dsdna  (New England Biolabs)


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    Name:
    NEBNext dsDNA Fragmentase
    Description:
    NEBNext dsDNA Fragmentase 250 rxns
    Catalog Number:
    M0348L
    Price:
    400
    Category:
    Other Endonucleases
    Size:
    250 rxns
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    Structured Review

    New England Biolabs dsdna
    NEBNext dsDNA Fragmentase
    NEBNext dsDNA Fragmentase 250 rxns
    https://www.bioz.com/result/dsdna/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dsdna - by Bioz Stars, 2021-05
    86/100 stars

    Images

    1) Product Images from "Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site"

    Article Title: Viroplasm Protein P9-1 of Rice Black-Streaked Dwarf Virus Preferentially Binds to Single-Stranded RNA in Its Octamer Form, and the Central Interior Structure Formed by This Octamer Constitutes the Major RNA Binding Site

    Journal: Journal of Virology

    doi: 10.1128/JVI.02264-13

    Competition and specificity assays of P9-1. (A) Increasing amounts of unlabeled competitor ssRNA, dsRNA, ssDNA, or dsDNA were mixed with 3.1 nmol DIG-labeled S9-1900nt ssRNA; 4.6 μmol of purified P9-1 was added to each sample, and the sample was
    Figure Legend Snippet: Competition and specificity assays of P9-1. (A) Increasing amounts of unlabeled competitor ssRNA, dsRNA, ssDNA, or dsDNA were mixed with 3.1 nmol DIG-labeled S9-1900nt ssRNA; 4.6 μmol of purified P9-1 was added to each sample, and the sample was

    Techniques Used: Labeling, Purification

    2) Product Images from "Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway"

    Article Title: Regulation of HLTF-mediated PCNA polyubiquitination by RFC and PCNA monoubiquitination levels determines choice of damage tolerance pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky943

    Effects of various types of DNA. Chain-formation activities of wild-type HLTF ( A – E ) and his HLTF ΔN ( F, G ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Poly(dA)-oligo(dT) is a 2:1 mixture of poly(dA) and 18-mer oligo(dT) as nucleotides. (B, F) The indicated DNA was titrated as shown. (C, G) Poly(dA) was annealed to 18-mer oligo(dT) modified with biotin at the 5′- or 3′-end at a 2:1 ratio as nucleotides. (D) Poly(dA) was annealed to 18-mer oligo(dT) modified with phosphate at the 5′-OH or 3′-OH at a 2:1 ratio as nucleotides. ( E ) Poly(dA) was annealed to 18-mer oligo(dT) with one (-C1), two (-C2), or four (-C4) additional dCs at the 3′-end at a 2:1 ratio as nucleotides. The same data with poly(dA)-oligo(dT) were plotted in graphs for the wild type (A–E) and for his HLTF ΔN (F, G) as controls. Error bars from at least two experiments are shown with the symbols. ( H ) DNA-binding assay. HLTF (upper panel) or his HLTF ΔN (middle panel) was incubated with M13mp18 ssDNA (ss) or dsDNA (ds) tethered with magnetic beads, or magnetic beads only (–), at 4°C for 2 min, and the beads were separated from the supernatants. Each fraction was analyzed by western blotting with an anti-HLTF antibody, and band intensities were measured. The relative values of binding fractions normalized by the amount of the input were plotted in a graph (bottom panel).
    Figure Legend Snippet: Effects of various types of DNA. Chain-formation activities of wild-type HLTF ( A – E ) and his HLTF ΔN ( F, G ) were analyzed under standard assay conditions containing E1, MMS2-UBC13, and ubiquitin at 30°C for 10 min with the indicated DNA. The total amounts of ubiquitin in chains in each 25 μl reaction mixture were plotted. (A) Poly(dA)-oligo(dT) is a 2:1 mixture of poly(dA) and 18-mer oligo(dT) as nucleotides. (B, F) The indicated DNA was titrated as shown. (C, G) Poly(dA) was annealed to 18-mer oligo(dT) modified with biotin at the 5′- or 3′-end at a 2:1 ratio as nucleotides. (D) Poly(dA) was annealed to 18-mer oligo(dT) modified with phosphate at the 5′-OH or 3′-OH at a 2:1 ratio as nucleotides. ( E ) Poly(dA) was annealed to 18-mer oligo(dT) with one (-C1), two (-C2), or four (-C4) additional dCs at the 3′-end at a 2:1 ratio as nucleotides. The same data with poly(dA)-oligo(dT) were plotted in graphs for the wild type (A–E) and for his HLTF ΔN (F, G) as controls. Error bars from at least two experiments are shown with the symbols. ( H ) DNA-binding assay. HLTF (upper panel) or his HLTF ΔN (middle panel) was incubated with M13mp18 ssDNA (ss) or dsDNA (ds) tethered with magnetic beads, or magnetic beads only (–), at 4°C for 2 min, and the beads were separated from the supernatants. Each fraction was analyzed by western blotting with an anti-HLTF antibody, and band intensities were measured. The relative values of binding fractions normalized by the amount of the input were plotted in a graph (bottom panel).

    Techniques Used: Modification, DNA Binding Assay, Incubation, Magnetic Beads, Western Blot, Binding Assay

    3) Product Images from "RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin"

    Article Title: RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin

    Journal: eLife

    doi: 10.7554/eLife.25299

    Characterization of SUV39H1-RNA binding in vitro. ( A ) Competitive binding assay measuring the ability of unlabeled 19mer ssRNA to compete with the interaction of labeled 19mer ssRNA binding to MBP-SUV39H1 42–106. MBP-SUV39H1 42–106 present at 12.4 μM, labeled RNA at approximately 3 nM, and unlabeled RNA at concentrations ranging from 30 to 1500 nM. Percent labeled RNA bound is quantified and listed. ( B ) Various nucleic acids oligonucleotides consisting of the first 50 bases of E. coli MBP run out on a native polyacrylamide gel. Oligonucleotides were annealed to create various nucleic acids and end labeled with radioactive 32 P. ssRNA 1, sense MBP 1–50; ssRNA 2, anti-sense MBP 1–50; ssDNA, sense MBP 1–50. ( C ) Representative EMSAs showing the binding of purified MBP-SUV39H1 1–106 and to various nucleic acids, all composed of the E. coli MBP 1–50 sequence, shown in B. Protein is diluted 2-fold from 25 μM. Quantification in Figure 3E . ( D ) Left, binding curves showing binding of MBP-SUV39H1 1–106 and either sense or anti-sense 19mer ssRNA, protein diluted 2-fold from 100 µM. Error bars are standard deviation from two independent experiments. Right, dissociation constants (K d , μM) determined by non-linear fitting of the binding curves. Standard error represents the error of the curve fitting to the average of two experimental replicates. ( E ) Representative EMSAs showing binding of purified MBP-SUV39H1 1–106 to 180 bases of either α-satellite or β-actin ssRNA. Protein diluted 2-fold from 2.5 μM. Quantification in Figure 3F . ( F ) SUV39H1 affinity increases as length of nucleic acid increases. Binding curves compiled from Figure 3E and F (50mers and 180mers, respectively) and D ) (19mers) showing the binding of MBP-SUV39H1 1–106 to various nucleic acid types. 19mer random sequence: sense and antisense ssRNA; 50mer MBP 1–50: sense and antisense ssRNA, ssDNA, dsRNA, dsDNA, and RNA/DNA hybrid; 180mers: α-satellite and β-actin ssRNA. All sequences are described in the materials and methods. Error bars are standard deviation from two independent experiments. Dissociation constants (K d , μM) displayed on graph are determined by non-linear fitting of the binding curves. Standard error represents the error of the curve fitting to the average of two experimental replicates. DOI: http://dx.doi.org/10.7554/eLife.25299.009
    Figure Legend Snippet: Characterization of SUV39H1-RNA binding in vitro. ( A ) Competitive binding assay measuring the ability of unlabeled 19mer ssRNA to compete with the interaction of labeled 19mer ssRNA binding to MBP-SUV39H1 42–106. MBP-SUV39H1 42–106 present at 12.4 μM, labeled RNA at approximately 3 nM, and unlabeled RNA at concentrations ranging from 30 to 1500 nM. Percent labeled RNA bound is quantified and listed. ( B ) Various nucleic acids oligonucleotides consisting of the first 50 bases of E. coli MBP run out on a native polyacrylamide gel. Oligonucleotides were annealed to create various nucleic acids and end labeled with radioactive 32 P. ssRNA 1, sense MBP 1–50; ssRNA 2, anti-sense MBP 1–50; ssDNA, sense MBP 1–50. ( C ) Representative EMSAs showing the binding of purified MBP-SUV39H1 1–106 and to various nucleic acids, all composed of the E. coli MBP 1–50 sequence, shown in B. Protein is diluted 2-fold from 25 μM. Quantification in Figure 3E . ( D ) Left, binding curves showing binding of MBP-SUV39H1 1–106 and either sense or anti-sense 19mer ssRNA, protein diluted 2-fold from 100 µM. Error bars are standard deviation from two independent experiments. Right, dissociation constants (K d , μM) determined by non-linear fitting of the binding curves. Standard error represents the error of the curve fitting to the average of two experimental replicates. ( E ) Representative EMSAs showing binding of purified MBP-SUV39H1 1–106 to 180 bases of either α-satellite or β-actin ssRNA. Protein diluted 2-fold from 2.5 μM. Quantification in Figure 3F . ( F ) SUV39H1 affinity increases as length of nucleic acid increases. Binding curves compiled from Figure 3E and F (50mers and 180mers, respectively) and D ) (19mers) showing the binding of MBP-SUV39H1 1–106 to various nucleic acid types. 19mer random sequence: sense and antisense ssRNA; 50mer MBP 1–50: sense and antisense ssRNA, ssDNA, dsRNA, dsDNA, and RNA/DNA hybrid; 180mers: α-satellite and β-actin ssRNA. All sequences are described in the materials and methods. Error bars are standard deviation from two independent experiments. Dissociation constants (K d , μM) displayed on graph are determined by non-linear fitting of the binding curves. Standard error represents the error of the curve fitting to the average of two experimental replicates. DOI: http://dx.doi.org/10.7554/eLife.25299.009

    Techniques Used: RNA Binding Assay, In Vitro, Competitive Binding Assay, Labeling, Binding Assay, Purification, Sequencing, Standard Deviation

    4) Product Images from "DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange"

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp200

    The strand-exchange assay with human RAD51. ( A ) A schematic diagram of the strand-exchange assay. ( B ) The strand exchange activity of RAD51 under the three different conditions. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Joint molecules and nicked circular DNA are indicated by jm and nc, respectively. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate experiments with RAD51 in the absence and presence of 0.2 M KCl, respectively. Lane 4 indicates an experiment with 5% methanol in the absence of 0.2 M KCl.
    Figure Legend Snippet: The strand-exchange assay with human RAD51. ( A ) A schematic diagram of the strand-exchange assay. ( B ) The strand exchange activity of RAD51 under the three different conditions. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Joint molecules and nicked circular DNA are indicated by jm and nc, respectively. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate experiments with RAD51 in the absence and presence of 0.2 M KCl, respectively. Lane 4 indicates an experiment with 5% methanol in the absence of 0.2 M KCl.

    Techniques Used: Activity Assay, Incubation, Recombinase Polymerase Amplification, Agarose Gel Electrophoresis, Staining, Negative Control

    Effects of the DIDS reaction order on RAD51-mediated strand exchange. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA containing 2 μM 32 P-labeled ϕX174 linear dsDNA, in the absence of 0.2 M KCl. The reactions were stopped and deproteinized at the indicated times, and the products were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The jm products and dsDNA labeled by 32 P were visualized and quantified using an FLA-7000 imaging analyzer (Fujifilm). ( A ) RAD51 was incubated with DIDS (10 μM) at 37°C for 10 min. After the addition of the ϕX174 circular ssDNA, RPA was added to the reaction mixture. The ϕX174 linear dsDNA was then added to initiate the reaction. The reactions were continued for the indicated times. Lanes 1–4 indicate positive control experiments with RAD51 and 5% methanol. Lanes 5–8 indicate experiments with RAD51 and DIDS (and 5% methanol). Reaction times were 0 min (lanes 1 and 5), 30 min (lanes 2 and 6), 60 min (lanes 3 and 7) and 90 min (lanes 4 and 8). ( B ) Graphic representation of the experiments shown in (A). Closed and open circles indicate experiments with and without DIDS, respectively. ( C ) RAD51 was incubated with the ϕX174 circular ssDNA at 37°C for 10 min. After this incubation, DIDS (10 μM) was added, followed by the addition of RPA. The ϕX174 linear dsDNA was then added to initiate the reaction. ( D ) Graphic representation of the experiments shown in (C). Closed and open circles indicate experiments with and without DIDS, respectively. ( E ) RAD51 was incubated with the ϕX174 circular ssDNA at 37°C for 10 min, and RPA was added. DIDS was added, and then ϕX174 linear dsDNA was added to initiate the reaction. ( F ) Graphic representation of the experiments shown in (E). Closed and open circles indicate experiments with and without DIDS, respectively.
    Figure Legend Snippet: Effects of the DIDS reaction order on RAD51-mediated strand exchange. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA containing 2 μM 32 P-labeled ϕX174 linear dsDNA, in the absence of 0.2 M KCl. The reactions were stopped and deproteinized at the indicated times, and the products were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The jm products and dsDNA labeled by 32 P were visualized and quantified using an FLA-7000 imaging analyzer (Fujifilm). ( A ) RAD51 was incubated with DIDS (10 μM) at 37°C for 10 min. After the addition of the ϕX174 circular ssDNA, RPA was added to the reaction mixture. The ϕX174 linear dsDNA was then added to initiate the reaction. The reactions were continued for the indicated times. Lanes 1–4 indicate positive control experiments with RAD51 and 5% methanol. Lanes 5–8 indicate experiments with RAD51 and DIDS (and 5% methanol). Reaction times were 0 min (lanes 1 and 5), 30 min (lanes 2 and 6), 60 min (lanes 3 and 7) and 90 min (lanes 4 and 8). ( B ) Graphic representation of the experiments shown in (A). Closed and open circles indicate experiments with and without DIDS, respectively. ( C ) RAD51 was incubated with the ϕX174 circular ssDNA at 37°C for 10 min. After this incubation, DIDS (10 μM) was added, followed by the addition of RPA. The ϕX174 linear dsDNA was then added to initiate the reaction. ( D ) Graphic representation of the experiments shown in (C). Closed and open circles indicate experiments with and without DIDS, respectively. ( E ) RAD51 was incubated with the ϕX174 circular ssDNA at 37°C for 10 min, and RPA was added. DIDS was added, and then ϕX174 linear dsDNA was added to initiate the reaction. ( F ) Graphic representation of the experiments shown in (E). Closed and open circles indicate experiments with and without DIDS, respectively.

    Techniques Used: Incubation, Recombinase Polymerase Amplification, Labeling, Agarose Gel Electrophoresis, Imaging, Positive Control

    DIDS efficiently inhibits RAD51-mediated strand exchange. ( A ) DIDS titration experiments in the absence of 0.2 M KCl. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) in the presence of DIDS at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Joint molecule is indicated by jm. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 and 5% methanol in the absence of DIDS. DIDS concentrations were 0.01 μM (lane 3), 0.1 μM (lane 4), 1 μM (lane 5) and 10 μM (lane 6). Lane 7 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( B ) Graphic representation of the experiments shown in (A). The band intensities of the jm product were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (A, lane 2) were plotted against the DIDS concentration. ( C ) The strand-exchange assay in the presence of 0.2 M KCl. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate control experiments without DIDS with RAD51 in the absence and presence of 5% methanol, respectively. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( D ) Graphic representation of the experiments shown in (C). The band intensities of the jm products were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (C, lane 3) were plotted against the DIDS concentration.
    Figure Legend Snippet: DIDS efficiently inhibits RAD51-mediated strand exchange. ( A ) DIDS titration experiments in the absence of 0.2 M KCl. The ϕX174 circular ssDNA (20 μM) was incubated with RAD51 (6 μM) in the presence of DIDS at 37°C for 10 min. After this incubation, 2 μM RPA was added to the reaction mixture, which was incubated at 37°C for 10 min. The reactions were then initiated by the addition of 20 μM ϕX174 linear dsDNA. The DNA products were then deproteinized, and were separated by 1% agarose gel electrophoresis in 1× TAE buffer at 3.3 V/cm for 4 h. The products were visualized by SYBR Gold (Invitrogen) staining. Joint molecule is indicated by jm. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 and 5% methanol in the absence of DIDS. DIDS concentrations were 0.01 μM (lane 3), 0.1 μM (lane 4), 1 μM (lane 5) and 10 μM (lane 6). Lane 7 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( B ) Graphic representation of the experiments shown in (A). The band intensities of the jm product were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (A, lane 2) were plotted against the DIDS concentration. ( C ) The strand-exchange assay in the presence of 0.2 M KCl. Lane 1 indicates a negative control experiment without RAD51. Lanes 2 and 3 indicate control experiments without DIDS with RAD51 in the absence and presence of 5% methanol, respectively. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( D ) Graphic representation of the experiments shown in (C). The band intensities of the jm products were quantified as the peak volumes of densitometric scans. The jm peak volumes relative to that in the reaction without DIDS (C, lane 3) were plotted against the DIDS concentration.

    Techniques Used: Titration, Incubation, Recombinase Polymerase Amplification, Agarose Gel Electrophoresis, Staining, Negative Control, Concentration Assay

    The strand-exchange assay in the presence of chemical compounds. ( A ) Each chemical compound (10 μM, lanes 1–185) was incubated with RAD51 (6 μM), and the strand-exchange reaction was performed under the conditions containing 5% methanol at 37°C for 1 h with 20 μM ssDNA, 20 μM dsDNA and 2 μM RPA. Lanes C, R and M indicate negative control reactions without RAD51, complete reactions and complete reactions with 5% methanol, respectively. The chemical compounds are listed in Supplementary Table 1 . ( B ) Structure of 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS).
    Figure Legend Snippet: The strand-exchange assay in the presence of chemical compounds. ( A ) Each chemical compound (10 μM, lanes 1–185) was incubated with RAD51 (6 μM), and the strand-exchange reaction was performed under the conditions containing 5% methanol at 37°C for 1 h with 20 μM ssDNA, 20 μM dsDNA and 2 μM RPA. Lanes C, R and M indicate negative control reactions without RAD51, complete reactions and complete reactions with 5% methanol, respectively. The chemical compounds are listed in Supplementary Table 1 . ( B ) Structure of 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS).

    Techniques Used: Incubation, Recombinase Polymerase Amplification, Negative Control

    DIDS inhibits DNA binding by RAD51. ( A ) The ssDNA-binding experiments in the presence of ATP. The ϕX174 circular ssDNA (40 μM) was incubated with RAD51 (2 μM) in the presence of DIDS at 37°C for 15 min. The samples were analyzed by 0.8% agarose gel electrophoresis in 1× TAE buffer. The bands were visualized by ethidium bromide staining. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 alone. Lane 3 indicates an experiment with RAD51 and 5% methanol. DIDS concentrations were 0.1 μM (lane 4), 1 μM (lane 5), 10 μM (lane 6) and 20 μM (lane 7). Lane 8 indicates an experiment with 20 μM DIDS in the absence of RAD51. ( B ) The dsDNA-binding experiments in the presence of ATP. The linear ϕX174 dsDNA (10 μM) was incubated with RAD51 (1 μM) in the presence of DIDS at 37°C for 15 min. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 alone. Lane 3 indicates an experiment with RAD51 and 5% methanol. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( C ) The ssDNA-binding experiments in the presence of ATP, AMPPNP and ATPγS. The ϕX174 circular ssDNA (40 μM) was incubated with RAD51 (2 μM) at 37°C for 15 min. Lanes 1–4, lanes 5–8 and lanes 9–12 represent experiments with ATP, AMPPNP and ATPγS, respectively. Lanes 1, 5 and 9 indicate negative control experiments without RAD51. Lanes 2, 6 and 10 indicate experiments with RAD51 alone. Lanes 3, 7 and 11 indicate experiments with RAD51 and 5% methanol. Lanes 4, 8 and 12 indicate experiments with RAD51 and DIDS (20 μM). ( D ) The dsDNA-binding experiments in the presence of ATP, AMPPNP and ATPγS. The linear ϕX174 dsDNA (10 μM) was incubated with RAD51 (1 μM) in the presence of DIDS at 37°C for 15 min. Lanes correspond to those in (C). The DIDS concentration was 10 μM. ( E ) Effect of DIDS on the ssDNA binding of RPA. The ϕX174 circular ssDNA (40 μM) was incubated with RPA (0.5 μM) in the presence of DIDS at 37°C for 15 min. Lanes 1 and 8 indicate negative control experiments without RPA. Lanes 2 and 3 indicate experiments with RPA in the absence and presence of 5% methanol, respectively. The DIDS concentrations were 0.1 μM (lane 4), 1 μM (lane 5), 10 μM (lane 6) and 20 μM (lanes 7 and 8). ( F ) The ssDNA-binding and dsDNA-binding experiments with RAD51 were performed in the presence of methanol. Lanes 1–6 and lanes 7–12 indicate experiments with ssDNA and dsDNA, respectively. Lanes 1, 6, 7 and 12 are negative controls without RAD51, and lanes 2 and 8 are positive controls with RAD51 in the absence of methanol. Methanol concentrations were 2.5% (lanes 3 and 9), 5% (lanes 4 and 10) and 10% (lanes 5, 6, 11 and 12).
    Figure Legend Snippet: DIDS inhibits DNA binding by RAD51. ( A ) The ssDNA-binding experiments in the presence of ATP. The ϕX174 circular ssDNA (40 μM) was incubated with RAD51 (2 μM) in the presence of DIDS at 37°C for 15 min. The samples were analyzed by 0.8% agarose gel electrophoresis in 1× TAE buffer. The bands were visualized by ethidium bromide staining. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 alone. Lane 3 indicates an experiment with RAD51 and 5% methanol. DIDS concentrations were 0.1 μM (lane 4), 1 μM (lane 5), 10 μM (lane 6) and 20 μM (lane 7). Lane 8 indicates an experiment with 20 μM DIDS in the absence of RAD51. ( B ) The dsDNA-binding experiments in the presence of ATP. The linear ϕX174 dsDNA (10 μM) was incubated with RAD51 (1 μM) in the presence of DIDS at 37°C for 15 min. Lane 1 indicates a negative control experiment without RAD51. Lane 2 indicates an experiment with RAD51 alone. Lane 3 indicates an experiment with RAD51 and 5% methanol. DIDS concentrations were 0.01 μM (lane 4), 0.1 μM (lane 5), 1 μM (lane 6) and 10 μM (lane 7). Lane 8 indicates an experiment with 10 μM DIDS in the absence of RAD51. ( C ) The ssDNA-binding experiments in the presence of ATP, AMPPNP and ATPγS. The ϕX174 circular ssDNA (40 μM) was incubated with RAD51 (2 μM) at 37°C for 15 min. Lanes 1–4, lanes 5–8 and lanes 9–12 represent experiments with ATP, AMPPNP and ATPγS, respectively. Lanes 1, 5 and 9 indicate negative control experiments without RAD51. Lanes 2, 6 and 10 indicate experiments with RAD51 alone. Lanes 3, 7 and 11 indicate experiments with RAD51 and 5% methanol. Lanes 4, 8 and 12 indicate experiments with RAD51 and DIDS (20 μM). ( D ) The dsDNA-binding experiments in the presence of ATP, AMPPNP and ATPγS. The linear ϕX174 dsDNA (10 μM) was incubated with RAD51 (1 μM) in the presence of DIDS at 37°C for 15 min. Lanes correspond to those in (C). The DIDS concentration was 10 μM. ( E ) Effect of DIDS on the ssDNA binding of RPA. The ϕX174 circular ssDNA (40 μM) was incubated with RPA (0.5 μM) in the presence of DIDS at 37°C for 15 min. Lanes 1 and 8 indicate negative control experiments without RPA. Lanes 2 and 3 indicate experiments with RPA in the absence and presence of 5% methanol, respectively. The DIDS concentrations were 0.1 μM (lane 4), 1 μM (lane 5), 10 μM (lane 6) and 20 μM (lanes 7 and 8). ( F ) The ssDNA-binding and dsDNA-binding experiments with RAD51 were performed in the presence of methanol. Lanes 1–6 and lanes 7–12 indicate experiments with ssDNA and dsDNA, respectively. Lanes 1, 6, 7 and 12 are negative controls without RAD51, and lanes 2 and 8 are positive controls with RAD51 in the absence of methanol. Methanol concentrations were 2.5% (lanes 3 and 9), 5% (lanes 4 and 10) and 10% (lanes 5, 6, 11 and 12).

    Techniques Used: Binding Assay, Incubation, Agarose Gel Electrophoresis, Staining, Negative Control, Concentration Assay, Recombinase Polymerase Amplification

    5) Product Images from "A novel nuclease-ATPase (Nar71) from archaea is part of a proposed thermophilic DNA repair system"

    Article Title: A novel nuclease-ATPase (Nar71) from archaea is part of a proposed thermophilic DNA repair system

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh960

    ( A ) ATPase activity of Nar71 measured in malachite green assays. Reactions contained (i) 50 nM or (ii) 100 nM Nar71 and (iii) 3.3 mM Mg 2+ and 5 mM ATP. dsDNA or ssDNA (100 ng) was added as indicated. RecG (50 nM) was used as a comparison in 5 mM Mg 2+ and 5 mM ATP. In each panel, data are a mean of reactions in duplicate. ( B ) Time course reaction on 100 nM Nar71 containing 100 nM Nar71 and 100 ng ssDNA in 3.3 mM Mg 2+ and 5 mM ATP. Data are a mean of reactions in duplicate. ( C ) Effect of pre-incubating His-tag monoclonal antibody with tagged Nar71 (250 nM) prior to mixing with 5 nM 3′ flap DNA in 10 mM Mg 2+ and 15 mM ATP.
    Figure Legend Snippet: ( A ) ATPase activity of Nar71 measured in malachite green assays. Reactions contained (i) 50 nM or (ii) 100 nM Nar71 and (iii) 3.3 mM Mg 2+ and 5 mM ATP. dsDNA or ssDNA (100 ng) was added as indicated. RecG (50 nM) was used as a comparison in 5 mM Mg 2+ and 5 mM ATP. In each panel, data are a mean of reactions in duplicate. ( B ) Time course reaction on 100 nM Nar71 containing 100 nM Nar71 and 100 ng ssDNA in 3.3 mM Mg 2+ and 5 mM ATP. Data are a mean of reactions in duplicate. ( C ) Effect of pre-incubating His-tag monoclonal antibody with tagged Nar71 (250 nM) prior to mixing with 5 nM 3′ flap DNA in 10 mM Mg 2+ and 15 mM ATP.

    Techniques Used: Activity Assay

    6) Product Images from "Senataxin Mutation Reveals How R-loops Promote Transcription by Blocking DNA Methylation at Gene Promoters"

    Article Title: Senataxin Mutation Reveals How R-loops Promote Transcription by Blocking DNA Methylation at Gene Promoters

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2017.12.030

    DNMT1 favors binding and methylation of dsDNA over RNA/DNA hybrid ( A ) DNMT1 activity is higher with dsDNA than RNA-DNA hybrid as substrates; error bars represent SEM of triplicates. ( B ) Binding affinity of DNMT1 for dsDNA is higher than for RNA/DNA hybrid. EMSA was used to measure binding of DNMT1 to BAMBI promoter represented as biotinylated dsDNA or corresponding RNA/DNA hybrid. Unlabeled dsDNA was added as competitor to show specificity of binding. ( C ) More binding of DNMT1 to dsDNA than RNA/DNA hybrid. Binding with a dilution series of DNMT1 to 2.5nM dsDNA (solid lines) or RNA/DNA hybrid (dotted lines) assayed by biolayer interferometry. Baseline was recorded from 0 to 300 seconds, association of DNMT1 with dsDNA or RNA/DNA hybrid from 300-900 seconds, followed by dissociation. ( D ) ALS4 patients have less nascent transcripts of BAMBI . In nuclear run-on (NRO) of primary fibroblasts (n = 2 controls 2 ALS4 patients), nascent transcript level was measured by quantitative PCR using primers specific to promoter of BAMBI , and normalized to promoter of a housekeeping gene ABLI that has no R-loops in S9.6 pull-down assay.
    Figure Legend Snippet: DNMT1 favors binding and methylation of dsDNA over RNA/DNA hybrid ( A ) DNMT1 activity is higher with dsDNA than RNA-DNA hybrid as substrates; error bars represent SEM of triplicates. ( B ) Binding affinity of DNMT1 for dsDNA is higher than for RNA/DNA hybrid. EMSA was used to measure binding of DNMT1 to BAMBI promoter represented as biotinylated dsDNA or corresponding RNA/DNA hybrid. Unlabeled dsDNA was added as competitor to show specificity of binding. ( C ) More binding of DNMT1 to dsDNA than RNA/DNA hybrid. Binding with a dilution series of DNMT1 to 2.5nM dsDNA (solid lines) or RNA/DNA hybrid (dotted lines) assayed by biolayer interferometry. Baseline was recorded from 0 to 300 seconds, association of DNMT1 with dsDNA or RNA/DNA hybrid from 300-900 seconds, followed by dissociation. ( D ) ALS4 patients have less nascent transcripts of BAMBI . In nuclear run-on (NRO) of primary fibroblasts (n = 2 controls 2 ALS4 patients), nascent transcript level was measured by quantitative PCR using primers specific to promoter of BAMBI , and normalized to promoter of a housekeeping gene ABLI that has no R-loops in S9.6 pull-down assay.

    Techniques Used: Binding Assay, Methylation, Activity Assay, Real-time Polymerase Chain Reaction, Pull Down Assay

    Related Articles

    Sequencing:

    Article Title: Generation of a transgenic ORFeome library in Drosophila
    Article Snippet: .. More recently, we have moved to Illumina MiSeq for full insert sequencing using dsDNA fragmentase (NEB) as a convenient and scalable method to fragment plasmid DNA ( ). ..

    Article Title: Generation of a transgenic ORFeome library in Drosophila
    Article Snippet: ORF cloning from pooled cDNA libraries has more variable success and may be dependent on variables such as the expression level of the gene of interest, the size of the gene or GC content. .. For sequencing library preparation, a time course with the dsDNA fragmentase is useful for optimal digestion ( ). ..

    Plasmid Preparation:

    Article Title: Generation of a transgenic ORFeome library in Drosophila
    Article Snippet: .. More recently, we have moved to Illumina MiSeq for full insert sequencing using dsDNA fragmentase (NEB) as a convenient and scalable method to fragment plasmid DNA ( ). ..

    Article Title: A Bumpy Ride on the Diagnostic Bench of Massive Parallel Sequencing, the Case of the Mitochondrial Genome
    Article Snippet: The use of the same pUC19 DNA sample also allowed a comparison of sequencing results across platforms. .. One µg of pUC19 plasmid DNA (Thermo Fisher, Erembodegem-Aalst, Belgium) was sheared by the Covaris or NEBNext dsDNA Fragmentase. .. Subsequently, samples were processed using the TruSeq DNA PCR-Free Sample Preparation protocol, and sequenced on the MiSeq.

    Article Title: A Bumpy Ride on the Diagnostic Bench of Massive Parallel Sequencing, the Case of the Mitochondrial Genome
    Article Snippet: This value was based on the determination of the sequencing error and the sensitivity and specificity experiments previously performed . .. To set the detection threshold for the MiSeq, the same pUC19 plasmid DNA sample was sheared with two different methods, once using the Covaris M220 sonicator and secondly using the NEBNext dsDNA Fragmentase. .. Both differentially sheared samples were sequenced on the MiSeq following TruSeq PCR free library preparation and a 100% coverage was obtained with an average read depth of 30 440 and 30 966 respectively.

    Article Title: Generation of a transgenic ORFeome library in Drosophila
    Article Snippet: • 50 mg/ml Kanamycin (Formedium, cat. no. KAN0025). .. • Gateway BP Clonase II Enzyme mix (Life Technologies, cat. no. 11789-020) • Gateway LR Clonase II Enzyme mix (Life Technologies, cat. no. 11791-020) • pDONR221 (Life Technologies, cat. no. 12536-017) • NEBNext dsDNA Fragmentase (NEB, cat. no. M0348S) • NEBNext End Repair Module (NEB, cat. no. E6050S) • NEBNext dA-Tailing Module (NEB, cat. no. E6053S) • T4 DNA ligase (Fermentas, cat. no. EL0011 ) • Wizard SV 96 Plasmid DNA Purification System (Promega, cat. no. A2250) • Plasmid midi kit (Qiagen, cat. no. 12143) for cleaning up pooled plasmid DNA • ORF-specific primer pairs for first-round PCR amplification (Sigma-Aldrich) (see Reagent Setup) • Barcode oligo pair, both 5′ phosphorylated (IDT). .. When annealed (see Reagent Setup) will have sticky ends (bold nucleotides) compatible with XhoI and HindIII digested vector. barcode_up: TCGAG ANNTGNNNACNNNNTGANNNNACNNNATNNNGANN G barcode_down: AGCTC NNTCNNNATNNNGTNNNNTCANNNNGTNNNCANNT C .

    Positive Control:

    Article Title: Suboptimal extracellular pH values alter DNA damage response to induced double‐strand breaks
    Article Snippet: .. As a positive control, digestion of the DNA was performed with 2 μL of fragmentase (M0348, NEB, Ipswich, MA, USA). .. Intracellular pH measurement Intracellular pH (pHi) was determined by confocal microscopy using the fluorescent pH‐sensitive probe BCECF (2,7‐bis‐(2‐carboxyethyl)‐5‐(and‐6)‐carboxyfluorescein, acetoxy‐methyl ester) (B1170, ThermoFisher) as described in Ref. .

    DNA Purification:

    Article Title: Generation of a transgenic ORFeome library in Drosophila
    Article Snippet: • 50 mg/ml Kanamycin (Formedium, cat. no. KAN0025). .. • Gateway BP Clonase II Enzyme mix (Life Technologies, cat. no. 11789-020) • Gateway LR Clonase II Enzyme mix (Life Technologies, cat. no. 11791-020) • pDONR221 (Life Technologies, cat. no. 12536-017) • NEBNext dsDNA Fragmentase (NEB, cat. no. M0348S) • NEBNext End Repair Module (NEB, cat. no. E6050S) • NEBNext dA-Tailing Module (NEB, cat. no. E6053S) • T4 DNA ligase (Fermentas, cat. no. EL0011 ) • Wizard SV 96 Plasmid DNA Purification System (Promega, cat. no. A2250) • Plasmid midi kit (Qiagen, cat. no. 12143) for cleaning up pooled plasmid DNA • ORF-specific primer pairs for first-round PCR amplification (Sigma-Aldrich) (see Reagent Setup) • Barcode oligo pair, both 5′ phosphorylated (IDT). .. When annealed (see Reagent Setup) will have sticky ends (bold nucleotides) compatible with XhoI and HindIII digested vector. barcode_up: TCGAG ANNTGNNNACNNNNTGANNNNACNNNATNNNGANN G barcode_down: AGCTC NNTCNNNATNNNGTNNNNTCANNNNGTNNNCANNT C .

    Polymerase Chain Reaction:

    Article Title: Generation of a transgenic ORFeome library in Drosophila
    Article Snippet: • 50 mg/ml Kanamycin (Formedium, cat. no. KAN0025). .. • Gateway BP Clonase II Enzyme mix (Life Technologies, cat. no. 11789-020) • Gateway LR Clonase II Enzyme mix (Life Technologies, cat. no. 11791-020) • pDONR221 (Life Technologies, cat. no. 12536-017) • NEBNext dsDNA Fragmentase (NEB, cat. no. M0348S) • NEBNext End Repair Module (NEB, cat. no. E6050S) • NEBNext dA-Tailing Module (NEB, cat. no. E6053S) • T4 DNA ligase (Fermentas, cat. no. EL0011 ) • Wizard SV 96 Plasmid DNA Purification System (Promega, cat. no. A2250) • Plasmid midi kit (Qiagen, cat. no. 12143) for cleaning up pooled plasmid DNA • ORF-specific primer pairs for first-round PCR amplification (Sigma-Aldrich) (see Reagent Setup) • Barcode oligo pair, both 5′ phosphorylated (IDT). .. When annealed (see Reagent Setup) will have sticky ends (bold nucleotides) compatible with XhoI and HindIII digested vector. barcode_up: TCGAG ANNTGNNNACNNNNTGANNNNACNNNATNNNGANN G barcode_down: AGCTC NNTCNNNATNNNGTNNNNTCANNNNGTNNNCANNT C .

    Article Title: From cheek swabs to consensus sequences: an A to Z protocol for high-throughput DNA sequencing of complete human mitochondrial genomes
    Article Snippet: .. If samples of different length are pooled for library construction then the mass of DNA used for each sample should be adjusted accordingly to ensure coverage levels are the same across all samples (see ‘Fragmentation of PCR products using NEBNext dsDNA fragmentase’ above). ..

    Amplification:

    Article Title: Generation of a transgenic ORFeome library in Drosophila
    Article Snippet: • 50 mg/ml Kanamycin (Formedium, cat. no. KAN0025). .. • Gateway BP Clonase II Enzyme mix (Life Technologies, cat. no. 11789-020) • Gateway LR Clonase II Enzyme mix (Life Technologies, cat. no. 11791-020) • pDONR221 (Life Technologies, cat. no. 12536-017) • NEBNext dsDNA Fragmentase (NEB, cat. no. M0348S) • NEBNext End Repair Module (NEB, cat. no. E6050S) • NEBNext dA-Tailing Module (NEB, cat. no. E6053S) • T4 DNA ligase (Fermentas, cat. no. EL0011 ) • Wizard SV 96 Plasmid DNA Purification System (Promega, cat. no. A2250) • Plasmid midi kit (Qiagen, cat. no. 12143) for cleaning up pooled plasmid DNA • ORF-specific primer pairs for first-round PCR amplification (Sigma-Aldrich) (see Reagent Setup) • Barcode oligo pair, both 5′ phosphorylated (IDT). .. When annealed (see Reagent Setup) will have sticky ends (bold nucleotides) compatible with XhoI and HindIII digested vector. barcode_up: TCGAG ANNTGNNNACNNNNTGANNNNACNNNATNNNGANN G barcode_down: AGCTC NNTCNNNATNNNGTNNNNTCANNNNGTNNNCANNT C .

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    New England Biolabs circular dsdna
    DNA Binding Induces a Conformational Change within SPRTN (A) SPRTN undergoes a conformational change upon DNA binding. Catalytically inactive GST-SPRTN-Strep E112Q was subjected to limited proteolytic digestion by trypsin in the presence or absence of <t>ssDNA</t> or <t>dsDNA.</t> (B) Quantification of specific proteolytic fragments observed in (A). Values represent mean ± SEM of three independent experiments. (C) SAXS analysis indicates that ssDNA binding increases the flexibility of SPRTN. Electron pair distribution shows an increase in Rg and Dmax upon ssDNA (15-mer) binding. (D) Heatmap showing H/D exchange mass spectrometry indicating differences in deuterium incorporation between SPRTN and SPRTN + ssDNA. Regions of increased protection are shown in blue and increased exposure in red. Deuterium labeling was carried out at three time points (0.3, 3, and 30 s) in triplicates. See also Figure S4 .
    Circular Dsdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs psti linearized φx 174 dsdna
    DNA strand exchange activity of hRad51 and hRad51-Δex9. ( A ) Western blot analysis of the purified recombinant hRad51 and hRad51-Δex9 protein using a commercial hRad51 antibody. Lane 1, hRad51; Lane 2, hRad51-Δex9. ( B ) Schematic diagram of DNA strand exchange between circular ssDNA and linear <t>dsDNA</t> of φX 174. ( C ) DNA strand exchange reactions mediated by the purified recombinant hRad51 and hRad51-Δex9 proteins. After incubation with 3.5 μM of either the hRad51 or hRad51-Δex9 protein for a series of time-intervals (15, 30, 60, 120 and 240 min), the DNA was analyzed by 0.8% agarose gel electrophoresis, followed by staining with Syber green. When the hRad51 or hRad51-Δex9 protein was not included in the strand-exchange reactions, no bands corresponding to the forms of joint molecules or nicked circles were detected at 240 min of incubation (the first lane in each panel).
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    New England Biolabs double stranded pbr322 plasmid dna
    Sequence-specific labeling of DNA with an oligodeoxynucleotide (ODN). (a) Modification of the DNA at the sequence 5′-TCGA-3′ using M. Taq I and AdoYnODN11 cofactor. The site was covalently labeled with an ODN containing 11 nucleotides (5′-TTATACATCTA-3′). (b) Distribution of the target sequence (5′-TCGA-3′) sites on <t>pBR322</t> plasmid DNA. (c) Confirmation of the modification using restriction enzymes. The left panel shows the distribution of the sites of the restriction enzymes. The right panel shows the analysis by agarose gel electrophoresis. (d) Linearization of the pBR322 DNA for nanopore measurement. The labeled DNA was linearized with the restriction endonuclease R. Ahd I which cleaves the pBR322 plasmid at a single site.
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    New England Biolabs dsdna fragmentase
    ORF cloning and sequencing strategy (a) Illustration of the two-step PCR for amplification of ORFs using Act5C gene as an example. (b) Anticipated PCR results from ORF cloning. Example of eight different ORFs (1.2-1.5 kb) amplified using the two-step PCR strategy; a 5 μl aliquot of the final PCR product for each ORF was run on a 1.2% agarose gel. Each ORF is visible as single bright band without additional non-specific bands. Note that some genes may produce more than one specific band due to alternative transcripts. (c) Fragmentation of plasmids for high-throughput sequencing. Time-scale of ORF entry clone plasmid pool digestion using <t>dsDNA</t> <t>fragmentase</t> enzyme mixture. In this case, 45 minute digestion yields ideal fragmentation of the plasmids, with the majority of the plasmid pool being fragmented into small molecular weight fragments. (d) Strategy for high-throughput sequencing of ORFs. Individual ORF entry clones are pooled and fragmented followed by high-throughput sequencing library preparation. We prefer to use a “beads-in” protocol where paramagnetic beads used to purify the DNA are kept in the reaction mix to increase the final yield of the library. (e) Illustration of the Illumina sequencing library preparation. Inclusion of barcoded sequencing adapters (optional) during library preparation allows multiplexing of sequencing libraries or association of different plasmid pools with specific plates or wells.
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    Image Search Results


    DNA Binding Induces a Conformational Change within SPRTN (A) SPRTN undergoes a conformational change upon DNA binding. Catalytically inactive GST-SPRTN-Strep E112Q was subjected to limited proteolytic digestion by trypsin in the presence or absence of ssDNA or dsDNA. (B) Quantification of specific proteolytic fragments observed in (A). Values represent mean ± SEM of three independent experiments. (C) SAXS analysis indicates that ssDNA binding increases the flexibility of SPRTN. Electron pair distribution shows an increase in Rg and Dmax upon ssDNA (15-mer) binding. (D) Heatmap showing H/D exchange mass spectrometry indicating differences in deuterium incorporation between SPRTN and SPRTN + ssDNA. Regions of increased protection are shown in blue and increased exposure in red. Deuterium labeling was carried out at three time points (0.3, 3, and 30 s) in triplicates. See also Figure S4 .

    Journal: Molecular Cell

    Article Title: Mechanism and Regulation of DNA-Protein Crosslink Repair by the DNA-Dependent Metalloprotease SPRTN

    doi: 10.1016/j.molcel.2016.09.031

    Figure Lengend Snippet: DNA Binding Induces a Conformational Change within SPRTN (A) SPRTN undergoes a conformational change upon DNA binding. Catalytically inactive GST-SPRTN-Strep E112Q was subjected to limited proteolytic digestion by trypsin in the presence or absence of ssDNA or dsDNA. (B) Quantification of specific proteolytic fragments observed in (A). Values represent mean ± SEM of three independent experiments. (C) SAXS analysis indicates that ssDNA binding increases the flexibility of SPRTN. Electron pair distribution shows an increase in Rg and Dmax upon ssDNA (15-mer) binding. (D) Heatmap showing H/D exchange mass spectrometry indicating differences in deuterium incorporation between SPRTN and SPRTN + ssDNA. Regions of increased protection are shown in blue and increased exposure in red. Deuterium labeling was carried out at three time points (0.3, 3, and 30 s) in triplicates. See also Figure S4 .

    Article Snippet: Either circular ssDNA (ΦX174 virion, New England Biolabs) or circular dsDNA (ΦX174 RF I, New England Biolabs) was used for activation.

    Techniques: Binding Assay, Mass Spectrometry, Labeling

    DNA strand exchange activity of hRad51 and hRad51-Δex9. ( A ) Western blot analysis of the purified recombinant hRad51 and hRad51-Δex9 protein using a commercial hRad51 antibody. Lane 1, hRad51; Lane 2, hRad51-Δex9. ( B ) Schematic diagram of DNA strand exchange between circular ssDNA and linear dsDNA of φX 174. ( C ) DNA strand exchange reactions mediated by the purified recombinant hRad51 and hRad51-Δex9 proteins. After incubation with 3.5 μM of either the hRad51 or hRad51-Δex9 protein for a series of time-intervals (15, 30, 60, 120 and 240 min), the DNA was analyzed by 0.8% agarose gel electrophoresis, followed by staining with Syber green. When the hRad51 or hRad51-Δex9 protein was not included in the strand-exchange reactions, no bands corresponding to the forms of joint molecules or nicked circles were detected at 240 min of incubation (the first lane in each panel).

    Journal: Nucleic Acids Research

    Article Title: Identification of a novel human Rad51 variant that promotes DNA strand exchange

    doi: 10.1093/nar/gkn171

    Figure Lengend Snippet: DNA strand exchange activity of hRad51 and hRad51-Δex9. ( A ) Western blot analysis of the purified recombinant hRad51 and hRad51-Δex9 protein using a commercial hRad51 antibody. Lane 1, hRad51; Lane 2, hRad51-Δex9. ( B ) Schematic diagram of DNA strand exchange between circular ssDNA and linear dsDNA of φX 174. ( C ) DNA strand exchange reactions mediated by the purified recombinant hRad51 and hRad51-Δex9 proteins. After incubation with 3.5 μM of either the hRad51 or hRad51-Δex9 protein for a series of time-intervals (15, 30, 60, 120 and 240 min), the DNA was analyzed by 0.8% agarose gel electrophoresis, followed by staining with Syber green. When the hRad51 or hRad51-Δex9 protein was not included in the strand-exchange reactions, no bands corresponding to the forms of joint molecules or nicked circles were detected at 240 min of incubation (the first lane in each panel).

    Article Snippet: After 5 min of incubation at 37°C, 120 ng (final concentration, 8.4 μM in base pairs) of PstI-linearized φX 174 dsDNA (New England Biolabs) in 1 μl and 1 μl of 100 mM MgCl2 were added to the reaction mixture.

    Techniques: Activity Assay, Western Blot, Purification, Recombinant, Incubation, Agarose Gel Electrophoresis, Staining

    Sequence-specific labeling of DNA with an oligodeoxynucleotide (ODN). (a) Modification of the DNA at the sequence 5′-TCGA-3′ using M. Taq I and AdoYnODN11 cofactor. The site was covalently labeled with an ODN containing 11 nucleotides (5′-TTATACATCTA-3′). (b) Distribution of the target sequence (5′-TCGA-3′) sites on pBR322 plasmid DNA. (c) Confirmation of the modification using restriction enzymes. The left panel shows the distribution of the sites of the restriction enzymes. The right panel shows the analysis by agarose gel electrophoresis. (d) Linearization of the pBR322 DNA for nanopore measurement. The labeled DNA was linearized with the restriction endonuclease R. Ahd I which cleaves the pBR322 plasmid at a single site.

    Journal: ACS Nano

    Article Title: Electrical DNA Sequence Mapping Using Oligodeoxynucleotide Labels and Nanopores

    doi: 10.1021/acsnano.0c07947

    Figure Lengend Snippet: Sequence-specific labeling of DNA with an oligodeoxynucleotide (ODN). (a) Modification of the DNA at the sequence 5′-TCGA-3′ using M. Taq I and AdoYnODN11 cofactor. The site was covalently labeled with an ODN containing 11 nucleotides (5′-TTATACATCTA-3′). (b) Distribution of the target sequence (5′-TCGA-3′) sites on pBR322 plasmid DNA. (c) Confirmation of the modification using restriction enzymes. The left panel shows the distribution of the sites of the restriction enzymes. The right panel shows the analysis by agarose gel electrophoresis. (d) Linearization of the pBR322 DNA for nanopore measurement. The labeled DNA was linearized with the restriction endonuclease R. Ahd I which cleaves the pBR322 plasmid at a single site.

    Article Snippet: Sequence-Specific Labeling with ODN ODN-labeled DNA was prepared by incubating double-stranded pBR322 plasmid DNA (100 ng/μL, New England BioLabs (NEB), Ipswich, MA), ODN-modified AdoMet analogue AdoYnODN11 (10 μM) and M.Taq I (2.43 μM, 10 equiv of M.Taq I with respect to 5′-TCGA-3′ recognition sequences on the plasmid) in NEB buffer 4 (110 μL, 20 mM Tris–HCl, 50 mM KOAc, 10 mM Mg(OAc)2 , 1 mM DTT, pH 7.9) at 65 °C for 1 h. Plasmids were purified using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany) according to the instructions of the manufacturer.

    Techniques: Sequencing, Labeling, Modification, Plasmid Preparation, Agarose Gel Electrophoresis

    ORF cloning and sequencing strategy (a) Illustration of the two-step PCR for amplification of ORFs using Act5C gene as an example. (b) Anticipated PCR results from ORF cloning. Example of eight different ORFs (1.2-1.5 kb) amplified using the two-step PCR strategy; a 5 μl aliquot of the final PCR product for each ORF was run on a 1.2% agarose gel. Each ORF is visible as single bright band without additional non-specific bands. Note that some genes may produce more than one specific band due to alternative transcripts. (c) Fragmentation of plasmids for high-throughput sequencing. Time-scale of ORF entry clone plasmid pool digestion using dsDNA fragmentase enzyme mixture. In this case, 45 minute digestion yields ideal fragmentation of the plasmids, with the majority of the plasmid pool being fragmented into small molecular weight fragments. (d) Strategy for high-throughput sequencing of ORFs. Individual ORF entry clones are pooled and fragmented followed by high-throughput sequencing library preparation. We prefer to use a “beads-in” protocol where paramagnetic beads used to purify the DNA are kept in the reaction mix to increase the final yield of the library. (e) Illustration of the Illumina sequencing library preparation. Inclusion of barcoded sequencing adapters (optional) during library preparation allows multiplexing of sequencing libraries or association of different plasmid pools with specific plates or wells.

    Journal: Nature protocols

    Article Title: Generation of a transgenic ORFeome library in Drosophila

    doi: 10.1038/nprot.2014.105

    Figure Lengend Snippet: ORF cloning and sequencing strategy (a) Illustration of the two-step PCR for amplification of ORFs using Act5C gene as an example. (b) Anticipated PCR results from ORF cloning. Example of eight different ORFs (1.2-1.5 kb) amplified using the two-step PCR strategy; a 5 μl aliquot of the final PCR product for each ORF was run on a 1.2% agarose gel. Each ORF is visible as single bright band without additional non-specific bands. Note that some genes may produce more than one specific band due to alternative transcripts. (c) Fragmentation of plasmids for high-throughput sequencing. Time-scale of ORF entry clone plasmid pool digestion using dsDNA fragmentase enzyme mixture. In this case, 45 minute digestion yields ideal fragmentation of the plasmids, with the majority of the plasmid pool being fragmented into small molecular weight fragments. (d) Strategy for high-throughput sequencing of ORFs. Individual ORF entry clones are pooled and fragmented followed by high-throughput sequencing library preparation. We prefer to use a “beads-in” protocol where paramagnetic beads used to purify the DNA are kept in the reaction mix to increase the final yield of the library. (e) Illustration of the Illumina sequencing library preparation. Inclusion of barcoded sequencing adapters (optional) during library preparation allows multiplexing of sequencing libraries or association of different plasmid pools with specific plates or wells.

    Article Snippet: More recently, we have moved to Illumina MiSeq for full insert sequencing using dsDNA fragmentase (NEB) as a convenient and scalable method to fragment plasmid DNA ( ).

    Techniques: Clone Assay, Sequencing, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Next-Generation Sequencing, Plasmid Preparation, Molecular Weight, Multiplexing