λ exonuclease  (New England Biolabs)


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    Name:
    Lambda Exonuclease
    Description:
    Lambda Exonuclease 5 000 units
    Catalog Number:
    m0262l
    Price:
    281
    Size:
    5 000 units
    Category:
    Exonucleases
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    Structured Review

    New England Biolabs λ exonuclease
    Lambda Exonuclease
    Lambda Exonuclease 5 000 units
    https://www.bioz.com/result/λ exonuclease/product/New England Biolabs
    Average 99 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    λ exonuclease - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Nucleotidyl transferase assisted DNA labeling with different click chemistries"

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv544

    Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with λ-exonuclease (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.
    Figure Legend Snippet: Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with λ-exonuclease (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.

    Techniques Used: Modification, Polyacrylamide Gel Electrophoresis, Staining

    2) Product Images from "Human Heart Mitochondrial DNA Is Organized in Complex Catenated Networks Containing Abundant Four-way Junctions and Replication Forks *"

    Article Title: Human Heart Mitochondrial DNA Is Organized in Complex Catenated Networks Containing Abundant Four-way Junctions and Replication Forks *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.016600

    Electrophoretic and TEM analysis of human heart mtDNA. a , one-dimensional AGE of undigested cardiac muscle mtDNA treated with the enzymes as indicated (topoisomerase ( Topo ) I, topoisomerase IV, T7 endonuclease ( endo ) I, and λ-exonuclease ( exo )). The identity of the various species is inferred by the effects of enzymatic treatments on their relative amounts and by their migration properties on two-dimensional AGE ( b ). Bands t and f are each composites of several species. λ-Exonuclease digests molecules with exposed 5′ phosphorylated ends. Because it removes or modifies the residual material running just behind open circles after topoisomerase IV treatment, i.e. 2n linear molecules plus species x ( b , panel ii ), we infer that species x has exposed ends. b , two-dimensional AGE of cardiac muscle mtDNA untreated ( panel i ), treated with topoisomerase IV ( panel ii ), treated with T7 endonuclease I ( panel iii ), and treated with topoisomerase IV + T7 endonuclease I ( panel iv ). The topology of the various forms, annotated on the gel images and inferred from the treatments and electrophoretic mobilities, is shown below the gel panels. tgl , tangled complexes (see Fig. 3 ); cat , catenanes (can also include > 2 monomeric or dimeric circles); 1n and 2n , monomeric and dimeric linear molecules, respectively; c and 2nc , monomeric and dimeric open circles, respectively; sc and 2nsc , supercoiled monomeric and dimeric circles, respectively. xc , circular molecules joined by four-way junctions; x , suggested to be circles joined to linear molecules by four-way junctions. Arrows indicate directions of first and second dimension electrophoresis. c and d , examples of forms seen by TEM following topoisomerase IV treatment of heart mtDNA alongside interpretations. Distinct circles and linear segments are indicated in different colors with inferred contour lengths in kb or (for circles) genome lengths. Scale bars , 200 nm.
    Figure Legend Snippet: Electrophoretic and TEM analysis of human heart mtDNA. a , one-dimensional AGE of undigested cardiac muscle mtDNA treated with the enzymes as indicated (topoisomerase ( Topo ) I, topoisomerase IV, T7 endonuclease ( endo ) I, and λ-exonuclease ( exo )). The identity of the various species is inferred by the effects of enzymatic treatments on their relative amounts and by their migration properties on two-dimensional AGE ( b ). Bands t and f are each composites of several species. λ-Exonuclease digests molecules with exposed 5′ phosphorylated ends. Because it removes or modifies the residual material running just behind open circles after topoisomerase IV treatment, i.e. 2n linear molecules plus species x ( b , panel ii ), we infer that species x has exposed ends. b , two-dimensional AGE of cardiac muscle mtDNA untreated ( panel i ), treated with topoisomerase IV ( panel ii ), treated with T7 endonuclease I ( panel iii ), and treated with topoisomerase IV + T7 endonuclease I ( panel iv ). The topology of the various forms, annotated on the gel images and inferred from the treatments and electrophoretic mobilities, is shown below the gel panels. tgl , tangled complexes (see Fig. 3 ); cat , catenanes (can also include > 2 monomeric or dimeric circles); 1n and 2n , monomeric and dimeric linear molecules, respectively; c and 2nc , monomeric and dimeric open circles, respectively; sc and 2nsc , supercoiled monomeric and dimeric circles, respectively. xc , circular molecules joined by four-way junctions; x , suggested to be circles joined to linear molecules by four-way junctions. Arrows indicate directions of first and second dimension electrophoresis. c and d , examples of forms seen by TEM following topoisomerase IV treatment of heart mtDNA alongside interpretations. Distinct circles and linear segments are indicated in different colors with inferred contour lengths in kb or (for circles) genome lengths. Scale bars , 200 nm.

    Techniques Used: Transmission Electron Microscopy, Migration, Electrophoresis

    3) Product Images from "The hSNM1 protein is a DNA 5?-exonuclease"

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm530

    hSNM1 is a single-strand-specific exonuclease. ( A ) Assay of hSNM1 IMAC fraction 3 on 20-dT substrate. Lanes: 1, RecJ f ; 2, substrate only; 3, ySNM1; Lanes 4, 116 ng protein 5, 29 ng protein; 6, 7.25 ng protein; 7, 1.8 ng protein; 8, 0.45 ng protein. ( B ) Assay of IMAC fraction 2 on internally labeled double-stranded substrate. Lanes: 1, λ exonuclease control; 2, ySNM1; 3, 140 ng protein; 4, 35 ng protein; 5, 4.4 ng protein. ( C ) Assay of FPLC fraction 16 on 20-dT substrate. Lanes: 1, substrate only; 2, RecJ f  control; 3, fraction 16 (∼10 ng); 4, ∼2.5 ng; 5, ∼0.65 ng; 6, ∼0.17 ng. ( D ) Assay of FPLC fraction 16 on internally labeled ds substrate. Lanes: 1, fraction 16 (∼10 ng); 2, ∼2.5 ng; 3, ∼0.65ng.
    Figure Legend Snippet: hSNM1 is a single-strand-specific exonuclease. ( A ) Assay of hSNM1 IMAC fraction 3 on 20-dT substrate. Lanes: 1, RecJ f ; 2, substrate only; 3, ySNM1; Lanes 4, 116 ng protein 5, 29 ng protein; 6, 7.25 ng protein; 7, 1.8 ng protein; 8, 0.45 ng protein. ( B ) Assay of IMAC fraction 2 on internally labeled double-stranded substrate. Lanes: 1, λ exonuclease control; 2, ySNM1; 3, 140 ng protein; 4, 35 ng protein; 5, 4.4 ng protein. ( C ) Assay of FPLC fraction 16 on 20-dT substrate. Lanes: 1, substrate only; 2, RecJ f control; 3, fraction 16 (∼10 ng); 4, ∼2.5 ng; 5, ∼0.65 ng; 6, ∼0.17 ng. ( D ) Assay of FPLC fraction 16 on internally labeled ds substrate. Lanes: 1, fraction 16 (∼10 ng); 2, ∼2.5 ng; 3, ∼0.65ng.

    Techniques Used: Labeling, Fast Protein Liquid Chromatography

    4) Product Images from "Transcription-generated torsional stress destabilizes nucleosomes"

    Article Title: Transcription-generated torsional stress destabilizes nucleosomes

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2723

    High resolution detection of supercoiling states ( a ) Strategy for paired-end sequencing of TMP cross-linked DNA fragments. I. Illumina barcoded adapters are ligated to cross-linked fragments. II. The 5′ strand is digested with λ exonuclease. III. Using a primer complementary to the paired-end adapter, 10 rounds of primer extension were performed. IV. Ribo-Gs were added at the 3′ end using Terminal Transferase, V. A double stranded adapter with a 5′ CCC overhang was ligated. VI. One round of primer extension followed by cycles of library amplification were performed. Libraries were sequenced from the CCC overhang end. ( b ) TMP-seq was performed on control samples with two replicates. Reads were normalized for DNA sequence bias. The average normalized TMP-seq (y-axis Ave. sample/DNA) signal for every 10 bp in a 4 kb region surrounding the transcription start site (TSS) and transcription end site (TES) of all genes is plotted (top), and for expressed and silent genes separately (bottom). n.c. Normalized counts.
    Figure Legend Snippet: High resolution detection of supercoiling states ( a ) Strategy for paired-end sequencing of TMP cross-linked DNA fragments. I. Illumina barcoded adapters are ligated to cross-linked fragments. II. The 5′ strand is digested with λ exonuclease. III. Using a primer complementary to the paired-end adapter, 10 rounds of primer extension were performed. IV. Ribo-Gs were added at the 3′ end using Terminal Transferase, V. A double stranded adapter with a 5′ CCC overhang was ligated. VI. One round of primer extension followed by cycles of library amplification were performed. Libraries were sequenced from the CCC overhang end. ( b ) TMP-seq was performed on control samples with two replicates. Reads were normalized for DNA sequence bias. The average normalized TMP-seq (y-axis Ave. sample/DNA) signal for every 10 bp in a 4 kb region surrounding the transcription start site (TSS) and transcription end site (TES) of all genes is plotted (top), and for expressed and silent genes separately (bottom). n.c. Normalized counts.

    Techniques Used: Sequencing, Countercurrent Chromatography, Amplification

    5) Product Images from "Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes"

    Article Title: Efficient enzymatic synthesis and dual-colour fluorescent labelling of DNA probes using long chain azido-dUTP and BCN dyes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw028

    λ-exonuclease digestion of PCR amplicons (81 bp, primer P6 and P7p) containing different percentages of AHP dU modifications and re-annealing of the modified single strands to complementary template T8. 2% agarose gel analysis containing SYBR Gold stain. PCR lanes: PCR amplicons; single strand lanes: PCR amplicons after exonuclease digestion; re-annealing lanes, ssDNA re-annealed with T8. Lane L: 25 bp ladder. D = duplex products; T + P = excess primer annealed to template T8; T = template T8.
    Figure Legend Snippet: λ-exonuclease digestion of PCR amplicons (81 bp, primer P6 and P7p) containing different percentages of AHP dU modifications and re-annealing of the modified single strands to complementary template T8. 2% agarose gel analysis containing SYBR Gold stain. PCR lanes: PCR amplicons; single strand lanes: PCR amplicons after exonuclease digestion; re-annealing lanes, ssDNA re-annealed with T8. Lane L: 25 bp ladder. D = duplex products; T + P = excess primer annealed to template T8; T = template T8.

    Techniques Used: Polymerase Chain Reaction, Modification, Agarose Gel Electrophoresis, Staining

    6) Product Images from "Method to eliminate linear DNA from mixture containing nicked-circular, supercoiled, and linear plasmid DNA"

    Article Title: Method to eliminate linear DNA from mixture containing nicked-circular, supercoiled, and linear plasmid DNA

    Journal: Analytical biochemistry

    doi: 10.1016/j.ab.2008.06.037

    (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with λ exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).
    Figure Legend Snippet: (A) Nicking of supercoiled (SC) pUC19 by Eco R1 in presence of ethidium bromide assessed by agarose gel electrophoresis after overnight incubation at 37°C. (B) After incubation continued an additional 24 h. Control supercoiled DNA (lane 1); aliquot drawn from nicking reaction (lane 2). Note that at end of second incubation (B, lane 2) supercoiled DNA is completely converted into nicked-circular (N) and linear (L) forms. (C) Agarose gel showing digestion of nicked-circular (N) DNA preparation with λ exonuclease and RecJ f to remove undesired linear DNA. Control supercoiled (SC) pUC19 DNA (lane 1); control linear (N) pUC19 DNA (lane 2); nicked-circular DNA preparation before exonuclease digestion (note presence of contaminating linear DNA) (lane 3); nicked-circular DNA preparation after exonuclease digestion (note disappearance of contaminating linear DNA) (lane 4); DNA size marker (lane M).

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Marker

    Agarose gel electrophoresis demonstrating potential of λ exonuclease and RecJ f to selectively digest linear (L) form of plasmid DNA. Mixture of supercoiled (SC) and linear forms of pUC19 before (lane 1) and after (lane 2) digestion with λ exonuclease and RecJ f . DNA size marker (lane 3). Note that after digestion linear form disappears completely leaving only supercoiled DNA.
    Figure Legend Snippet: Agarose gel electrophoresis demonstrating potential of λ exonuclease and RecJ f to selectively digest linear (L) form of plasmid DNA. Mixture of supercoiled (SC) and linear forms of pUC19 before (lane 1) and after (lane 2) digestion with λ exonuclease and RecJ f . DNA size marker (lane 3). Note that after digestion linear form disappears completely leaving only supercoiled DNA.

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Marker

    7) Product Images from "A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition"

    Article Title: A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s18082441

    Relative fluorescence intensity of the reaction systems upon addition of ADA, hoGG I, UDG, RNase H and λexo. Error bars were estimated from three replicate measurements.
    Figure Legend Snippet: Relative fluorescence intensity of the reaction systems upon addition of ADA, hoGG I, UDG, RNase H and λexo. Error bars were estimated from three replicate measurements.

    Techniques Used: Fluorescence

    8) Product Images from "Functional interactions of DNA topoisomerases with a human replication origin"

    Article Title: Functional interactions of DNA topoisomerases with a human replication origin

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7601578

    Interaction of topo I and II with the lamin B2 origin in vitro . ( A ) Detection of the in vitro topo I cleavages stabilized on the lower strand by CPT (lane 3), 7-[CH2–Tris] CPT (lane 5) or gimatecan (lane 6), and on the upper strand by CPT (lane 10); lanes 7 and 12: Maxam–Gilbert sequencing reactions; the position of the cleavages also present in vivo is indicated by an asterisk. ( B ) Effect of base substitution mutations in the lamin B2 origin on topo I-mediated cleavage. ( C ) Sequence of the origin portion covered by the replicative complexes; the position of substituted bases is highlighted; the position of in vitro topo I-cleavable complexes is indicated by filled triangles; the asterisks indicate the position of the topo I cleavages also present in vivo . ( D ) Detection of the in vitro VP16-induced topo II cleavages introduced by the enzyme alone (lanes 1–5) or by topo II as part of a complex with nuclear proteins (lanes 6–14); lane 9: the origin DNA incubated with the nuclear extract and VP16 was immunopurified using anti-topo II antibody; black vertical bars indicate the region protected in vivo ; the arrows indicate the borders of the region protected in vitro by the origin binding proteins (OBP) as determined by λ-exonuclease digestion; lanes 10 and 14: Maxam–Gilbert sequencing reactions.
    Figure Legend Snippet: Interaction of topo I and II with the lamin B2 origin in vitro . ( A ) Detection of the in vitro topo I cleavages stabilized on the lower strand by CPT (lane 3), 7-[CH2–Tris] CPT (lane 5) or gimatecan (lane 6), and on the upper strand by CPT (lane 10); lanes 7 and 12: Maxam–Gilbert sequencing reactions; the position of the cleavages also present in vivo is indicated by an asterisk. ( B ) Effect of base substitution mutations in the lamin B2 origin on topo I-mediated cleavage. ( C ) Sequence of the origin portion covered by the replicative complexes; the position of substituted bases is highlighted; the position of in vitro topo I-cleavable complexes is indicated by filled triangles; the asterisks indicate the position of the topo I cleavages also present in vivo . ( D ) Detection of the in vitro VP16-induced topo II cleavages introduced by the enzyme alone (lanes 1–5) or by topo II as part of a complex with nuclear proteins (lanes 6–14); lane 9: the origin DNA incubated with the nuclear extract and VP16 was immunopurified using anti-topo II antibody; black vertical bars indicate the region protected in vivo ; the arrows indicate the borders of the region protected in vitro by the origin binding proteins (OBP) as determined by λ-exonuclease digestion; lanes 10 and 14: Maxam–Gilbert sequencing reactions.

    Techniques Used: In Vitro, Cycling Probe Technology, Sequencing, In Vivo, Incubation, Binding Assay

    9) Product Images from "Novel system for detecting SARS coronavirus nucleocapsid protein using an ssDNA aptamer"

    Article Title: Novel system for detecting SARS coronavirus nucleocapsid protein using an ssDNA aptamer

    Journal: Journal of Bioscience and Bioengineering

    doi: 10.1016/j.jbiosc.2011.08.014

    A schematic showing the sequence of the ssDNA library used for in vitro selection and the design of the SELEX procedure. A random ssDNA library was obtained by PCR, which contains 45 random sequences, using 5′-FAM TM labeled forward primer and 5′-phosphorylated reverse primer, which was followed by λ-exonuclease digestion. Ball symbol at the 5′ end of the forward primer represents FAM TM . After eliminating non-specific binding to Ni–NTA beads, the ssDNA pool was incubated with the His-tagged N proteins, which were immobilized on Ni–NTA sepharose beads. Unbound DNA was discarded, and bound DNA was eluted by the addition of imidazole. The DNA was enriched after twelve cycles of selection, and the selected DNA was amplified by PCR, cloned and sequenced.
    Figure Legend Snippet: A schematic showing the sequence of the ssDNA library used for in vitro selection and the design of the SELEX procedure. A random ssDNA library was obtained by PCR, which contains 45 random sequences, using 5′-FAM TM labeled forward primer and 5′-phosphorylated reverse primer, which was followed by λ-exonuclease digestion. Ball symbol at the 5′ end of the forward primer represents FAM TM . After eliminating non-specific binding to Ni–NTA beads, the ssDNA pool was incubated with the His-tagged N proteins, which were immobilized on Ni–NTA sepharose beads. Unbound DNA was discarded, and bound DNA was eluted by the addition of imidazole. The DNA was enriched after twelve cycles of selection, and the selected DNA was amplified by PCR, cloned and sequenced.

    Techniques Used: Sequencing, In Vitro, Selection, Polymerase Chain Reaction, Labeling, Binding Assay, Incubation, Amplification, Clone Assay

    10) Product Images from "Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection"

    Article Title: Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00166-17

    Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and λ-exonuclease treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.
    Figure Legend Snippet: Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and λ-exonuclease treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.

    Techniques Used: In Situ, Infection, Plasmid Purification

    11) Product Images from "Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1"

    Article Title: Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1

    Journal: Genes & Development

    doi: 10.1101/gad.310995.117

    The nucleosome exclusion reaction counteracts DNA synthesis-coupled chromatin assembly. ( A ) Schematic diagram of the primer extension assay. A 92-nucleotide (nt) primer carrying either no mismatch or an A:C mismatch is annealed on a single-stranded pMM1. Upon incubation in NPE, complementary DNA is synthesized depending on the primer, converting the substrate into covalently closed circular DNA. ( B ) The requirements of canonical MMR factors for primer extension-coupled mismatch correction. The primer extension assay was performed in mock-treated, Mlh1-depleted (ΔMlh1), Msh2-depleted (ΔMsh2), or Msh2/Mlh1 doubly depleted (ΔMsh2ΔMlh1) NPE. The ratio of XhoI-sensitive molecules that correspond to the C-to-T repair products is plotted in a graph. Mean ± one SD is shown. n = 4. P -values were calculated by the unpaired t , for the details of quantification. ( C ) Nucleosome exclusion on the primer extension products. The products described in B were separated by agarose gel without any treatment (lanes 2 – 5 ), after digestion of incomplete intermediates by S1 nuclease and ExoV (lanes 6 – 9 ), or after digestion of C-to-T repair products and incomplete intermediates by XhoI, S1 nuclease, and λ exonuclease (lanes 10 – 13 ). (ss) ssDNA; (IM) primer extension intermediates. ( D ) The assay presented in C was repeated in NPE depleted of Mlh1 and HIRA (lanes 3 , 4 ) or Mlh1, HIRA, and Smarcad1 (lanes 5 – 8 ) supplemented with either buffer (lanes 3 – 6 ) or recombinant Smarcad1 (lanes 7 , 8 ). The linking number of each band relative to the open circular or relaxed DNA (oc/r) position (ΔL) is indicated at the right of the gel. ( E ) The ratio of the plasmids of the indicated ΔL in D was quantified and is presented as a graph. Mean ± one SD is shown. n = 3.
    Figure Legend Snippet: The nucleosome exclusion reaction counteracts DNA synthesis-coupled chromatin assembly. ( A ) Schematic diagram of the primer extension assay. A 92-nucleotide (nt) primer carrying either no mismatch or an A:C mismatch is annealed on a single-stranded pMM1. Upon incubation in NPE, complementary DNA is synthesized depending on the primer, converting the substrate into covalently closed circular DNA. ( B ) The requirements of canonical MMR factors for primer extension-coupled mismatch correction. The primer extension assay was performed in mock-treated, Mlh1-depleted (ΔMlh1), Msh2-depleted (ΔMsh2), or Msh2/Mlh1 doubly depleted (ΔMsh2ΔMlh1) NPE. The ratio of XhoI-sensitive molecules that correspond to the C-to-T repair products is plotted in a graph. Mean ± one SD is shown. n = 4. P -values were calculated by the unpaired t , for the details of quantification. ( C ) Nucleosome exclusion on the primer extension products. The products described in B were separated by agarose gel without any treatment (lanes 2 – 5 ), after digestion of incomplete intermediates by S1 nuclease and ExoV (lanes 6 – 9 ), or after digestion of C-to-T repair products and incomplete intermediates by XhoI, S1 nuclease, and λ exonuclease (lanes 10 – 13 ). (ss) ssDNA; (IM) primer extension intermediates. ( D ) The assay presented in C was repeated in NPE depleted of Mlh1 and HIRA (lanes 3 , 4 ) or Mlh1, HIRA, and Smarcad1 (lanes 5 – 8 ) supplemented with either buffer (lanes 3 – 6 ) or recombinant Smarcad1 (lanes 7 , 8 ). The linking number of each band relative to the open circular or relaxed DNA (oc/r) position (ΔL) is indicated at the right of the gel. ( E ) The ratio of the plasmids of the indicated ΔL in D was quantified and is presented as a graph. Mean ± one SD is shown. n = 3.

    Techniques Used: DNA Synthesis, Primer Extension Assay, Incubation, Synthesized, Agarose Gel Electrophoresis, Recombinant

    12) Product Images from "Simplified ChIP-exo assays"

    Article Title: Simplified ChIP-exo assays

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05265-7

    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )
    Figure Legend Snippet: ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Techniques Used: Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced

    13) Product Images from "Simplified ChIP-exo assays"

    Article Title: Simplified ChIP-exo assays

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05265-7

    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )
    Figure Legend Snippet: ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Techniques Used: Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced

    14) Product Images from "Simplified ChIP-exo assays"

    Article Title: Simplified ChIP-exo assays

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05265-7

    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )
    Figure Legend Snippet: ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Techniques Used: Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced

    15) Product Images from "A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition"

    Article Title: A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s18082441

    Relative fluorescence intensity of the reaction systems upon addition of ADA, hoGG I, UDG, RNase H and λexo. Error bars were estimated from three replicate measurements.
    Figure Legend Snippet: Relative fluorescence intensity of the reaction systems upon addition of ADA, hoGG I, UDG, RNase H and λexo. Error bars were estimated from three replicate measurements.

    Techniques Used: Fluorescence

    16) Product Images from "Functional interactions of DNA topoisomerases with a human replication origin"

    Article Title: Functional interactions of DNA topoisomerases with a human replication origin

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7601578

    Interaction of topo I and II with the lamin B2 origin in vitro . ( A ) Detection of the in vitro topo I cleavages stabilized on the lower strand by CPT (lane 3), 7-[CH2–Tris] CPT (lane 5) or gimatecan (lane 6), and on the upper strand by CPT (lane 10); lanes 7 and 12: Maxam–Gilbert sequencing reactions; the position of the cleavages also present in vivo is indicated by an asterisk. ( B ) Effect of base substitution mutations in the lamin B2 origin on topo I-mediated cleavage. ( C ) Sequence of the origin portion covered by the replicative complexes; the position of substituted bases is highlighted; the position of in vitro topo I-cleavable complexes is indicated by filled triangles; the asterisks indicate the position of the topo I cleavages also present in vivo . ( D ) Detection of the in vitro VP16-induced topo II cleavages introduced by the enzyme alone (lanes 1–5) or by topo II as part of a complex with nuclear proteins (lanes 6–14); lane 9: the origin DNA incubated with the nuclear extract and VP16 was immunopurified using anti-topo II antibody; black vertical bars indicate the region protected in vivo ; the arrows indicate the borders of the region protected in vitro by the origin binding proteins (OBP) as determined by λ-exonuclease digestion; lanes 10 and 14: Maxam–Gilbert sequencing reactions.
    Figure Legend Snippet: Interaction of topo I and II with the lamin B2 origin in vitro . ( A ) Detection of the in vitro topo I cleavages stabilized on the lower strand by CPT (lane 3), 7-[CH2–Tris] CPT (lane 5) or gimatecan (lane 6), and on the upper strand by CPT (lane 10); lanes 7 and 12: Maxam–Gilbert sequencing reactions; the position of the cleavages also present in vivo is indicated by an asterisk. ( B ) Effect of base substitution mutations in the lamin B2 origin on topo I-mediated cleavage. ( C ) Sequence of the origin portion covered by the replicative complexes; the position of substituted bases is highlighted; the position of in vitro topo I-cleavable complexes is indicated by filled triangles; the asterisks indicate the position of the topo I cleavages also present in vivo . ( D ) Detection of the in vitro VP16-induced topo II cleavages introduced by the enzyme alone (lanes 1–5) or by topo II as part of a complex with nuclear proteins (lanes 6–14); lane 9: the origin DNA incubated with the nuclear extract and VP16 was immunopurified using anti-topo II antibody; black vertical bars indicate the region protected in vivo ; the arrows indicate the borders of the region protected in vitro by the origin binding proteins (OBP) as determined by λ-exonuclease digestion; lanes 10 and 14: Maxam–Gilbert sequencing reactions.

    Techniques Used: In Vitro, Cycling Probe Technology, Sequencing, In Vivo, Incubation, Binding Assay

    Related Articles

    Binding Assay:

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends
    Article Snippet: .. For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction. ..

    In Vitro:

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    Ligation:

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: .. Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5). .. Purified DNA (ds or ss) was subjected to CuAAC (1 μM DNA, 500 μM biotin azide, 500 μM CuSO4 , 2.5 mM THPTA, 5 mM sodium ascorbate) at 50°C for 2 h and reactions were purified by ethanol precipitation.

    Ethanol Precipitation:

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: .. Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5). .. Purified DNA (ds or ss) was subjected to CuAAC (1 μM DNA, 500 μM biotin azide, 500 μM CuSO4 , 2.5 mM THPTA, 5 mM sodium ascorbate) at 50°C for 2 h and reactions were purified by ethanol precipitation.

    Purification:

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: .. Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5). .. Purified DNA (ds or ss) was subjected to CuAAC (1 μM DNA, 500 μM biotin azide, 500 μM CuSO4 , 2.5 mM THPTA, 5 mM sodium ascorbate) at 50°C for 2 h and reactions were purified by ethanol precipitation.

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair
    Article Snippet: .. The substrates were then incubated with lambda exonuclease (0.025 unit/μl; NEB, MA) or purified recombinant Xenopus Exo1 (0.25ng/μl) at 22ºC. .. To assay the effect of the MRN complexes on the nucleases, the reactions also contained recombinant wild-type or mutant MRN proteins at 16 ng/μl.

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    Incubation:

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair
    Article Snippet: .. The substrates were then incubated with lambda exonuclease (0.025 unit/μl; NEB, MA) or purified recombinant Xenopus Exo1 (0.25ng/μl) at 22ºC. .. To assay the effect of the MRN complexes on the nucleases, the reactions also contained recombinant wild-type or mutant MRN proteins at 16 ng/μl.

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    other:

    Article Title: Human Heart Mitochondrial DNA Is Organized in Complex Catenated Networks Containing Abundant Four-way Junctions and Replication Forks *
    Article Snippet: Subsequent treatments with topoisomerase IV (John Innes Enterprises), topoisomerase I, T7 endonuclease I, or λ-exonuclease (all New England Biolabs) used the manufacturers' recommended conditions.

    Nuclease Assay:

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    Polymerase Chain Reaction:

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences
    Article Snippet: .. The digested PCR product was treated with 0.1 units lambda exonuclease (New England Biolabs) at 37°C for 15 min in the same restriction enzyme buffer. .. The probes were phosphorylated with 5 units of T4 polynucleotide kinase (NEB) in 50 mM Tris·HCl, pH7.9; 10 mM MgCl2 . dHPLC analysis was used to monitor the efficiency of lambda exonuclease digestion.

    Affinity Purification:

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends
    Article Snippet: .. For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction. ..

    Recombinant:

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair
    Article Snippet: .. The substrates were then incubated with lambda exonuclease (0.025 unit/μl; NEB, MA) or purified recombinant Xenopus Exo1 (0.25ng/μl) at 22ºC. .. To assay the effect of the MRN complexes on the nucleases, the reactions also contained recombinant wild-type or mutant MRN proteins at 16 ng/μl.

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    New England Biolabs λ exonuclease
    Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with <t>λ-exonuclease</t> (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.
    λ Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with λ-exonuclease (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.

    Journal: Nucleic Acids Research

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries

    doi: 10.1093/nar/gkv544

    Figure Lengend Snippet: Internal modification of DNA. DNA is first tailed with either 5- E -UTP or N 6 - P -ATP, and then elongated by primer extension. The 5′-monophosphorylated template (shown in gray) is optionally digested with λ-exonuclease (λ-Exo) and the alkyne is reacted in CuAAC, to attach biotin to the single-stranded (ss) or double-stranded (ds) DNA. 12% denaturing PAGE, visualization by SYBR Gold staining.

    Article Snippet: Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5).

    Techniques: Modification, Polyacrylamide Gel Electrophoresis, Staining

    Electrophoretic and TEM analysis of human heart mtDNA. a , one-dimensional AGE of undigested cardiac muscle mtDNA treated with the enzymes as indicated (topoisomerase ( Topo ) I, topoisomerase IV, T7 endonuclease ( endo ) I, and λ-exonuclease ( exo )). The identity of the various species is inferred by the effects of enzymatic treatments on their relative amounts and by their migration properties on two-dimensional AGE ( b ). Bands t and f are each composites of several species. λ-Exonuclease digests molecules with exposed 5′ phosphorylated ends. Because it removes or modifies the residual material running just behind open circles after topoisomerase IV treatment, i.e. 2n linear molecules plus species x ( b , panel ii ), we infer that species x has exposed ends. b , two-dimensional AGE of cardiac muscle mtDNA untreated ( panel i ), treated with topoisomerase IV ( panel ii ), treated with T7 endonuclease I ( panel iii ), and treated with topoisomerase IV + T7 endonuclease I ( panel iv ). The topology of the various forms, annotated on the gel images and inferred from the treatments and electrophoretic mobilities, is shown below the gel panels. tgl , tangled complexes (see Fig. 3 ); cat , catenanes (can also include > 2 monomeric or dimeric circles); 1n and 2n , monomeric and dimeric linear molecules, respectively; c and 2nc , monomeric and dimeric open circles, respectively; sc and 2nsc , supercoiled monomeric and dimeric circles, respectively. xc , circular molecules joined by four-way junctions; x , suggested to be circles joined to linear molecules by four-way junctions. Arrows indicate directions of first and second dimension electrophoresis. c and d , examples of forms seen by TEM following topoisomerase IV treatment of heart mtDNA alongside interpretations. Distinct circles and linear segments are indicated in different colors with inferred contour lengths in kb or (for circles) genome lengths. Scale bars , 200 nm.

    Journal: The Journal of Biological Chemistry

    Article Title: Human Heart Mitochondrial DNA Is Organized in Complex Catenated Networks Containing Abundant Four-way Junctions and Replication Forks *

    doi: 10.1074/jbc.M109.016600

    Figure Lengend Snippet: Electrophoretic and TEM analysis of human heart mtDNA. a , one-dimensional AGE of undigested cardiac muscle mtDNA treated with the enzymes as indicated (topoisomerase ( Topo ) I, topoisomerase IV, T7 endonuclease ( endo ) I, and λ-exonuclease ( exo )). The identity of the various species is inferred by the effects of enzymatic treatments on their relative amounts and by their migration properties on two-dimensional AGE ( b ). Bands t and f are each composites of several species. λ-Exonuclease digests molecules with exposed 5′ phosphorylated ends. Because it removes or modifies the residual material running just behind open circles after topoisomerase IV treatment, i.e. 2n linear molecules plus species x ( b , panel ii ), we infer that species x has exposed ends. b , two-dimensional AGE of cardiac muscle mtDNA untreated ( panel i ), treated with topoisomerase IV ( panel ii ), treated with T7 endonuclease I ( panel iii ), and treated with topoisomerase IV + T7 endonuclease I ( panel iv ). The topology of the various forms, annotated on the gel images and inferred from the treatments and electrophoretic mobilities, is shown below the gel panels. tgl , tangled complexes (see Fig. 3 ); cat , catenanes (can also include > 2 monomeric or dimeric circles); 1n and 2n , monomeric and dimeric linear molecules, respectively; c and 2nc , monomeric and dimeric open circles, respectively; sc and 2nsc , supercoiled monomeric and dimeric circles, respectively. xc , circular molecules joined by four-way junctions; x , suggested to be circles joined to linear molecules by four-way junctions. Arrows indicate directions of first and second dimension electrophoresis. c and d , examples of forms seen by TEM following topoisomerase IV treatment of heart mtDNA alongside interpretations. Distinct circles and linear segments are indicated in different colors with inferred contour lengths in kb or (for circles) genome lengths. Scale bars , 200 nm.

    Article Snippet: Subsequent treatments with topoisomerase IV (John Innes Enterprises), topoisomerase I, T7 endonuclease I, or λ-exonuclease (all New England Biolabs) used the manufacturers' recommended conditions.

    Techniques: Transmission Electron Microscopy, Migration, Electrophoresis

    ( A ) Fluorescence responses of Nanoprobe A (0.1 mg/ml) to APE1 at different concentrations. ( B ) Linear calibration curve for detection of the activity of APE1. The linear regression equation is F = 0.20 c (U/ml) – 2.1 × 10 −4 , and the detection limit is 0.01 U/ml. ( C ) Selectivity of Nanoprobe A toward APE1 (2.0 U/ml) over other nucleases. (DNase I: 5.0 U/ml; Exo III: 4.0 U/ml; lambda exo: 66.7 U/ml; Exo I: 12.5 U/ml; T5: 5.0 U/ml; T7: 50 U/ml). All experiments were repeated at least three times.

    Journal: Nucleic Acids Research

    Article Title: A specific DNA-nanoprobe for tracking the activities of human apurinic/apyrimidinic endonuclease 1 in living cells

    doi: 10.1093/nar/gkw1205

    Figure Lengend Snippet: ( A ) Fluorescence responses of Nanoprobe A (0.1 mg/ml) to APE1 at different concentrations. ( B ) Linear calibration curve for detection of the activity of APE1. The linear regression equation is F = 0.20 c (U/ml) – 2.1 × 10 −4 , and the detection limit is 0.01 U/ml. ( C ) Selectivity of Nanoprobe A toward APE1 (2.0 U/ml) over other nucleases. (DNase I: 5.0 U/ml; Exo III: 4.0 U/ml; lambda exo: 66.7 U/ml; Exo I: 12.5 U/ml; T5: 5.0 U/ml; T7: 50 U/ml). All experiments were repeated at least three times.

    Article Snippet: Apurinic/apyrimidinic endonuclease I (APE1), uracil–DNA glycocasylase (UDG), deoxyribonuclease I (DNase I), exonuclease III (Exo III), exonuclease I (Exo I), lambda exonuclease (λ exo), T5 exonuclease (T5 Exo), T7 exonuclease (T7 Exo) and their corresponding buffers ( ) were all purchased from New England Biolabs (NEB, USA).

    Techniques: Fluorescence, Activity Assay

    hSNM1 is a single-strand-specific exonuclease. ( A ) Assay of hSNM1 IMAC fraction 3 on 20-dT substrate. Lanes: 1, RecJ f ; 2, substrate only; 3, ySNM1; Lanes 4, 116 ng protein 5, 29 ng protein; 6, 7.25 ng protein; 7, 1.8 ng protein; 8, 0.45 ng protein. ( B ) Assay of IMAC fraction 2 on internally labeled double-stranded substrate. Lanes: 1, λ exonuclease control; 2, ySNM1; 3, 140 ng protein; 4, 35 ng protein; 5, 4.4 ng protein. ( C ) Assay of FPLC fraction 16 on 20-dT substrate. Lanes: 1, substrate only; 2, RecJ f  control; 3, fraction 16 (∼10 ng); 4, ∼2.5 ng; 5, ∼0.65 ng; 6, ∼0.17 ng. ( D ) Assay of FPLC fraction 16 on internally labeled ds substrate. Lanes: 1, fraction 16 (∼10 ng); 2, ∼2.5 ng; 3, ∼0.65ng.

    Journal: Nucleic Acids Research

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease

    doi: 10.1093/nar/gkm530

    Figure Lengend Snippet: hSNM1 is a single-strand-specific exonuclease. ( A ) Assay of hSNM1 IMAC fraction 3 on 20-dT substrate. Lanes: 1, RecJ f ; 2, substrate only; 3, ySNM1; Lanes 4, 116 ng protein 5, 29 ng protein; 6, 7.25 ng protein; 7, 1.8 ng protein; 8, 0.45 ng protein. ( B ) Assay of IMAC fraction 2 on internally labeled double-stranded substrate. Lanes: 1, λ exonuclease control; 2, ySNM1; 3, 140 ng protein; 4, 35 ng protein; 5, 4.4 ng protein. ( C ) Assay of FPLC fraction 16 on 20-dT substrate. Lanes: 1, substrate only; 2, RecJ f control; 3, fraction 16 (∼10 ng); 4, ∼2.5 ng; 5, ∼0.65 ng; 6, ∼0.17 ng. ( D ) Assay of FPLC fraction 16 on internally labeled ds substrate. Lanes: 1, fraction 16 (∼10 ng); 2, ∼2.5 ng; 3, ∼0.65ng.

    Article Snippet: In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier.

    Techniques: Labeling, Fast Protein Liquid Chromatography