ecorv restriction endonuclease  (New England Biolabs)


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    Name:
    Endonuclease V
    Description:
    Endonuclease V 250 units
    Catalog Number:
    m0305s
    Price:
    76
    Size:
    250 units
    Category:
    Other Endonucleases
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    Structured Review

    New England Biolabs ecorv restriction endonuclease
    Endonuclease V
    Endonuclease V 250 units
    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
    ecorv restriction endonuclease - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV"

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

    Journal: The FEBS journal

    doi: 10.1111/j.1742-4658.2011.08198.x

    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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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,
    Figure Legend 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,

    Techniques Used: Binding Assay, Concentration Assay

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

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: Binding Assay, Concentration Assay

    2) Product Images from "Wild-type p53 binds to MYC promoter G-quadruplex"

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

    Journal: Bioscience Reports

    doi: 10.1042/BSR20160232

    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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Binding Assay, Construct, Incubation

    Related Articles

    Agarose Gel Electrophoresis:

    Article Title: Upregulation of KLHDC4 Predicts a Poor Prognosis in Human Nasopharyngeal Carcinoma
    Article Snippet: .. The mixture of duplexes was treated with T7E1 endonuclease for 20 minutes at 37°C (New England Biolabs); the reaction was stopped using 1.5 μl of 0.25 M EDTA, and the products were analyzed on a 3% agarose gel. .. Establishment of a KLHDC4 knockout cell line CNE2 cells were cultured in 6-well dishes to 70–80% confluence and then cotransfected with 1 μg of KLHDC4 sgRNA#2 plasmid plus 1 μg of pSpCas9(BB)-2A-GFP plasmid and 5 μl of Lipofectamine 2000 per well.

    Methylation:

    Article Title: Transcriptome analysis of an apple (Malus × domestica) yellow fruit somatic mutation identifies a gene network module highly associated with anthocyanin and epigenetic regulation
    Article Snippet: .. One microgram (µg) of genomic DNA (g DNA) isolated from KID and BLO fruit skin samples collected in 2013 and 2014 were digested with methylation-specific endonuclease enzyme McrBC (New England Biolabs), according to the manufacturer’s instructions, with three biological replicates. .. For the negative control, Guanosine-5′-triphosphate (GTP) was replaced by water in the reaction.

    Isolation:

    Article Title: Transcriptome analysis of an apple (Malus × domestica) yellow fruit somatic mutation identifies a gene network module highly associated with anthocyanin and epigenetic regulation
    Article Snippet: .. One microgram (µg) of genomic DNA (g DNA) isolated from KID and BLO fruit skin samples collected in 2013 and 2014 were digested with methylation-specific endonuclease enzyme McrBC (New England Biolabs), according to the manufacturer’s instructions, with three biological replicates. .. For the negative control, Guanosine-5′-triphosphate (GTP) was replaced by water in the reaction.

    Purification:

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV
    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. .. Active protein concentrations of the EcoRV were determined by the direct titration with the specific site 310 bp DNA fragment under conditions of stoichiometric binding as described previously [ , ].

    Polymerase Chain Reaction:

    Article Title: Efficient genome editing in hematopoietic stem cells with helper-dependent Ad5/35 vectors expressing site-specific endonucleases under microRNA regulation
    Article Snippet: .. PCR products were hybridized and treated with 2.5 units of T7E1 endonuclease (NEB, Ipswich, MA). .. Digested PCR products were resolved by 10% Tris-borate EDTA buffer (TBE) polyacrylamide gel electrophoresis (Biorad) and stained with ethidium bromide.

    Article Title: Promoter Methylation Regulates SAMHD1 Gene Expression in Human CD4+ T Cells *
    Article Snippet: .. 2) For HpaII digestion of genomic DNA, followed by SAMHD1 promoter-specific PCR, genomic DNA (1 μg) was digested with 10 units of HpaII endonuclease (New England Biolabs) for 16 h at 37 °C. .. The enzyme was heat-inactivated, and DNA was purified using DNA Clean and ConcentratorTM -5.

    Sonication:

    Article Title: Dependence of antibody gene diversification on uracil excision
    Article Snippet: .. Total cell extracts of vertebrate cells were prepared by sonication and samples (2–20 μg of protein) assayed for their ability to excise uracil from a FITC-labeled oligonucleotide substrate essentially as previously described , except that 0.2 U of apyrimidinic endonuclease (New England Biolabs) was included in each reaction. .. When measuring SMUG1 activity, the endogenous UNG enzyme (if present) was inhibited using 0.5 U of Ugi (New England Biolabs, Inc.) where indicated.

    Binding Assay:

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV
    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. .. Active protein concentrations of the EcoRV were determined by the direct titration with the specific site 310 bp DNA fragment under conditions of stoichiometric binding as described previously [ , ].

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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
    ecorv restriction endonuclease - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

    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
    restriction endonucleases ecorv - by Bioz Stars, 2020-07
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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
    https://www.bioz.com/result/restriction endonucleases eco ri/product/New England Biolabs
    Average 88 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
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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