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and T4 DNA ligase (NEB), followed by transformation into chemically competent DH5α cells.

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: Promoter Transfection Experiments with Luciferase Vector A construct of the murine AQP5 promoter (2021 bp upstream of the ATG start codon) was produced by the fusion of three overlapping fragments treated with restriction enzymes followed by ligation. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C.

    Electrophoresis:

    Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs
    Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using T4 DNA ligase (New England Biolab). .. The resulting products were separated by electrophoresis in a urea-15% polyacrylamide TBE gel (Invitrogen) with Dynamarker Small RNA plus (BioDynamics Laboratory, Tokyo, Japan) as a size marker and exposed to X-ray film (Kodak).

    Incubation:

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: .. Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation. ..

    Article Title: Rapid Identification of Major QTLs Associated with Rice Grain Weight and Their Utilization
    Article Snippet: .. In short, genomic DNA of two DNA bulks and two parents were incubated at 37°C with Mse I (New England Biolabs, NEB), T4 DNA ligase (NEB), ATP (NEB), and Mse I adapter. ..

    Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression
    Article Snippet: .. 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of T4 DNA ligase (200 U, New England Biolabs). .. Reactions were impeded and de-proteinated by adding Proteinase K enzyme (Invitrogen) followed by 15 min incubation at 37°C.

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C. .. Half of the ligation reaction (10 μ L) was transformed into XL1-Blue competent E. coli cells (Agilent) according to manufacturer's instructions and plated on LB-agar containing 100 μ g/mL ampicillin.

    Luciferase:

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: Paragraph title: 2.8. Promoter Transfection Experiments with Luciferase Vector ... The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C.

    Expressing:

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: Paragraph title: Expression plasmids. ... The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol.

    Article Title: A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide
    Article Snippet: M15 and BL21 were used for the expression of the proteins. .. Restriction enzymes, Taq polymerase, factor Xa and T4 DNA ligase were purchased from New England Biolabs.

    Modification:

    Article Title: Rapid Identification of Major QTLs Associated with Rice Grain Weight and Their Utilization
    Article Snippet: SLAF library construction and sequencing The construction of SLAF library followed the methods proposed by Sun et al [ ] with little modification. .. In short, genomic DNA of two DNA bulks and two parents were incubated at 37°C with Mse I (New England Biolabs, NEB), T4 DNA ligase (NEB), ATP (NEB), and Mse I adapter.

    Transformation Assay:

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol. .. The plasmid was then transformed following the high-efficiency transformation protocol using NEB 10-beta Competent Escherichia coli (NEB; C3019).

    Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation
    Article Snippet: .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and T4 DNA ligase (NEB), followed by transformation into chemically competent DH5α cells. .. All the constructs used in this study were sequenced at the regions of interest using Sanger DNA sequencing.

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C. .. Half of the ligation reaction (10 μ L) was transformed into XL1-Blue competent E. coli cells (Agilent) according to manufacturer's instructions and plated on LB-agar containing 100 μ g/mL ampicillin.

    Activated Clotting Time Assay:

    Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs
    Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using T4 DNA ligase (New England Biolab). .. The following bridge oligonucleotides were used: let-7i, 5′-GAA TGT CAT AAG CGA ACA GCA CAA ACT ACT ACC TCA-3′; miR-214, 5′-GAA TGT CAT AAG CGG CAC AGC AAG TGT AGA CAG GCA-3′; miR-15a, 5′-GAA TGT CAT AAG CGC ACA AAC CAT TAT GTG CTG CTA-3′; miR-30c, 5′-GAA TGT CAT AAG CGG CTG AGA GTG TAG GAT GTT TAC A-3′; miR-485, 5′-GAA TGT CAT AAG CGG AAT TCA TCA CGG CCA GCC TCT-3′; and miR-666, 5′-GAA TGT CAT AAG CGG GCT CTC ACA GCT GTG CCC GCT-3′.

    Countercurrent Chromatography:

    Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs
    Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using T4 DNA ligase (New England Biolab). .. The following bridge oligonucleotides were used: let-7i, 5′-GAA TGT CAT AAG CGA ACA GCA CAA ACT ACT ACC TCA-3′; miR-214, 5′-GAA TGT CAT AAG CGG CAC AGC AAG TGT AGA CAG GCA-3′; miR-15a, 5′-GAA TGT CAT AAG CGC ACA AAC CAT TAT GTG CTG CTA-3′; miR-30c, 5′-GAA TGT CAT AAG CGG CTG AGA GTG TAG GAT GTT TAC A-3′; miR-485, 5′-GAA TGT CAT AAG CGG AAT TCA TCA CGG CCA GCC TCT-3′; and miR-666, 5′-GAA TGT CAT AAG CGG GCT CTC ACA GCT GTG CCC GCT-3′.

    Transfection:

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: Paragraph title: 2.8. Promoter Transfection Experiments with Luciferase Vector ... The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C.

    Ligation:

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: .. Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation. ..

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: .. The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol. .. The plasmid was then transformed following the high-efficiency transformation protocol using NEB 10-beta Competent Escherichia coli (NEB; C3019).

    Article Title: Rapid Identification of Major QTLs Associated with Rice Grain Weight and Their Utilization
    Article Snippet: In short, genomic DNA of two DNA bulks and two parents were incubated at 37°C with Mse I (New England Biolabs, NEB), T4 DNA ligase (NEB), ATP (NEB), and Mse I adapter. .. The PCR reaction was performed using diluted restriction ligation samples, dNTP, Taq DNA polymerase (NEB) and Mse I primer containing barcode 1.

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: Promoter Transfection Experiments with Luciferase Vector A construct of the murine AQP5 promoter (2021 bp upstream of the ATG start codon) was produced by the fusion of three overlapping fragments treated with restriction enzymes followed by ligation. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C.

    Article Title: Assessing long-distance RNA sequence connectivity via RNA-templated DNA–DNA ligation
    Article Snippet: .. Final ligation conditions in were (left panel) 1.5 μM ssDNA or RNA template, 5′-32 P-labeled oligos (10 μM each), and 1 μl of neat indicated enzyme (specific units varied according to the manufacturer and enzyme) in manufacturer's recommended buffer; (right panel) 250 nM RNA template, 5′-32 P-labeled oligos (500 nM each), and 10 U/μl Rnl2 or 20 U/μl T4 DNA ligase. .. Reactions in contained 1.25 μM DDX1 RNA template, 5 μM each 5′-32 P-labeled oligo, and 10 U/μl Rnl2 were incubated for 4 hr at 37°C and separated on a 11.25% denaturing polyacrylamide gel.

    DNA Sequencing:

    Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation
    Article Snippet: The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and T4 DNA ligase (NEB), followed by transformation into chemically competent DH5α cells. .. All the constructs used in this study were sequenced at the regions of interest using Sanger DNA sequencing.

    Polymerase Chain Reaction:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: KK8235) PEG/NaCl SPRI solution, supplied with HTP Library Preparation Kit PCR-free (96rxn) (Kapa Biosystems) 2× Kapa HiFi HotStart Ready Mix (Kapa Biosystems, cat.no. .. M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA Ligase USER Enzyme (New England BioLabs, cat.no.

    Article Title: Using specific length amplified fragment sequencing to construct the high-density genetic map for Vitis (Vitis vinifera L. × Vitis amurensis Rupr.)
    Article Snippet: The genomic DNA from each sample was treated with Rsa I, Hae III (NEB, Ipswich, MA, USA), T4 DNA ligase (NEB), and ATP (NEB), and maintained at 37°C. .. The restriction-ligation reaction solutions were diluted and mixed with dNTP, Taq DNA polymerase (NEB), and Hae III primer for polymerase chain reaction (PCR) analyses.

    Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection
    Article Snippet: PCR-amplified fragments and digested fragments separated on 0.8% agarose gels were purified with kits from Qiagen (France). .. Restriction enzymes, T4 DNA ligase, and Taq or Phusion high-fidelity polymerase (New England Biolabs, France) were used in accordance with the manufacturer’s recommendations.

    Article Title: Rapid Identification of Major QTLs Associated with Rice Grain Weight and Their Utilization
    Article Snippet: In short, genomic DNA of two DNA bulks and two parents were incubated at 37°C with Mse I (New England Biolabs, NEB), T4 DNA ligase (NEB), ATP (NEB), and Mse I adapter. .. The PCR reaction was performed using diluted restriction ligation samples, dNTP, Taq DNA polymerase (NEB) and Mse I primer containing barcode 1.

    Article Title: A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide
    Article Snippet: Plasmids pCR®2.1-TOPO® (Invitrogen), pQE40 (Qiagen) and pET28a+ (Novagen) were used for cloning and production of the proteins linked to a 6His-tag. .. Restriction enzymes, Taq polymerase, factor Xa and T4 DNA ligase were purchased from New England Biolabs.

    Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation
    Article Snippet: .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and T4 DNA ligase (NEB), followed by transformation into chemically competent DH5α cells. .. All the constructs used in this study were sequenced at the regions of interest using Sanger DNA sequencing.

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: After digestion of the three DNA fragments and the target vector (pGL3-basic, Promega) using the respective restriction enzymes (gBlock1: Xho I and Pst I, gBlock2: Pst I and Pvu I, PCR amplicon 3: Pvu I and Hind III, pGL3-basic: Xho I and Hind III, NEB, 90 min 37°C) the DNA fragments and the vector were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) according manufacturer's instructions. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C.

    Binding Assay:

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation. .. Biotinylated Hi-C DNA in 1X binding buffer (5 mM Tris-HCl, pH 8, 0.5 mM EDTA, 1 M NaCl) was pulled down using Dynabeads MyOne Streptavidin C1 (Life Technologies, 650.01), using 5 µl of beads per microgram of DNA, pre-washed in 1X binding buffer.

    Cellular Antioxidant Activity Assay:

    Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs
    Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using T4 DNA ligase (New England Biolab). .. The following bridge oligonucleotides were used: let-7i, 5′-GAA TGT CAT AAG CGA ACA GCA CAA ACT ACT ACC TCA-3′; miR-214, 5′-GAA TGT CAT AAG CGG CAC AGC AAG TGT AGA CAG GCA-3′; miR-15a, 5′-GAA TGT CAT AAG CGC ACA AAC CAT TAT GTG CTG CTA-3′; miR-30c, 5′-GAA TGT CAT AAG CGG CTG AGA GTG TAG GAT GTT TAC A-3′; miR-485, 5′-GAA TGT CAT AAG CGG AAT TCA TCA CGG CCA GCC TCT-3′; and miR-666, 5′-GAA TGT CAT AAG CGG GCT CTC ACA GCT GTG CCC GCT-3′.

    Hi-C:

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: Paragraph title: In situ Hi-C of knockdown and control cells ... Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation.

    Marker:

    Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs
    Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using T4 DNA ligase (New England Biolab). .. The resulting products were separated by electrophoresis in a urea-15% polyacrylamide TBE gel (Invitrogen) with Dynamarker Small RNA plus (BioDynamics Laboratory, Tokyo, Japan) as a size marker and exposed to X-ray film (Kodak).

    Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation
    Article Snippet: This lentiviral vector does not have a selection marker, but as lentiviruses were used to transduce cells the efficiencies should be high. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and T4 DNA ligase (NEB), followed by transformation into chemically competent DH5α cells.

    Mutagenesis:

    Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation
    Article Snippet: To generate the ΔNTD mutant of human TMEM120A-SBP (lacking amino acids 2–105), site directed mutagenesis was done using the NEB Q5 mutagenesis kit strategy with minor modifications. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and T4 DNA ligase (NEB), followed by transformation into chemically competent DH5α cells.

    Subcloning:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: 28704) XL1-Blue subcloning grade competent cells (Agilent, cat.no. .. M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA ligase LB agar (Miller powder) (Fisher Scientific, cat.no.

    Size-exclusion Chromatography:

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: The amplicon was produced with genomic DNA from EpH4 cells extracted using the Gentra Puregene Cell Kit (QIAGEN) according to manufacturer's instructions and amplified using 0.125 U OneTaq Hot Start DNA polymerase (NEB) with an initial heating step of 94°C for 5 min followed by 45 cycles at 94°C for 15 sec, 58°C for 15 sec, and 68°C for 45 sec (primer sequences are shown in Supplementary Table 1). .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C.

    Labeling:

    Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs
    Article Snippet: Splinted-ligation-mediated miRNA detection MiRNAs were detected using the miRtect-IT miRNA Labeling and Detection Kit (Affymetrix) according to the manufacturer’s instructions. .. In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using T4 DNA ligase (New England Biolab).

    Purification:

    Article Title: Using specific length amplified fragment sequencing to construct the high-density genetic map for Vitis (Vitis vinifera L. × Vitis amurensis Rupr.)
    Article Snippet: The genomic DNA from each sample was treated with Rsa I, Hae III (NEB, Ipswich, MA, USA), T4 DNA ligase (NEB), and ATP (NEB), and maintained at 37°C. .. The PCR products were purified using E.Z.N.A.

    Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection
    Article Snippet: PCR-amplified fragments and digested fragments separated on 0.8% agarose gels were purified with kits from Qiagen (France). .. Restriction enzymes, T4 DNA ligase, and Taq or Phusion high-fidelity polymerase (New England Biolabs, France) were used in accordance with the manufacturer’s recommendations.

    Article Title: Rapid Identification of Major QTLs Associated with Rice Grain Weight and Their Utilization
    Article Snippet: In short, genomic DNA of two DNA bulks and two parents were incubated at 37°C with Mse I (New England Biolabs, NEB), T4 DNA ligase (NEB), ATP (NEB), and Mse I adapter. .. The PCR products were purified using EZNAH Cycle Pure Kit (Omega) and pooled.

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: After digestion of the three DNA fragments and the target vector (pGL3-basic, Promega) using the respective restriction enzymes (gBlock1: Xho I and Pst I, gBlock2: Pst I and Pvu I, PCR amplicon 3: Pvu I and Hind III, pGL3-basic: Xho I and Hind III, NEB, 90 min 37°C) the DNA fragments and the vector were purified using the Wizard SV Gel and PCR Clean-Up System (Promega) according manufacturer's instructions. .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C.

    Sequencing:

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol. .. All the X-expressing plasmids were checked by Sanger sequencing using eGFP-F primer, 5′-CAT GGT CCT GCT GGA GTT CGT G-3′, and pWPT-R, 5′-GTC AGC AAA CAC AGT GCA CAC CA-3′.

    Article Title: Rapid Identification of Major QTLs Associated with Rice Grain Weight and Their Utilization
    Article Snippet: Paragraph title: SLAF library construction and sequencing ... In short, genomic DNA of two DNA bulks and two parents were incubated at 37°C with Mse I (New England Biolabs, NEB), T4 DNA ligase (NEB), ATP (NEB), and Mse I adapter.

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C. .. The integrity of the final pGL3-construct containing the AQP5 promoter upstream of the luc + gene (Firefly) was verified by sequencing.

    Gel Extraction:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: R3535L) 10× CutSmart Buffer (New England BioLabs) supplied with Bsa I-HF QIAquick Gel extraction kit (Qiagen, cat.no. .. M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA ligase LB agar (Miller powder) (Fisher Scientific, cat.no.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs
    Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using T4 DNA ligase (New England Biolab). .. The following bridge oligonucleotides were used: let-7i, 5′-GAA TGT CAT AAG CGA ACA GCA CAA ACT ACT ACC TCA-3′; miR-214, 5′-GAA TGT CAT AAG CGG CAC AGC AAG TGT AGA CAG GCA-3′; miR-15a, 5′-GAA TGT CAT AAG CGC ACA AAC CAT TAT GTG CTG CTA-3′; miR-30c, 5′-GAA TGT CAT AAG CGG CTG AGA GTG TAG GAT GTT TAC A-3′; miR-485, 5′-GAA TGT CAT AAG CGG AAT TCA TCA CGG CCA GCC TCT-3′; and miR-666, 5′-GAA TGT CAT AAG CGG GCT CTC ACA GCT GTG CCC GCT-3′.

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: .. M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA ligase LB agar (Miller powder) (Fisher Scientific, cat.no. .. DF0445174) Luria Broth base (Miller’s LB Broth Base) (Thermo Fisher Scientific, cat.no.

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: .. M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA Ligase USER Enzyme (New England BioLabs, cat.no. .. M5505L) Exonuclease I ( E. coli ) (New England BioLabs, cat.no.

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol. .. The pWPT-ΔGFP-X plasmids encoding the native form of the X proteins were obtained after digestion of the pWPT-GFP-X vectors using MluI and NotI and amplification of each X using Mlu1-pWPT-X-F primer (5′-GCT TAC GCG TTC TGC AGT CGA CGA ATT CAC CAT G-3′) and pWPT-R primer (5′-GTC AGC AAA CAC AGT GCA CAC CA-3′).

    Plasmid Preparation:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA Ligase USER Enzyme (New England BioLabs, cat.no. .. M0262L) Plasmid-Safe ATP-dependent DNase (Epicentre, cat.no.

    Article Title: Smc5/6 Antagonism by HBx Is an Evolutionarily Conserved Function of Hepatitis B Virus Infection in Mammals
    Article Snippet: The X coding regions (synthesized by Genewiz) from hepadnaviruses infecting the New World wooly monkey ( Lagothrix ) (WMHBx) and three distant bat species, including the roundleaf bat ( Hipposideros cf. ruber ), the horseshoe bat ( Rhinolophus Alcyone ), and the tent-making bat ( Uroderma bilobatum ) (RBHBx, HBHBx, TBHBx, respectively) , were expressed from the same vector. .. The X insert was ligated to the pWPT-GFP backbone between the PstI and NotI sites following the T4 DNA (New England BioLabs [NEB]; M0202) ligation protocol.

    Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection
    Article Snippet: Plasmid DNA was extracted from E. coli by a standard alkaline lysis procedure with QIAprep spin columns (Qiagen, France). .. Restriction enzymes, T4 DNA ligase, and Taq or Phusion high-fidelity polymerase (New England Biolabs, France) were used in accordance with the manufacturer’s recommendations.

    Article Title: A comparison between the recombinant expression and chemical synthesis of a short cysteine-rich insecticidal spider peptide
    Article Snippet: Bacterial strains, enzymes and plasmids E. coli XL1 Blue was used for plasmid propagation. .. Restriction enzymes, Taq polymerase, factor Xa and T4 DNA ligase were purchased from New England Biolabs.

    Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation
    Article Snippet: Briefly, non-phosphorylated primers were used (forward 5’-GGATTGTACCTGAGCCTGGTTCTG and reverse 5’-CATGGGTCGAGATCTGAGTCCG ) in a PCR reaction with Phusion polymerase and TMEM120A-SBP plasmid. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and T4 DNA ligase (NEB), followed by transformation into chemically competent DH5α cells.

    Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression
    Article Snippet: Linearized pUC19 DNA vector (Takara biotechnology, Dalia, CO., LTD) was prepared as previously described. .. 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of T4 DNA ligase (200 U, New England Biolabs).

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C. .. Half of the ligation reaction (10 μ L) was transformed into XL1-Blue competent E. coli cells (Agilent) according to manufacturer's instructions and plated on LB-agar containing 100 μ g/mL ampicillin.

    In Situ:

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: Paragraph title: In situ Hi-C of knockdown and control cells ... Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation.

    Selection:

    Article Title: TMEM120A and B: Nuclear Envelope Transmembrane Proteins Important for Adipocyte Differentiation
    Article Snippet: This lentiviral vector does not have a selection marker, but as lentiviruses were used to transduce cells the efficiencies should be high. .. The resulting PCR product was gel-purified and simultaneously phosphorylated and ligated using PNK and T4 DNA ligase (NEB), followed by transformation into chemically competent DH5α cells.

    Agarose Gel Electrophoresis:

    Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression
    Article Snippet: 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of T4 DNA ligase (200 U, New England Biolabs). .. DNA ligation products were recovered by extraction with phenol:chloroform (1:1 v/v) and ethanol precipitation and separated by 1% agarose gel electrophoresis.

    In Vitro:

    Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression
    Article Snippet: Paragraph title: T4 DNA ligase in vitro assay ... 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of T4 DNA ligase (200 U, New England Biolabs).

    Ethanol Precipitation:

    Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression
    Article Snippet: 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of T4 DNA ligase (200 U, New England Biolabs). .. DNA ligation products were recovered by extraction with phenol:chloroform (1:1 v/v) and ethanol precipitation and separated by 1% agarose gel electrophoresis.

    Next-Generation Sequencing:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: Paragraph title: CIRCLE-seq library preparation and NGS ... M0202L) 10× T4 DNA ligase Buffer (New England BioLabs), supplied with T4 DNA Ligase USER Enzyme (New England BioLabs, cat.no.

    Produced:

    Article Title: Aquaporin 5 Expression in Mouse Mammary Gland Cells Is Not Driven by Promoter Methylation
    Article Snippet: The amplicon was produced with genomic DNA from EpH4 cells extracted using the Gentra Puregene Cell Kit (QIAGEN) according to manufacturer's instructions and amplified using 0.125 U OneTaq Hot Start DNA polymerase (NEB) with an initial heating step of 94°C for 5 min followed by 45 cycles at 94°C for 15 sec, 58°C for 15 sec, and 68°C for 45 sec (primer sequences are shown in Supplementary Table 1). .. The inserts and the vector were ligated in a 1 : 3 molar ratio using the T4 DNA ligase (NEB) in an overnight incubation at 16°C.

    Concentration Assay:

    Article Title: High-resolution TADs reveal DNA sequences underlying genome organization in flies
    Article Snippet: After 10 min incubation at room temperature, SDS was quenched adding 1% Triton X-100 (final concentration) and 1X of NEBuffer 3.1 (NEB, B7203S). .. Ligase mix was added (1X Ligation buffer NEB B0202, 0.8% Triton X-100, 0.1 mg/ml BSA, 2000 U T4 DNA ligase NEB M0202S, final sample volume 1.2 ml) and samples were incubated for 4 h at room temperature under rotation.

    Alkaline Lysis:

    Article Title: Cell Differentiation in a Bacillus thuringiensis Population during Planktonic Growth, Biofilm Formation, and Host Infection
    Article Snippet: Plasmid DNA was extracted from E. coli by a standard alkaline lysis procedure with QIAprep spin columns (Qiagen, France). .. Restriction enzymes, T4 DNA ligase, and Taq or Phusion high-fidelity polymerase (New England Biolabs, France) were used in accordance with the manufacturer’s recommendations.

    CTG Assay:

    Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs
    Article Snippet: In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using T4 DNA ligase (New England Biolab). .. The following bridge oligonucleotides were used: let-7i, 5′-GAA TGT CAT AAG CGA ACA GCA CAA ACT ACT ACC TCA-3′; miR-214, 5′-GAA TGT CAT AAG CGG CAC AGC AAG TGT AGA CAG GCA-3′; miR-15a, 5′-GAA TGT CAT AAG CGC ACA AAC CAT TAT GTG CTG CTA-3′; miR-30c, 5′-GAA TGT CAT AAG CGG CTG AGA GTG TAG GAT GTT TAC A-3′; miR-485, 5′-GAA TGT CAT AAG CGG AAT TCA TCA CGG CCA GCC TCT-3′; and miR-666, 5′-GAA TGT CAT AAG CGG GCT CTC ACA GCT GTG CCC GCT-3′.

    Staining:

    Article Title: Gain of function mutant p53 proteins cooperate with E2F4 to transcriptionally downregulate RAD17 and BRCA1 gene expression
    Article Snippet: 200 ng of this DNA was incubated with nuclear extracts for 1h at 25°C in a reaction mixture containing 1 × ligase buffer and 1 μl of T4 DNA ligase (200 U, New England Biolabs). .. The gel was visualized by staining with ethidium bromide and represented as an inverted image.

    Variant Assay:

    Article Title: Rbfox3 Controls the Biogenesis of a Subset of MicroRNAs
    Article Snippet: .. In brief, miRNAs were captured from 4 μg of total RNA using a bridge oligonucleotide that is designed to specifically detect one miRNA variant, and ligated to a 32 P-labeled detection oligonucleotide using T4 DNA ligase (New England Biolab). .. The following bridge oligonucleotides were used: let-7i, 5′-GAA TGT CAT AAG CGA ACA GCA CAA ACT ACT ACC TCA-3′; miR-214, 5′-GAA TGT CAT AAG CGG CAC AGC AAG TGT AGA CAG GCA-3′; miR-15a, 5′-GAA TGT CAT AAG CGC ACA AAC CAT TAT GTG CTG CTA-3′; miR-30c, 5′-GAA TGT CAT AAG CGG CTG AGA GTG TAG GAT GTT TAC A-3′; miR-485, 5′-GAA TGT CAT AAG CGG AAT TCA TCA CGG CCA GCC TCT-3′; and miR-666, 5′-GAA TGT CAT AAG CGG GCT CTC ACA GCT GTG CCC GCT-3′.

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