restriction endonucleases ecorv  (New England Biolabs)


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    Name:
    EcoRV
    Description:
    EcoRV 20 000 units
    Catalog Number:
    r0195l
    Price:
    249
    Size:
    20 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs restriction endonucleases ecorv
    EcoRV
    EcoRV 20 000 units
    https://www.bioz.com/result/restriction endonucleases ecorv/product/New England Biolabs
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    restriction endonucleases ecorv - by Bioz Stars, 2020-07
    99/100 stars

    Images

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

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

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

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Figure Legend Snippet: Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Techniques Used: Ligation, Agarose Gel Electrophoresis, Staining

    Related Articles

    Mutagenesis:

    Article Title: Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase
    Article Snippet: .. Therefore, to further confirm the mutation of hepP in PA14ΔhepP , we performed restriction enzyme digestion on the PCR products with EcoR V (New England Biolabs). ..

    Isolation:

    Article Title: Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases
    Article Snippet: .. Isolated C. perfringens DNA samples, prepared as previously described ( , ), were digested with EcoRV or HpaI (New England Biolabs), separated by electrophoresis on 1% agarose gels, and Southern transferred. .. The blots were hybridized with the DIG-labeled cpb2 probe, which was then detected using a DIG chemiluminescence detection system utilizing CSPD [disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5-chloro)tricyclo[3,3.1.13.7]decane}-4-yl)phenyl phosphate] ready-to-use substrate (Roche) as described earlier ( ).

    Purification:

    Article Title: A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting
    Article Snippet: .. The pGRFP plasmid was cut with EcoRV (NEB), 5′ dephosphorylated with CIAP, and gel purified. .. The linearized vector was blunt-end ligated to the SU66E20 BAC library inserts using T4 DNA ligase.

    Electrophoresis:

    Article Title: Integrative and Sequence Characteristics of a Novel Genetic Element, ICE6013, in Staphylococcus aureus
    Article Snippet: .. Genomic DNA was digested with EcoRV (New England Biolabs), and restriction fragments were separated by electrophoresis in 0.5% agarose. .. The fragments were blotted onto nylon membranes (Bio-Rad) by capillary transfer.

    Article Title: Regulated Expression of the Beta2-Toxin Gene (cpb2) in Clostridium perfringens Type A Isolates from Horses with Gastrointestinal Diseases
    Article Snippet: .. Isolated C. perfringens DNA samples, prepared as previously described ( , ), were digested with EcoRV or HpaI (New England Biolabs), separated by electrophoresis on 1% agarose gels, and Southern transferred. .. The blots were hybridized with the DIG-labeled cpb2 probe, which was then detected using a DIG chemiluminescence detection system utilizing CSPD [disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-(5-chloro)tricyclo[3,3.1.13.7]decane}-4-yl)phenyl phosphate] ready-to-use substrate (Roche) as described earlier ( ).

    Transgenic Assay:

    Article Title: Overexpression of Arabidopsis thaliana gibberellic acid 20 oxidase (AtGA20ox) gene enhance the vegetative growth and fiber quality in kenaf (Hibiscus cannabinus L.) plants
    Article Snippet: .. DNA from putative transgenic and UT plants was digested with EcoR V (NEB, UK). ..

    Molecular Weight:

    Article Title: Structural diversity of supercoiled DNA
    Article Snippet: .. BbvCI, EcoRV, Nb.BbvCI, NdeI, Nuclease Bal-31, T4 DNA Ligase, low molecular weight DNA ladder and 100 bp DNA ladder were purchased from New England Biolabs (Ipswich, MA). .. Proteinase K was purchased from Roche Molecular Biochemicals (Mannheim, Germany).

    Polymerase Chain Reaction:

    Article Title: Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase
    Article Snippet: .. Therefore, to further confirm the mutation of hepP in PA14ΔhepP , we performed restriction enzyme digestion on the PCR products with EcoR V (New England Biolabs). ..

    Plasmid Preparation:

    Article Title: A dual-fluorescence reporter system for high-throughput clone characterization and selection by cell sorting
    Article Snippet: .. The pGRFP plasmid was cut with EcoRV (NEB), 5′ dephosphorylated with CIAP, and gel purified. .. The linearized vector was blunt-end ligated to the SU66E20 BAC library inserts using T4 DNA ligase.

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    New England Biolabs ecorv restriction endonuclease
    pH dependence of the <t>EcoRV</t> specific-nonspecific free binding energy difference. The pH dependence of ln(K nsp-sp ) is shown for the range 5.5 to 8.0. Mixtures of EcoRV, the 310 bp specific site <t>DNA</t> fragment, and the nonspecific oligonucleotide competitor
    Ecorv Restriction Endonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ecorv restriction endonuclease/product/New England Biolabs
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs restriction endonucleases ecorv
    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by <t>EcoRV</t> (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM <t>ATP</t> and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Restriction Endonucleases Ecorv, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction endonucleases ecorv/product/New England Biolabs
    Average 99 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
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    88
    New England Biolabs restriction endonucleases eco ri
    Wild type DNA ligase λ DNA digest ligation assay. Agarose gel electrophoresis of λ DNA cut by <t>EcoRV</t> (A/T Blunt, 1 ), NruI (G/C Blunt, 2 ), BstNI (5′ SBO, 3 ), Hpy188I (3′SBO, 4 ), NdeI (2 BO, 5 ) and BamHI (4 BO, 6 ), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in the presence of T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM <t>ATP</t> and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2, 1 mM DTT, 1 mM ATP, 6% polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with T4 DNA ligase (A), T3 DNA ligase (B), PBCV1 DNA ligase (C) and, hLig3 (D), respectively. E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.
    Restriction Endonucleases Eco Ri, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    pH dependence of the EcoRV specific-nonspecific free binding energy difference. The pH dependence of ln(K nsp-sp ) is shown for the range 5.5 to 8.0. Mixtures of EcoRV, the 310 bp specific site DNA fragment, and the nonspecific oligonucleotide competitor

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: pH dependence of the EcoRV specific-nonspecific free binding energy difference. The pH dependence of ln(K nsp-sp ) is shown for the range 5.5 to 8.0. Mixtures of EcoRV, the 310 bp specific site DNA fragment, and the nonspecific oligonucleotide competitor

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay

    Kinetics of the EcoRV-DNA complex formation. The kinetics of DNA-protein complex formation was measured using the self-cleavage assay at different conditions of pH: pH 6.3 (▲); pH 7.6 (■). The binding of the EcoRV proceeds in at least

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: Kinetics of the EcoRV-DNA complex formation. The kinetics of DNA-protein complex formation was measured using the self-cleavage assay at different conditions of pH: pH 6.3 (▲); pH 7.6 (■). The binding of the EcoRV proceeds in at least

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Cleavage Assay, Binding Assay

    A direct comparison of EcoRV-DNA binding analyzed by the gel mobility shift assay and by the self-cleavage assay. (A ) A gel image is shown illustrating a direct comparison of the EcoRV-DNA binding by the gel mobility shift assay (left), and by the self-cleavage

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: A direct comparison of EcoRV-DNA binding analyzed by the gel mobility shift assay and by the self-cleavage assay. (A ) A gel image is shown illustrating a direct comparison of the EcoRV-DNA binding by the gel mobility shift assay (left), and by the self-cleavage

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay, Mobility Shift, Cleavage Assay

    Equilibrium competition between specific and nonspecific DNA sequences for the EcoRV binding. Mixtures of EcoRV, the 310 bp DNA fragment with a specific recognition site, and nonspecific oligonucleotide competitor were incubated at 20 °C overnight

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: Equilibrium competition between specific and nonspecific DNA sequences for the EcoRV binding. Mixtures of EcoRV, the 310 bp DNA fragment with a specific recognition site, and nonspecific oligonucleotide competitor were incubated at 20 °C overnight

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay, Incubation

    The dependence of the EcoRV specific-nonspecific binding free energy difference, ln(K nsp-sp ) in the units of kT, on solute osmolal concentration is shown for four neutral osmolytes. Mixtures of the specific site DNA fragment, nonspecific oligonucleotide,

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: The dependence of the EcoRV specific-nonspecific binding free energy difference, ln(K nsp-sp ) in the units of kT, on solute osmolal concentration is shown for four neutral osmolytes. Mixtures of the specific site DNA fragment, nonspecific oligonucleotide,

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay, Concentration Assay

    The pH dependence of Knsp-sp for EcoRV-DNA binding

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: The pH dependence of Knsp-sp for EcoRV-DNA binding

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay

    The dependence of the EcoRV specific-nonspecific binding free energy difference on triethylene glycol concentration is shown for different pH values. Mixtures of EcoRV, the 310 bp specific site DNA fragment, and the nonspecific oligonucleotide competitor

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: The dependence of the EcoRV specific-nonspecific binding free energy difference on triethylene glycol concentration is shown for different pH values. Mixtures of EcoRV, the 310 bp specific site DNA fragment, and the nonspecific oligonucleotide competitor

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay, Concentration Assay

    Full-length wild-type p53 binds to MYC G-quadruplex more efficiently than its isolated C-terminal and central regions The role of p53 domains in MYC G-quadruplex binding was studied by EMSA. Oligonucleotides (1 pmol) Pu52 ( A ), Pu33 ( B ) and Pu22 ( C ) were incubated with wtp53 (lanes 2–5; 50, 100, 200, 400 ng), p53-320 (lanes 6–9; 25, 50, 100, 200 ng) or p53CD (lanes 10–13; 12.5, 25, 50, 100 ng) in the presence of 10 ng of non-specific competitor pBSK/EcoRV. Binding of full-length wtp53, central DBD and C-terminal construct of p53 to double-stranded oligonucleotides containing p53CON was studied by EMSA. Oligonucleotides (0.25 pmol) ( D ) P1-50, ( E ) P1-30 and ( F ) P1-22 were incubated with wtp53 (lanes 2–5; 12.5, 25, 50, 100 ng), p53-320 (lanes 6–9; 6, 12.5, 25, 50 ng) or p53CD (lanes 10–13; 3, 6, 12.5, 25 ng) in the presence of 2.5 ng of non-specific competitor pBSK/EcoRV.

    Journal: Bioscience Reports

    Article Title: Wild-type p53 binds to MYC promoter G-quadruplex

    doi: 10.1042/BSR20160232

    Figure Lengend Snippet: Full-length wild-type p53 binds to MYC G-quadruplex more efficiently than its isolated C-terminal and central regions The role of p53 domains in MYC G-quadruplex binding was studied by EMSA. Oligonucleotides (1 pmol) Pu52 ( A ), Pu33 ( B ) and Pu22 ( C ) were incubated with wtp53 (lanes 2–5; 50, 100, 200, 400 ng), p53-320 (lanes 6–9; 25, 50, 100, 200 ng) or p53CD (lanes 10–13; 12.5, 25, 50, 100 ng) in the presence of 10 ng of non-specific competitor pBSK/EcoRV. Binding of full-length wtp53, central DBD and C-terminal construct of p53 to double-stranded oligonucleotides containing p53CON was studied by EMSA. Oligonucleotides (0.25 pmol) ( D ) P1-50, ( E ) P1-30 and ( F ) P1-22 were incubated with wtp53 (lanes 2–5; 12.5, 25, 50, 100 ng), p53-320 (lanes 6–9; 6, 12.5, 25, 50 ng) or p53CD (lanes 10–13; 3, 6, 12.5, 25 ng) in the presence of 2.5 ng of non-specific competitor pBSK/EcoRV.

    Article Snippet: Non-specific competitor plasmid pBSK/EcoRV was prepared by EcoRV restriction endonuclease (New England Biolabs) cleavage of pBSK.

    Techniques: Isolation, Binding Assay, Incubation, Construct

    Full-length wild-type p53 binding to the parallel G-quadruplex from MYC promoter NHE III 1 region is comparable with p53CON binding ( A ) Comparison of sequence-specific and MYC G-quadruplex Pu52 binding of p53 by EMSA. Oligonucelotides P1-50 (0.25 pmol, lanes 1–5), Pu52 (1 pmol, lanes 6–10) and A50 (0.25 pmol, lanes 11–15) were incubated with wtp53 protein (50, 100, 200, 400 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV. ( B ) CD spectra of Pu52 oligonucleotide measured in 5 mM Tris pH 7.6 (dotted line) and after addition of 10 mM KCl (dashed line) and 50 mM KCl respectively (solid line). ( C ) DMS footprinting of Pu52 oligonucleotide. Pu52 was annealed in 50 mM KCl without subsequent DMS treatment (lane 1), annealed in the absence of KCl and treated with DMS (lane 2) or annealed in 50 mM KCl and treated with DMS (lane 3). ( D ) Comparison of sequence-specific and MYC G-quadruplex Pu22 binding of p53 by EMSA. Oligonucelotides P1-22 (0.25 pmol, lanes 1–5), Pu22 (1 pmol, lanes 6–10) and A25 (0.25 pmol, lanes 11–15) were incubated with wtp53 protein (50, 100, 200, 400 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV. ( E ) CD spectra of Pu22 oligonucleotide measured in 5 mM Tris pH 7.6 (dotted line) and after addition of 10 mM KCl (dashed line) and 50 mM KCl respectively (solid line). ( F ) DMS footprinting of Pu22 oligonucleotide. Pu22 was annealed in 50 mM KCl without subsequent DMS treatment (lane 1), annealed in the absence of KCl and treated with DMS (lane 2) or annealed in 50 mM KCl and treated with DMS (lane 3).

    Journal: Bioscience Reports

    Article Title: Wild-type p53 binds to MYC promoter G-quadruplex

    doi: 10.1042/BSR20160232

    Figure Lengend Snippet: Full-length wild-type p53 binding to the parallel G-quadruplex from MYC promoter NHE III 1 region is comparable with p53CON binding ( A ) Comparison of sequence-specific and MYC G-quadruplex Pu52 binding of p53 by EMSA. Oligonucelotides P1-50 (0.25 pmol, lanes 1–5), Pu52 (1 pmol, lanes 6–10) and A50 (0.25 pmol, lanes 11–15) were incubated with wtp53 protein (50, 100, 200, 400 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV. ( B ) CD spectra of Pu52 oligonucleotide measured in 5 mM Tris pH 7.6 (dotted line) and after addition of 10 mM KCl (dashed line) and 50 mM KCl respectively (solid line). ( C ) DMS footprinting of Pu52 oligonucleotide. Pu52 was annealed in 50 mM KCl without subsequent DMS treatment (lane 1), annealed in the absence of KCl and treated with DMS (lane 2) or annealed in 50 mM KCl and treated with DMS (lane 3). ( D ) Comparison of sequence-specific and MYC G-quadruplex Pu22 binding of p53 by EMSA. Oligonucelotides P1-22 (0.25 pmol, lanes 1–5), Pu22 (1 pmol, lanes 6–10) and A25 (0.25 pmol, lanes 11–15) were incubated with wtp53 protein (50, 100, 200, 400 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV. ( E ) CD spectra of Pu22 oligonucleotide measured in 5 mM Tris pH 7.6 (dotted line) and after addition of 10 mM KCl (dashed line) and 50 mM KCl respectively (solid line). ( F ) DMS footprinting of Pu22 oligonucleotide. Pu22 was annealed in 50 mM KCl without subsequent DMS treatment (lane 1), annealed in the absence of KCl and treated with DMS (lane 2) or annealed in 50 mM KCl and treated with DMS (lane 3).

    Article Snippet: Non-specific competitor plasmid pBSK/EcoRV was prepared by EcoRV restriction endonuclease (New England Biolabs) cleavage of pBSK.

    Techniques: Binding Assay, Sequencing, Incubation, Footprinting

    Wild-type p53 and C-terminal region of p53 bind G-quadruplex Pu52 with higher affinity than double-stranded Pu52/Py52 Binding of various p53 protein constructs to MYC promoter G-quadruplexes from was studied by EMSA. Oligonucleotides ( A ) P1-40 (0.5 pmol), ( B ) Pu52 (1 pmol) and ( C ) dsPu52/Py52 (0.5 pmol) were incubated with wtp53 (lanes 2–4; 25, 50, 100 ng/1 pmol of oligonucleotide), CΔ30 (lanes 5–7; 25, 50, 100 ng/1 pmol of oligonucleotide), p53CT (lanes 8–10; 50, 100, 200 ng/1 pmol of oligonucleotide), p53T (lanes 11–13; 50, 100, 200 ng/1 pmol of oligonucleotide) or p53CD (lanes 14 and 15; 100, 200 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV.

    Journal: Bioscience Reports

    Article Title: Wild-type p53 binds to MYC promoter G-quadruplex

    doi: 10.1042/BSR20160232

    Figure Lengend Snippet: Wild-type p53 and C-terminal region of p53 bind G-quadruplex Pu52 with higher affinity than double-stranded Pu52/Py52 Binding of various p53 protein constructs to MYC promoter G-quadruplexes from was studied by EMSA. Oligonucleotides ( A ) P1-40 (0.5 pmol), ( B ) Pu52 (1 pmol) and ( C ) dsPu52/Py52 (0.5 pmol) were incubated with wtp53 (lanes 2–4; 25, 50, 100 ng/1 pmol of oligonucleotide), CΔ30 (lanes 5–7; 25, 50, 100 ng/1 pmol of oligonucleotide), p53CT (lanes 8–10; 50, 100, 200 ng/1 pmol of oligonucleotide), p53T (lanes 11–13; 50, 100, 200 ng/1 pmol of oligonucleotide) or p53CD (lanes 14 and 15; 100, 200 ng/1 pmol of oligonucleotide) in the presence of 20 ng (per 1 pmol of oligonucleotide) of non-specific competitor pBSK/EcoRV.

    Article Snippet: Non-specific competitor plasmid pBSK/EcoRV was prepared by EcoRV restriction endonuclease (New England Biolabs) cleavage of pBSK.

    Techniques: Binding Assay, Construct, Incubation

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0190062

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

    Article Snippet: General materials T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) as a 10x stock, NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) as a 5x stock, restriction endonucleases EcoRV, NruI, BstNI, Hpy188I, NdeI, BamHI, as well as λ DNA, 1 M DTT, 6x Purple Loading Dye with SDS, Proteinase K and 10 mM ATP were obtained from New England Biolabs (NEB, Ipswich, MA).

    Techniques: Ligation, Agarose Gel Electrophoresis, Staining

    Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0190062

    Figure Lengend Snippet: Effect of DBDs on blunt/cohesive end λ DNA Re-ligation. Agarose gel electrophoresis of λ DNA cut by EcoRV (A/T Blunt, 1), NruI (G/C Blunt, 2), BstNI (5′ SBO, 3), Hpy188I (3′SBO, 4), NdeI (2 BO, 5) and BamHI (4 BO, 6), generating DNA fragments with ligatable ends. 0.5 ng of the cut DNA was ligated in T4 ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl 2 ) or NEBNext ® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl 2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) and 7 μM of the indicated DNA ligase for 1 hour at 25°C. Ligation assays performed with PBCV1-Nterm-Sso7d (A), PBCV1-Cterm-Sso7d terminus (B), PBCV1-Nterm-ZnF (C), PBCV1-Nterm-T4NTD (D). (E) Gel of restriction enzyme digested λ DNA samples as well as a schematic depiction of each substrate. The DNA fragments were visualized using ethidium bromide stain.

    Article Snippet: General materials T4 DNA ligase reaction buffer (50 mM Tris-HCl pH 7.5 @ 25°C, 1 mM ATP and 10 mM MgCl2 ) as a 10x stock, NEBNext® Quick Ligation reaction buffer (66 mM Tris pH 7.6 @ 25°C, 10 mM MgCl2 , 1 mM DTT, 1 mM ATP, 6% Polyethylene glycol (PEG 6000)) as a 5x stock, restriction endonucleases EcoRV, NruI, BstNI, Hpy188I, NdeI, BamHI, as well as λ DNA, 1 M DTT, 6x Purple Loading Dye with SDS, Proteinase K and 10 mM ATP were obtained from New England Biolabs (NEB, Ipswich, MA).

    Techniques: Ligation, Agarose Gel Electrophoresis, Staining