t4 dna ligase reaction buffer  (New England Biolabs)


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    T4 DNA Ligase Reaction Buffer
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    T4 DNA Ligase Reaction Buffer 6 0 ml
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    b0202s
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    6 0 ml
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    New England Biolabs t4 dna ligase reaction buffer
    T4 DNA Ligase Reaction Buffer
    T4 DNA Ligase Reaction Buffer 6 0 ml
    https://www.bioz.com/result/t4 dna ligase reaction buffer/product/New England Biolabs
    Average 95 stars, based on 273 article reviews
    Price from $9.99 to $1999.99
    t4 dna ligase reaction buffer - by Bioz Stars, 2020-02
    95/100 stars

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    1) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    2) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.008

    YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .
    Figure Legend Snippet: YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Techniques Used: In Vitro, Nucleic Acid Electrophoresis, DNA Ligation, Incubation

    3) Product Images from "Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination"

    Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.03.011

    Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.
    Figure Legend Snippet: Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    Techniques Used: Sequencing, Agarose Gel Electrophoresis, Labeling, Recombinase Polymerase Amplification, Injection, Flow Cytometry

    4) 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

    5) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    6) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    7) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.008

    YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .
    Figure Legend Snippet: YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Techniques Used: In Vitro, Nucleic Acid Electrophoresis, DNA Ligation, Incubation

    8) Product Images from "S1-seq assay for mapping processed DNA ends"

    Article Title: S1-seq assay for mapping processed DNA ends

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.11.031

    Schematic of the key steps employed in S1-seq: DNA from meiotic cells bearing processed Spo11 DSBs is embedded in agarose plugs to protect from shearing. Treatment with S1 nuclease and T4 DNA polymerase removes the 3′ overhang and prepares the ends for ligation with the 5′ biotinylated adaptor. Following extraction from the plugs and sonication, the biotinylated fragments are bound on streptavidin beads and the second adaptor is ligated. Low-cycle amplification by PCR creates the library that is submitted for next generation sequencing and eventually mapped to the reference genome.
    Figure Legend Snippet: Schematic of the key steps employed in S1-seq: DNA from meiotic cells bearing processed Spo11 DSBs is embedded in agarose plugs to protect from shearing. Treatment with S1 nuclease and T4 DNA polymerase removes the 3′ overhang and prepares the ends for ligation with the 5′ biotinylated adaptor. Following extraction from the plugs and sonication, the biotinylated fragments are bound on streptavidin beads and the second adaptor is ligated. Low-cycle amplification by PCR creates the library that is submitted for next generation sequencing and eventually mapped to the reference genome.

    Techniques Used: Ligation, Sonication, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing

    9) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

    Techniques Used: Clone Assay, Plasmid Preparation, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay

    10) Product Images from "YY1 Is a Structural Regulator of Enhancer-Promoter Loops"

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops

    Journal: Cell

    doi: 10.1016/j.cell.2017.11.008

    YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .
    Figure Legend Snippet: YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Techniques Used: In Vitro, Nucleic Acid Electrophoresis, DNA Ligation, Incubation

    11) Product Images from "S1-seq assay for mapping processed DNA ends"

    Article Title: S1-seq assay for mapping processed DNA ends

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.11.031

    Schematic of the key steps employed in S1-seq: DNA from meiotic cells bearing processed Spo11 DSBs is embedded in agarose plugs to protect from shearing. Treatment with S1 nuclease and T4 DNA polymerase removes the 3′ overhang and prepares the ends for ligation with the 5′ biotinylated adaptor. Following extraction from the plugs and sonication, the biotinylated fragments are bound on streptavidin beads and the second adaptor is ligated. Low-cycle amplification by PCR creates the library that is submitted for next generation sequencing and eventually mapped to the reference genome.
    Figure Legend Snippet: Schematic of the key steps employed in S1-seq: DNA from meiotic cells bearing processed Spo11 DSBs is embedded in agarose plugs to protect from shearing. Treatment with S1 nuclease and T4 DNA polymerase removes the 3′ overhang and prepares the ends for ligation with the 5′ biotinylated adaptor. Following extraction from the plugs and sonication, the biotinylated fragments are bound on streptavidin beads and the second adaptor is ligated. Low-cycle amplification by PCR creates the library that is submitted for next generation sequencing and eventually mapped to the reference genome.

    Techniques Used: Ligation, Sonication, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing

    12) Product Images from "Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes"

    Article Title: Pyrite cloning: a single tube and programmed reaction cloning with restriction enzymes

    Journal: Plant Methods

    doi: 10.1186/s13007-018-0359-7

    Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts
    Figure Legend Snippet: Schematic diagram of Pyrite cloning and results. Diagram of Pyrite cloning. An intact plasmid vector and a DNA fragment (purified PCR product) with compatible restriction enzyme sites (RES1 and RES2) are incubated in a single tube together with the restriction enzymes (RE1 and RE2) and T4 DNA ligase. After the Pyrite reaction (incubation condition shown in box), the reaction can be directly transformed into E. coli without purification. Colony PCR will then screen for those colonies containing vectors with inserts

    Techniques Used: Clone Assay, Plasmid Preparation, Purification, Polymerase Chain Reaction, Incubation, Transformation Assay

    13) Product Images from "Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor"

    Article Title: Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19061608

    Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.
    Figure Legend Snippet: Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.

    Techniques Used: Ligation, Incubation, Electrophoresis, Staining

    Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.
    Figure Legend Snippet: Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.

    Techniques Used: Ligation, Staining

    14) Product Images from "Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains"

    Article Title: Assessing Protein Dynamics on Low-Complexity Single-Stranded DNA Curtains

    Journal: Langmuir : the ACS journal of surfaces and colloids

    doi: 10.1021/acs.langmuir.8b01812

    Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.
    Figure Legend Snippet: Assembly of low-complexity ssDNA curtains. (A) A phosphorylated template (black) and a biotinylated primer (green) are annealed and treated with T4 DNA ligase to make minicircles. Low-complexity ssDNA composed solely of thymidine and cytidine is synthesized via rolling circle replication by phi29 DNAP. (B) Low-complexity ssDNA curtains with fluorescent end labeling. The 3′ end of the ssDNA was labeled with a fluorescent antibody. (C) RPA-GFP (green)-coated ssDNA with fluorescent end labeling (magenta). (D) Kymograph of a representative ssDNA in panel (C) with buffer flow on and off, indicating that the ssDNA is anchored to the surface via the 5′-biotin tether.

    Techniques Used: Synthesized, End Labeling, Labeling, Recombinase Polymerase Amplification, Flow Cytometry

    15) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    16) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    Figure Legend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Techniques Used: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.
    Figure Legend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Techniques Used: Ligation, Produced, Standard Deviation

    17) Product Images from "Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor"

    Article Title: Biochemical Properties of a Decoy Oligodeoxynucleotide Inhibitor of STAT3 Transcription Factor

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19061608

    Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.
    Figure Legend Snippet: Ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D) is unaffected by biotinylation. ( A ) Structures of parental and biotinylated STAT3 decoy (S3D) and CS3D. ( B ) CS3D and biotinylated CS3D were incubated with T4 DNA ligase overnight, followed by electrophoresis on a urea/polyacrylamide gel and staining with SYBR Gold.

    Techniques Used: Ligation, Incubation, Electrophoresis, Staining

    Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.
    Figure Legend Snippet: Efficient ligation of cyclic signal transducer and activator of transcription 3 (STAT3) decoy (CS3D). ( A ) Schematic representation of CS3D ligation with T4 DNA ligase. The complementary segments of the single-stranded decoy molecule spontaneously self-anneal. Enzymatic ligation with T4 DNA ligase was used to complete cyclization. ( B ) Incubations were performed in the absence or presence of T4 DNA ligase overnight. Multiple identical ligations ( n = 5) were simultaneously performed. Samples from each reaction were then electrophoresed on a urea/polyacrylamide gel, stained with SYBR Gold, and quantified by densitometry.

    Techniques Used: Ligation, Staining

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    Filtration:

    Article Title: Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex
    Article Snippet: Briefly, 125 μg λ-DNA was mixed with 2 μM IF003 and IF004 in T4 DNA ligase reaction buffer (NEB) and heated to 70°C for 15 min followed by gradual cooling to 15°C for 2 h. After the oligomer hybridization, T4 DNA ligase (2000 units; NEB) was added to the mixture and incubated overnight at room temperature to seal nicks on DNA. .. The ligase was inactivated with 2 M NaCl, and the reaction was passed over an S-1000 gel filtration column (GE) to remove excess oligonucleotides and proteins.

    Synthesized:

    Article Title: Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs)
    Article Snippet: 70mer oligonucleotides synthesized at the 25 nanomole (nM) scale and hydrated to 100 micromole (μM) in 1× TE Buffer, pH 8.0. .. T4 DNA Ligase Reaction Buffer with 10 mM ATP (New England Biolabs).

    End-sequence Profiling:

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science
    Article Snippet: .. To each reaction, 1.5 μl of 10× T4 DNA ligase reaction buffer, 400 U of T4 DNA ligase (New England BioLabs), 5 U of Esp 3I (Thermo Fisher Scientific, Waltham, MA), and water were added to a final volume of 15 μl. .. Reactions were incubated in a thermocycler (37°C, 5 min; 16°C, 10 min) for five or six cycles (37°C 15 min, 80°C 5 min, and 4°C hold).

    Incubation:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Paragraph title: Supporting information Protein gels to evaluate ligase purity. Incubation of ligases with ssDNA to test for contaminating nuclease activity. Reaction endpoint and dsDNA nuclease contamination assessment for wild type ligases. Reaction endpoint and dsDNA nuclease contamination assessment for fusion ligases. Effect of monovalent cations on active blunt/cohesive-end ligases. Separate addition of DNA binding domains to PBCV1 DNA ligase. Fluorescence anisotropy DNA binding curves. Capillary gel electrophoresis panel raw data. ... Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). (TIF) Click here for additional data file.

    Article Title: Functional characteristics of novel pancreatic Pax6 regulatory elements
    Article Snippet: 3C samples were incubated with sodium dodecyl sulfate (SDS) at a final concentration of 0.2% for 1 h, before Triton X-100 was added to 2% for 1 h. 1000 U of concentrated Bgl II restriction enzyme (NEB Cat# R0144M) was added for overnight digestion at 37°C and 1200 rpm. .. Ligation of 3C library was performed in a final volume of 7 ml diluted in 1× T4 DNA ligase reaction buffer (NEB), with 1% Triton X-100.

    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: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: To form blunt ends the purified inserts were incubated with 36 μM of dNTP mix and 1 U/ μg of DNA of DNA Polymerase I, large (Klenow) fragment in NEBuffer 2 (New England Biolabs). .. Blunt end ligations of different POLGα inserts to pBABEpuro were done in reactions that contained 300-375 ng of insert, 30-75 ng of vector, 2000-3000 U of T4 DNA Ligase and T4 DNA Ligase Reaction Buffer (New England Biolabs) with polyethylene glycol (PEG) 8000 (Sigma).

    Article Title: Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo
    Article Snippet: The mixture was denatured at 95 °C for 5 min before cooling to ambient temperature over 2 h, before subsequent dialysis with water through 0.05 μm nitrocellulose filters (Millipore) for 30 min. Each oligomer for testing (0.02 pmol), as well as a positive (CTAGCGCCGTGCCATGCA) and negative (CTAGCGCCGCATGCA) oligomer control, was added to the dialysed heteroduplex mixture in 1× DNA ligase T4 reaction buffer (NEB) to 20 μL each. .. Ligation was performed by adding 1 mm ATP and 5 U T4 DNA Ligase (NEB) to the samples before overnight incubation at 16 °C.

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science
    Article Snippet: To each reaction, 1.5 μl of 10× T4 DNA ligase reaction buffer, 400 U of T4 DNA ligase (New England BioLabs), 5 U of Esp 3I (Thermo Fisher Scientific, Waltham, MA), and water were added to a final volume of 15 μl. .. Reactions were incubated in a thermocycler (37°C, 5 min; 16°C, 10 min) for five or six cycles (37°C 15 min, 80°C 5 min, and 4°C hold).

    Article Title: Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex
    Article Snippet: .. Briefly, 125 μg λ-DNA was mixed with 2 μM IF003 and IF004 in T4 DNA ligase reaction buffer (NEB) and heated to 70°C for 15 min followed by gradual cooling to 15°C for 2 h. After the oligomer hybridization, T4 DNA ligase (2000 units; NEB) was added to the mixture and incubated overnight at room temperature to seal nicks on DNA. .. The ligase was inactivated with 2 M NaCl, and the reaction was passed over an S-1000 gel filtration column (GE) to remove excess oligonucleotides and proteins.

    Article Title: Base pairing involving artificial bases in vitro and in vivo †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03474d
    Article Snippet: The oligomers (0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA) and negative d(CTAGCGCCG···CATGCA) oligomer controls, were added separately to the dialyzed heteroduplex mixture diluted into 1× DNA ligase T4 reaction buffer (NEB) for 20 μL per sample. .. Ligation was performed by adding 5 U T4 DNA ligase (NEB) supplemented with 1 mM ATP to the samples before overnight incubation (16 °C).

    Stripping Membranes:

    Article Title: Targeted Capture and High-Throughput Sequencing Using Molecular Inversion Probes (MIPs)
    Article Snippet: T4 DNA Ligase Reaction Buffer with 10 mM ATP (New England Biolabs). .. ABgene 8-Flat-Cap Strip Tubes.

    Activity Assay:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Paragraph title: Supporting information Protein gels to evaluate ligase purity. Incubation of ligases with ssDNA to test for contaminating nuclease activity. Reaction endpoint and dsDNA nuclease contamination assessment for wild type ligases. Reaction endpoint and dsDNA nuclease contamination assessment for fusion ligases. Effect of monovalent cations on active blunt/cohesive-end ligases. Separate addition of DNA binding domains to PBCV1 DNA ligase. Fluorescence anisotropy DNA binding curves. Capillary gel electrophoresis panel raw data. ... Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). (TIF) Click here for additional data file.

    Expressing:

    Article Title: Three Phosphatidylglycerol-phosphate Phosphatases in the Inner Membrane of Escherichia coli *
    Article Snippet: Paragraph title: Construction of Plasmids Expressing the pgp Genes ... The vector and the individual digested PCR products were mixed in a 1:3 molar ratio and ligated using T4 DNA ligase in T4 DNA ligase reaction buffer (New England Biolabs, Ipswich, MA) at room temperature for 1 h. The products were transferred into competent cells of E. coli ). ) ( ).

    Transformation Assay:

    Article Title: Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo
    Article Snippet: The mixture was denatured at 95 °C for 5 min before cooling to ambient temperature over 2 h, before subsequent dialysis with water through 0.05 μm nitrocellulose filters (Millipore) for 30 min. Each oligomer for testing (0.02 pmol), as well as a positive (CTAGCGCCGTGCCATGCA) and negative (CTAGCGCCGCATGCA) oligomer control, was added to the dialysed heteroduplex mixture in 1× DNA ligase T4 reaction buffer (NEB) to 20 μL each. .. Ligated mixtures were dialysed as before, and transformed by electroporation into a competent strain of E. coli K12 (ΔthyA :aadA ).

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science
    Article Snippet: To each reaction, 1.5 μl of 10× T4 DNA ligase reaction buffer, 400 U of T4 DNA ligase (New England BioLabs), 5 U of Esp 3I (Thermo Fisher Scientific, Waltham, MA), and water were added to a final volume of 15 μl. .. Chemically competent DH5α cells (minimal efficiency, 107 ) were transformed with 2–5 μl of the reaction and screened via blue/white selection on LB-agar + carbenicillin + X-Gal + IPTG.

    Article Title: Base pairing involving artificial bases in vitro and in vivo †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03474d
    Article Snippet: The oligomers (0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA) and negative d(CTAGCGCCG···CATGCA) oligomer controls, were added separately to the dialyzed heteroduplex mixture diluted into 1× DNA ligase T4 reaction buffer (NEB) for 20 μL per sample. .. Ligated mixtures were dialyzed as before, and transformed by electroporation into electro-competent E. coli K12 (ΔthyA :aadA ).

    Hybridization:

    Article Title: Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo
    Article Snippet: Oligomers were tested inside a gapped heteroduplex vector generated through the enzymatic digestion and PCR assisted denaturation and hybridisation of the ampicillin resistance gene containing pAK1 and pAK2 plasmids. .. The mixture was denatured at 95 °C for 5 min before cooling to ambient temperature over 2 h, before subsequent dialysis with water through 0.05 μm nitrocellulose filters (Millipore) for 30 min. Each oligomer for testing (0.02 pmol), as well as a positive (CTAGCGCCGTGCCATGCA) and negative (CTAGCGCCGCATGCA) oligomer control, was added to the dialysed heteroduplex mixture in 1× DNA ligase T4 reaction buffer (NEB) to 20 μL each.

    Article Title: Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex
    Article Snippet: .. Briefly, 125 μg λ-DNA was mixed with 2 μM IF003 and IF004 in T4 DNA ligase reaction buffer (NEB) and heated to 70°C for 15 min followed by gradual cooling to 15°C for 2 h. After the oligomer hybridization, T4 DNA ligase (2000 units; NEB) was added to the mixture and incubated overnight at room temperature to seal nicks on DNA. .. The ligase was inactivated with 2 M NaCl, and the reaction was passed over an S-1000 gel filtration column (GE) to remove excess oligonucleotides and proteins.

    Article Title: Base pairing involving artificial bases in vitro and in vivo †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03474d
    Article Snippet: Testing was performed with a gapped heteroduplex vector produced through the enzymatic digestion and PCR assisted denaturation and hybridization of the pAK1 and pAK2 plasmids. .. The oligomers (0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA) and negative d(CTAGCGCCG···CATGCA) oligomer controls, were added separately to the dialyzed heteroduplex mixture diluted into 1× DNA ligase T4 reaction buffer (NEB) for 20 μL per sample.

    Electroporation:

    Article Title: Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo
    Article Snippet: The mixture was denatured at 95 °C for 5 min before cooling to ambient temperature over 2 h, before subsequent dialysis with water through 0.05 μm nitrocellulose filters (Millipore) for 30 min. Each oligomer for testing (0.02 pmol), as well as a positive (CTAGCGCCGTGCCATGCA) and negative (CTAGCGCCGCATGCA) oligomer control, was added to the dialysed heteroduplex mixture in 1× DNA ligase T4 reaction buffer (NEB) to 20 μL each. .. Ligated mixtures were dialysed as before, and transformed by electroporation into a competent strain of E. coli K12 (ΔthyA :aadA ).

    Article Title: Base pairing involving artificial bases in vitro and in vivo †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03474d
    Article Snippet: The oligomers (0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA) and negative d(CTAGCGCCG···CATGCA) oligomer controls, were added separately to the dialyzed heteroduplex mixture diluted into 1× DNA ligase T4 reaction buffer (NEB) for 20 μL per sample. .. Ligated mixtures were dialyzed as before, and transformed by electroporation into electro-competent E. coli K12 (ΔthyA :aadA ).

    Ligation:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: .. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). (TIF) Click here for additional data file. .. Fluorescence anisotropy DNA binding curves.

    Article Title: Functional characteristics of novel pancreatic Pax6 regulatory elements
    Article Snippet: .. Ligation of 3C library was performed in a final volume of 7 ml diluted in 1× T4 DNA ligase reaction buffer (NEB), with 1% Triton X-100. .. Ligation was performed at 16°C for 4 h at 300 rpm with 3.3 μl of high-concentration DNA ligase (NEB Cat# M0202 M).

    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: AFSM sequencing approach: a simple and rapid method for genome-wide SNP and methylation site discovery and genetic mapping
    Article Snippet: .. Ligation The ligation reaction was completed using NEB 1x T4 DNA Ligase Reaction Buffer with 50 mM Tris-HCl, 10 mM MgCl2 , 10 mM DTT, and 1 mM ATP, and 100 U of T4 DNA Ligase (New England BioLabs Inc., Ipswich, MA, M0202) for each sample. ..

    Article Title: Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo
    Article Snippet: The mixture was denatured at 95 °C for 5 min before cooling to ambient temperature over 2 h, before subsequent dialysis with water through 0.05 μm nitrocellulose filters (Millipore) for 30 min. Each oligomer for testing (0.02 pmol), as well as a positive (CTAGCGCCGTGCCATGCA) and negative (CTAGCGCCGCATGCA) oligomer control, was added to the dialysed heteroduplex mixture in 1× DNA ligase T4 reaction buffer (NEB) to 20 μL each. .. Ligation was performed by adding 1 mm ATP and 5 U T4 DNA Ligase (NEB) to the samples before overnight incubation at 16 °C.

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Reactions included 1 uM of the ligase and 100 nM of the substrate and T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBuffer 2 (10 mM Tris pH 7.9 @ 25°C, 50 mM NaCl, 10 mM MgCl2 , 1 mM DTT). .. A 1-hour reaction time was used to provide an extended timepoint beyond the reaction time used for ligation experiments.

    Article Title: Base pairing involving artificial bases in vitro and in vivo †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03474d
    Article Snippet: The oligomers (0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA) and negative d(CTAGCGCCG···CATGCA) oligomer controls, were added separately to the dialyzed heteroduplex mixture diluted into 1× DNA ligase T4 reaction buffer (NEB) for 20 μL per sample. .. Ligation was performed by adding 5 U T4 DNA ligase (NEB) supplemented with 1 mM ATP to the samples before overnight incubation (16 °C).

    Serial Dilution:

    Article Title: Base pairing involving artificial bases in vitro and in vivo †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03474d
    Article Snippet: The oligomers (0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA) and negative d(CTAGCGCCG···CATGCA) oligomer controls, were added separately to the dialyzed heteroduplex mixture diluted into 1× DNA ligase T4 reaction buffer (NEB) for 20 μL per sample. .. Incubation of the electroporated bacteria was made at 37 °C for 1 hour, before plating 100 μL of a serial dilution of the suspension onto Muller-Hinton (MH) media containing 100 μg mL–1 ampicillin (spreading the 100 and 10–1 dilutions) and onto the same media supplemented additionally with 0.3 mM thymidine (10–3 and 10–4 dilutions).

    Generated:

    Article Title: Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo
    Article Snippet: Oligomers were tested inside a gapped heteroduplex vector generated through the enzymatic digestion and PCR assisted denaturation and hybridisation of the ampicillin resistance gene containing pAK1 and pAK2 plasmids. .. The mixture was denatured at 95 °C for 5 min before cooling to ambient temperature over 2 h, before subsequent dialysis with water through 0.05 μm nitrocellulose filters (Millipore) for 30 min. Each oligomer for testing (0.02 pmol), as well as a positive (CTAGCGCCGTGCCATGCA) and negative (CTAGCGCCGCATGCA) oligomer control, was added to the dialysed heteroduplex mixture in 1× DNA ligase T4 reaction buffer (NEB) to 20 μL each.

    Imaging:

    Article Title: Intrinsically disordered regions regulate both catalytic and non-catalytic activities of the MutLα mismatch repair complex
    Article Snippet: Preparation of single-molecule DNA substrates DNA substrates for single-molecule imaging were prepared by modifying the cohesive ends of λ-DNA (New England Biolabs; NEB). .. Briefly, 125 μg λ-DNA was mixed with 2 μM IF003 and IF004 in T4 DNA ligase reaction buffer (NEB) and heated to 70°C for 15 min followed by gradual cooling to 15°C for 2 h. After the oligomer hybridization, T4 DNA ligase (2000 units; NEB) was added to the mixture and incubated overnight at room temperature to seal nicks on DNA.

    Sequencing:

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: Blunt end ligations of different POLGα inserts to pBABEpuro were done in reactions that contained 300-375 ng of insert, 30-75 ng of vector, 2000-3000 U of T4 DNA Ligase and T4 DNA Ligase Reaction Buffer (New England Biolabs) with polyethylene glycol (PEG) 8000 (Sigma). .. Correct orientation of the insert was verified by sequencing and PCR.

    Binding Assay:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Paragraph title: Supporting information Protein gels to evaluate ligase purity. Incubation of ligases with ssDNA to test for contaminating nuclease activity. Reaction endpoint and dsDNA nuclease contamination assessment for wild type ligases. Reaction endpoint and dsDNA nuclease contamination assessment for fusion ligases. Effect of monovalent cations on active blunt/cohesive-end ligases. Separate addition of DNA binding domains to PBCV1 DNA ligase. Fluorescence anisotropy DNA binding curves. Capillary gel electrophoresis panel raw data. ... Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). (TIF) Click here for additional data file.

    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.

    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.

    Nucleic Acid Electrophoresis:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Paragraph title: Supporting information Protein gels to evaluate ligase purity. Incubation of ligases with ssDNA to test for contaminating nuclease activity. Reaction endpoint and dsDNA nuclease contamination assessment for wild type ligases. Reaction endpoint and dsDNA nuclease contamination assessment for fusion ligases. Effect of monovalent cations on active blunt/cohesive-end ligases. Separate addition of DNA binding domains to PBCV1 DNA ligase. Fluorescence anisotropy DNA binding curves. Capillary gel electrophoresis panel raw data. ... Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). (TIF) Click here for additional data file.

    Article Title: Functional characteristics of novel pancreatic Pax6 regulatory elements
    Article Snippet: Digestion efficiency was analysed on 5 μl pre- and post-digested template by gel electrophoresis. .. Ligation of 3C library was performed in a final volume of 7 ml diluted in 1× T4 DNA ligase reaction buffer (NEB), with 1% Triton X-100.

    Article Title: Three Phosphatidylglycerol-phosphate Phosphatases in the Inner Membrane of Escherichia coli *
    Article Snippet: The PCR products were purified and subsequently digested with the restriction enzymes NdeI and HindIII in NEB buffer 2 (New England Biolabs, Ipswich, MA) at 37 °C for 2 h. The digested products were purified by gel electrophoresis. .. The vector and the individual digested PCR products were mixed in a 1:3 molar ratio and ligated using T4 DNA ligase in T4 DNA ligase reaction buffer (New England Biolabs, Ipswich, MA) at room temperature for 1 h. The products were transferred into competent cells of E. coli ). ) ( ).

    In Vivo:

    Article Title: Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo
    Article Snippet: Paragraph title: In vivo assay of XNA-oligonucleotides ... The mixture was denatured at 95 °C for 5 min before cooling to ambient temperature over 2 h, before subsequent dialysis with water through 0.05 μm nitrocellulose filters (Millipore) for 30 min. Each oligomer for testing (0.02 pmol), as well as a positive (CTAGCGCCGTGCCATGCA) and negative (CTAGCGCCGCATGCA) oligomer control, was added to the dialysed heteroduplex mixture in 1× DNA ligase T4 reaction buffer (NEB) to 20 μL each.

    Article Title: Base pairing involving artificial bases in vitro and in vivo †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03474d
    Article Snippet: Paragraph title: In vivo assay of XNA-oligonucleotides ... The oligomers (0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA) and negative d(CTAGCGCCG···CATGCA) oligomer controls, were added separately to the dialyzed heteroduplex mixture diluted into 1× DNA ligase T4 reaction buffer (NEB) for 20 μL per sample.

    Fluorescence:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Paragraph title: Supporting information Protein gels to evaluate ligase purity. Incubation of ligases with ssDNA to test for contaminating nuclease activity. Reaction endpoint and dsDNA nuclease contamination assessment for wild type ligases. Reaction endpoint and dsDNA nuclease contamination assessment for fusion ligases. Effect of monovalent cations on active blunt/cohesive-end ligases. Separate addition of DNA binding domains to PBCV1 DNA ligase. Fluorescence anisotropy DNA binding curves. Capillary gel electrophoresis panel raw data. ... Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). (TIF) Click here for additional data file.

    Mutagenesis:

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: Paragraph title: Generation of POLG-retroviral vectors with and without mutation(s) ... Blunt end ligations of different POLGα inserts to pBABEpuro were done in reactions that contained 300-375 ng of insert, 30-75 ng of vector, 2000-3000 U of T4 DNA Ligase and T4 DNA Ligase Reaction Buffer (New England Biolabs) with polyethylene glycol (PEG) 8000 (Sigma).

    Purification:

    Article Title: Functional characteristics of novel pancreatic Pax6 regulatory elements
    Article Snippet: Ligation of 3C library was performed in a final volume of 7 ml diluted in 1× T4 DNA ligase reaction buffer (NEB), with 1% Triton X-100. .. Samples were purified by two sequential phenol–chloroform extractions (Sigma-Aldrich) and ethanol precipitation and resuspended in 150 μl of 10 mm Tris pH 7.5.

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: 30 μg of pBABEpuro vector was digested with 0,5 U/ 1μg of plasmid DNA of SnaBI restriction enzyme (New England Biolabs) with 1 μg/ml of bovine serum albumin (BSA) (New England Biolabs) in NEBuffer 4. pBABEpuro was dephosphorylated with 0,5 U/1 μg of plasmid DNA of calf intestinal alkaline phosphatase (CIP) (Finnzymes) for one hour at 37°C and subsequently gel purified. .. Blunt end ligations of different POLGα inserts to pBABEpuro were done in reactions that contained 300-375 ng of insert, 30-75 ng of vector, 2000-3000 U of T4 DNA Ligase and T4 DNA Ligase Reaction Buffer (New England Biolabs) with polyethylene glycol (PEG) 8000 (Sigma).

    Article Title: Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo
    Article Snippet: [ , ] Here, a mix of equimolar (25 ng each) purified NheI and NsiI cut pAK1 and purified two-fold EcoRI cut and dephosphorylated pAK2 were diluted in 10 mm Tris-HCl (pH 7.5) with 100 mm NaCL. .. The mixture was denatured at 95 °C for 5 min before cooling to ambient temperature over 2 h, before subsequent dialysis with water through 0.05 μm nitrocellulose filters (Millipore) for 30 min. Each oligomer for testing (0.02 pmol), as well as a positive (CTAGCGCCGTGCCATGCA) and negative (CTAGCGCCGCATGCA) oligomer control, was added to the dialysed heteroduplex mixture in 1× DNA ligase T4 reaction buffer (NEB) to 20 μL each.

    Article Title: Three Phosphatidylglycerol-phosphate Phosphatases in the Inner Membrane of Escherichia coli *
    Article Snippet: Purified plasmid pBAD33.1 ( ) was similarly digested, gel-purified, and then treated with calf intestinal alkaline phosphatase in NEB buffer 3 (New England Biolabs, Ipswich, MA) at 37 °C. .. The vector and the individual digested PCR products were mixed in a 1:3 molar ratio and ligated using T4 DNA ligase in T4 DNA ligase reaction buffer (New England Biolabs, Ipswich, MA) at room temperature for 1 h. The products were transferred into competent cells of E. coli ). ) ( ).

    Article Title: Base pairing involving artificial bases in vitro and in vivo †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03474d
    Article Snippet: A mix of equimolar (25 ng each) purified pAK1 (NheI and NsiI cut) and purified pAK2 (two-fold EcoRI cut and dephosphorylated) was diluted into 10 mM Tris–HCl (pH 7.5) with 100 mM NaCl. .. The oligomers (0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA) and negative d(CTAGCGCCG···CATGCA) oligomer controls, were added separately to the dialyzed heteroduplex mixture diluted into 1× DNA ligase T4 reaction buffer (NEB) for 20 μL per sample.

    Polymerase Chain Reaction:

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: Mutated PCR products were purified from parental template with DpnI restriction endonuclease (New England Biolabs). .. Blunt end ligations of different POLGα inserts to pBABEpuro were done in reactions that contained 300-375 ng of insert, 30-75 ng of vector, 2000-3000 U of T4 DNA Ligase and T4 DNA Ligase Reaction Buffer (New England Biolabs) with polyethylene glycol (PEG) 8000 (Sigma).

    Article Title: Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo
    Article Snippet: Oligomers were tested inside a gapped heteroduplex vector generated through the enzymatic digestion and PCR assisted denaturation and hybridisation of the ampicillin resistance gene containing pAK1 and pAK2 plasmids. .. The mixture was denatured at 95 °C for 5 min before cooling to ambient temperature over 2 h, before subsequent dialysis with water through 0.05 μm nitrocellulose filters (Millipore) for 30 min. Each oligomer for testing (0.02 pmol), as well as a positive (CTAGCGCCGTGCCATGCA) and negative (CTAGCGCCGCATGCA) oligomer control, was added to the dialysed heteroduplex mixture in 1× DNA ligase T4 reaction buffer (NEB) to 20 μL each.

    Article Title: Three Phosphatidylglycerol-phosphate Phosphatases in the Inner Membrane of Escherichia coli *
    Article Snippet: .. The vector and the individual digested PCR products were mixed in a 1:3 molar ratio and ligated using T4 DNA ligase in T4 DNA ligase reaction buffer (New England Biolabs, Ipswich, MA) at room temperature for 1 h. The products were transferred into competent cells of E. coli ). ) ( ). ..

    Article Title: Base pairing involving artificial bases in vitro and in vivo †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03474d
    Article Snippet: Testing was performed with a gapped heteroduplex vector produced through the enzymatic digestion and PCR assisted denaturation and hybridization of the pAK1 and pAK2 plasmids. .. The oligomers (0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA) and negative d(CTAGCGCCG···CATGCA) oligomer controls, were added separately to the dialyzed heteroduplex mixture diluted into 1× DNA ligase T4 reaction buffer (NEB) for 20 μL per sample.

    Lysis:

    Article Title: Functional characteristics of novel pancreatic Pax6 regulatory elements
    Article Snippet: Glycine was added, and cells were centrifuged and lysed in cell lysis buffer [10 mm Tris–HCl, pH 7.5; 10 mm NaCl; 0.2% (v/v) Ipegal, PI]. .. Ligation of 3C library was performed in a final volume of 7 ml diluted in 1× T4 DNA ligase reaction buffer (NEB), with 1% Triton X-100.

    Plasmid Preparation:

    Article Title: High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer
    Article Snippet: Materials pETDUET-1™ DNA (Cat. 71146-3) vector was obtained from Novagen (Merck Biosciences Division, Darmstadt, Germany). .. High-Fidelity (HF® ) Restriction Endonucleases (BamHI, EcoRI, MfeI and XhoI), CutSmart® Buffer, T4 DNA Ligase and T4 DNA Ligase Reaction Buffer for the cloning procedures were purchased from New England Biolabs (Ipswich, MA, USA).

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: .. Blunt end ligations of different POLGα inserts to pBABEpuro were done in reactions that contained 300-375 ng of insert, 30-75 ng of vector, 2000-3000 U of T4 DNA Ligase and T4 DNA Ligase Reaction Buffer (New England Biolabs) with polyethylene glycol (PEG) 8000 (Sigma). .. Correct orientation of the insert was verified by sequencing and PCR.

    Article Title: Isoguanine and 5-Methyl-Isocytosine Bases, In Vitro and In Vivo
    Article Snippet: Oligomers were tested inside a gapped heteroduplex vector generated through the enzymatic digestion and PCR assisted denaturation and hybridisation of the ampicillin resistance gene containing pAK1 and pAK2 plasmids. .. The mixture was denatured at 95 °C for 5 min before cooling to ambient temperature over 2 h, before subsequent dialysis with water through 0.05 μm nitrocellulose filters (Millipore) for 30 min. Each oligomer for testing (0.02 pmol), as well as a positive (CTAGCGCCGTGCCATGCA) and negative (CTAGCGCCGCATGCA) oligomer control, was added to the dialysed heteroduplex mixture in 1× DNA ligase T4 reaction buffer (NEB) to 20 μL each.

    Article Title: Three Phosphatidylglycerol-phosphate Phosphatases in the Inner Membrane of Escherichia coli *
    Article Snippet: .. The vector and the individual digested PCR products were mixed in a 1:3 molar ratio and ligated using T4 DNA ligase in T4 DNA ligase reaction buffer (New England Biolabs, Ipswich, MA) at room temperature for 1 h. The products were transferred into competent cells of E. coli ). ) ( ). ..

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science
    Article Snippet: To each reaction, 1.5 μl of 10× T4 DNA ligase reaction buffer, 400 U of T4 DNA ligase (New England BioLabs), 5 U of Esp 3I (Thermo Fisher Scientific, Waltham, MA), and water were added to a final volume of 15 μl. .. To each reaction, 0.5 μl of ATP (25 mM ) and 5 U of plasmid-safe DNase (Epicentre) were added and incubated at 37°C for 1 hr, followed by inactivation at 70°C for 30 min.

    Article Title: Base pairing involving artificial bases in vitro and in vivo †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03474d
    Article Snippet: Testing was performed with a gapped heteroduplex vector produced through the enzymatic digestion and PCR assisted denaturation and hybridization of the pAK1 and pAK2 plasmids. .. The oligomers (0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA) and negative d(CTAGCGCCG···CATGCA) oligomer controls, were added separately to the dialyzed heteroduplex mixture diluted into 1× DNA ligase T4 reaction buffer (NEB) for 20 μL per sample.

    Selection:

    Article Title: FusX: A Rapid One-Step Transcription Activator-Like Effector Assembly System for Genome Science
    Article Snippet: To each reaction, 1.5 μl of 10× T4 DNA ligase reaction buffer, 400 U of T4 DNA ligase (New England BioLabs), 5 U of Esp 3I (Thermo Fisher Scientific, Waltham, MA), and water were added to a final volume of 15 μl. .. Chemically competent DH5α cells (minimal efficiency, 107 ) were transformed with 2–5 μl of the reaction and screened via blue/white selection on LB-agar + carbenicillin + X-Gal + IPTG.

    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.

    Ethanol Precipitation:

    Article Title: Functional characteristics of novel pancreatic Pax6 regulatory elements
    Article Snippet: Ligation of 3C library was performed in a final volume of 7 ml diluted in 1× T4 DNA ligase reaction buffer (NEB), with 1% Triton X-100. .. Samples were purified by two sequential phenol–chloroform extractions (Sigma-Aldrich) and ethanol precipitation and resuspended in 150 μl of 10 mm Tris pH 7.5.

    Produced:

    Article Title: Base pairing involving artificial bases in vitro and in vivo †Electronic supplementary information (ESI) available. See DOI: 10.1039/c5sc03474d
    Article Snippet: Testing was performed with a gapped heteroduplex vector produced through the enzymatic digestion and PCR assisted denaturation and hybridization of the pAK1 and pAK2 plasmids. .. The oligomers (0.02 pmol), as well as a positive d(CTAGCGCCGTGCCATGCA) and negative d(CTAGCGCCG···CATGCA) oligomer controls, were added separately to the dialyzed heteroduplex mixture diluted into 1× DNA ligase T4 reaction buffer (NEB) for 20 μL per sample.

    Concentration Assay:

    Article Title: Functional characteristics of novel pancreatic Pax6 regulatory elements
    Article Snippet: 3C samples were incubated with sodium dodecyl sulfate (SDS) at a final concentration of 0.2% for 1 h, before Triton X-100 was added to 2% for 1 h. 1000 U of concentrated Bgl II restriction enzyme (NEB Cat# R0144M) was added for overnight digestion at 37°C and 1200 rpm. .. Ligation of 3C library was performed in a final volume of 7 ml diluted in 1× T4 DNA ligase reaction buffer (NEB), with 1% Triton X-100.

    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.

    DNA Purification:

    Article Title: High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer
    Article Snippet: High-Fidelity (HF® ) Restriction Endonucleases (BamHI, EcoRI, MfeI and XhoI), CutSmart® Buffer, T4 DNA Ligase and T4 DNA Ligase Reaction Buffer for the cloning procedures were purchased from New England Biolabs (Ipswich, MA, USA). .. Plasmid DNA Purification Kit and QIAquick® Gel Extraction Kit were purchased from Qiagen (Hilden, Germany).

    Gel Extraction:

    Article Title: High yield bacterial expression, purification and characterisation of bioactive Human Tousled-like Kinase 1B involved in cancer
    Article Snippet: High-Fidelity (HF® ) Restriction Endonucleases (BamHI, EcoRI, MfeI and XhoI), CutSmart® Buffer, T4 DNA Ligase and T4 DNA Ligase Reaction Buffer for the cloning procedures were purchased from New England Biolabs (Ipswich, MA, USA). .. Plasmid DNA Purification Kit and QIAquick® Gel Extraction Kit were purchased from Qiagen (Hilden, Germany).

    Article Title: Functional analysis of H. sapiens DNA polymerase ? spacer mutation W748S with and without common variant E1143G
    Article Snippet: The digested DNA pieces were gel purified with QIAquick Gel Extraction Kit (Qiagen). .. Blunt end ligations of different POLGα inserts to pBABEpuro were done in reactions that contained 300-375 ng of insert, 30-75 ng of vector, 2000-3000 U of T4 DNA Ligase and T4 DNA Ligase Reaction Buffer (New England Biolabs) with polyethylene glycol (PEG) 8000 (Sigma).

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    New England Biolabs t4 dna ligase reaction buffer
    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of <t>T4</t> DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.
    T4 Dna Ligase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Schematic representation of DNA ligase fusions. All DNA ligases contain a catalytic core NTase domain (blue) and an OBD (red), which are fairly well conserved. Many ligases also have additional domains, such as the N-terminal ZnF (yellow) and DBD (green) in Human Lig3 and the N-terminal domain (NTD) of T4 DNA ligase (purple). Wild type PBCV1 ligase, which contains only the core NTase and OBD domains, was chosen for fusion to other binding domains: Sso7d (white) at both the N- and C-termini, the hLig3 ZnF domain, and the T4 DNA ligase NTD.

    Article Snippet: Reactions included 1 uM of the ligase and 100 nM of the substrate and T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBuffer 2 (10 mM Tris pH 7.9 @ 25°C, 50 mM NaCl, 10 mM MgCl2 , 1 mM DTT).

    Techniques: Binding Assay

    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Article Snippet: Reactions included 1 uM of the ligase and 100 nM of the substrate and T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBuffer 2 (10 mM Tris pH 7.9 @ 25°C, 50 mM NaCl, 10 mM MgCl2 , 1 mM DTT).

    Techniques: Ligation, Agarose Gel Electrophoresis, Staining

    Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Wild type DNA ligase blunt/cohesive capillary electrophoresis assay. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and hLig3 (D), respectively Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Article Snippet: Reactions included 1 uM of the ligase and 100 nM of the substrate and T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBuffer 2 (10 mM Tris pH 7.9 @ 25°C, 50 mM NaCl, 10 mM MgCl2 , 1 mM DTT).

    Techniques: Electrophoresis, Produced, Ligation, Standard Deviation

    Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Journal: PLoS ONE

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Effect of DBD on blunt/cohesive end ligation. Bar graphs depict the fraction of either ligated DNA (product, blue) or abortive adenylylation (App, red) produced in a 20-minute sealing reaction with the indicated DNA substrate. Reactions included 1 μM of the DNA ligase, 100 nM of the substrate and reaction conditions consisting of either T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)). Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). Experiments were performed in triplicate; the plotted value is the average and the error bars represent the standard deviation across replicates.

    Article Snippet: Reactions included 1 uM of the ligase and 100 nM of the substrate and T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) or NEBuffer 2 (10 mM Tris pH 7.9 @ 25°C, 50 mM NaCl, 10 mM MgCl2 , 1 mM DTT).

    Techniques: Ligation, Produced, Standard Deviation

    YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Journal: Cell

    Article Title: YY1 Is a Structural Regulator of Enhancer-Promoter Loops

    doi: 10.1016/j.cell.2017.11.008

    Figure Lengend Snippet: YY1 Can Enhance DNA Interactions In Vitro (A and D) Models depicting the in vitro DNA circularization assays used to detect the ability of YY1 to enhance DNA looping interactions with no motif control (A) or competitor DNA control (D). (B and E) Results of the in vitro DNA circularization assay visualized by gel electrophoresis with no motif control (B) or competitor DNA control (E). The dominant lower band reflects the starting linear DNA template, while the upper band corresponds to the circularized DNA ligation product. (C and F) Quantifications of DNA template circularization as a function of incubation time with T4 DNA ligase for no motif control (C) or competitor DNA control (F). Values correspond to the percent of DNA template that is circularized and represents the mean and SD of four experiments. .

    Article Snippet: Reactions were prepared on ice in 66 μL with the following components: BSA control: 0.25 nM DNA, 1× T4 DNA ligase buffer (NEB B0202S), H2 O 0.12 μg/μL of BSA.

    Techniques: In Vitro, Nucleic Acid Electrophoresis, DNA Ligation, Incubation

    Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    Journal: Methods in enzymology

    Article Title: Next-Generation DNA Curtains for Single-Molecule Studies of Homologous Recombination

    doi: 10.1016/bs.mie.2017.03.011

    Figure Lengend Snippet: Nucleoprotein filament dynamics on low sequence complexity ssDNA curtains. (A) Sequences of the two ssDNA oligonucleotides used for rolling circle replication. (B) Schematic of rolling circle replication (RCR) reaction. T4 DNA ligase ligates the template oligo to form a contiguous template strand. Next, phi29 DNA polymerase catalyzes the synthesis of long ssDNA molecules. (C) Agarose gel of several time points along the RCR synthesis reaction. The primer oligonucleotide was 32 P labeled on the 5 ′ -terminus phosphate ( gold star ). (D) Wide-field image of a microfabricated barrier set with double-tethered ssDNA curtains coated with RPA-TagRFP ( magenta ). Arrows and circles denote chromium barriers and pedestals, respectively. (E) Illustration and kymograph showing a single ssDNA molecule coated with ATTO488-RAD51(C319S) ( green ) replaced by RPA-TagRFP ( magenta ). Yellow dashed line denotes the injection of RPA–TagRFP into the flowcell. Buffer controls indicate when the buffer flow was toggled off and on to show that the florescent proteins retract to the Cr barriers simultaneously with the ssDNA molecule. This indicates that RAD51 and RPA are on the ssDNA molecule. Panel A: Adapted from Lee, K. S., Marciel, A. B., Kozlov, A. G., Schroeder, C. M., Lohman, T. M., Ha, T. (2014). Ultrafast redistribution of E. coli SSB along long single-stranded DNA via intersegment transfer. Journal of Molecular Biology, 426 , 2413 – 2421.

    Article Snippet: TE buffer: 10m M Tris–HCl [pH 8.0]; 0.1m M EDTA RAD51 buffer: 40m M Tris–HCl [pH 8.0]; 1m M MgCl2 ; 5m M CaCl2 ; 100m M KCl; 1m M DTT; 1m M ATP; 0.2 mgmL−1 BSA; 1m M Trolox (Sigma-Aldrich); 1.0% glucose (w/v); 500units catalase (Sigma-Aldrich); 70units glucose oxidase (Sigma-Aldrich) 10× T4 DNA ligase reaction buffer (B0202S; NEB) T4 DNA ligase (M0202; NEB) Primer oligo (/Biosg/TC TCC TCC TTC T—HPLC purified; Integrated DNA Technologies) Template oligo (/5Phos/AG GAG AAA AAG AAA AAA AGA AAA GAA GG—PAGE purified; Integrated DNA Technologies) Nuclease-free water BSA, Molecular Biology Grade (B9000S; NEB) Thermocycler (Mastercycler pro S; Eppendorf ) 10× phi29 DNA polymerase reaction buffer (B0269S; NEB) phi29 DNA polymerase (homemade 5 μ M stock) Deoxynucleotide (dNTP) solution set (N0446S; NEB)

    Techniques: Sequencing, Agarose Gel Electrophoresis, Labeling, Recombinase Polymerase Amplification, Injection, Flow Cytometry