restriction endonucleases ecorv  (New England Biolabs)


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    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: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Comparative analysis of the end-joining activity of several DNA ligases"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0190062

    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

    2) 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

    Agarose Gel Electrophoresis:

    Article Title: Characterization of human adenovirus 35 and derivation of complex vectors
    Article Snippet: Relative levels of viral genomes were determined following the isolation of total DNA from the mixed input viral particles of a 200 μl aliquot of lysate using the High Pure Nucleic Acid Kit from Roche Applied Science (catalog # 11858874001). .. To distinguish the E3 wild type and E3 deletion X and wild type from deletion HE, the viral genomes were restricted with EcoRV and BlpI endonucleases (New England BioLabs), respectively, before being resolved on a 0.8% agarose gel using 0.5% TBE buffer. .. Virion temperature lability Wild type Ad35, an rAd35 with the E1 deletion d2 and GFP expression cassette, or an rAd35 with the E1 deletion d5 and HIV gp140BΔCFIΔV1V2 expression cassette were diluted in triplicate to 8 × 1010 particles/ml in 10 mM Tris pH7.5, 150 mM NaCl to a final volume of 800 ul in 1.5 ml microcentrifuge tubes on ice, then shifted to 48°C in a water bath.

    other:

    Article Title: The abundance of Fob1 modulates the efficiency of rRFBs to stall replication forks
    Article Snippet: DNA treatments DNA was digested with the restriction endonucleases BamHI, EcoRV, SwaI, FspI and MluI (New England Biolabs) for at least 2 h at 37°C except for SwaI that was digested at 25°C.

    BAC Assay:

    Article Title: Intracellular Trafficking of the Human Cytomegalovirus-Encoded 7-trans-Membrane Protein Homologs pUS27 and pUL78 during Viral Infection: A Comparative Analysis
    Article Snippet: .. Resulting BAC clones, termed TB40/E-US27-EYFP (US27-EYFP), TB40/E-Flag-US27 (Flag-US27), or TB40/E-UL78-EYFP (UL78-EYFP), were verified by distinct PCR reactions and subsequent nucleotide sequence analyses, as well as restriction fragment length polymorphism analysis (RFLP, using restriction endonuclease EcoRV-HF, NEB). .. To generate double-tagged TB40/E-UL78-EYFP/Flag-US27 (UL78-EYFP/Flag-US27), the parental BAC TB40/E-Flag-US27 (Flag-US27) was used to transform E.coli GS1783.

    Clone Assay:

    Article Title: Intracellular Trafficking of the Human Cytomegalovirus-Encoded 7-trans-Membrane Protein Homologs pUS27 and pUL78 during Viral Infection: A Comparative Analysis
    Article Snippet: .. Resulting BAC clones, termed TB40/E-US27-EYFP (US27-EYFP), TB40/E-Flag-US27 (Flag-US27), or TB40/E-UL78-EYFP (UL78-EYFP), were verified by distinct PCR reactions and subsequent nucleotide sequence analyses, as well as restriction fragment length polymorphism analysis (RFLP, using restriction endonuclease EcoRV-HF, NEB). .. To generate double-tagged TB40/E-UL78-EYFP/Flag-US27 (UL78-EYFP/Flag-US27), the parental BAC TB40/E-Flag-US27 (Flag-US27) was used to transform E.coli GS1783.

    Polymerase Chain Reaction:

    Article Title: Intracellular Trafficking of the Human Cytomegalovirus-Encoded 7-trans-Membrane Protein Homologs pUS27 and pUL78 during Viral Infection: A Comparative Analysis
    Article Snippet: .. Resulting BAC clones, termed TB40/E-US27-EYFP (US27-EYFP), TB40/E-Flag-US27 (Flag-US27), or TB40/E-UL78-EYFP (UL78-EYFP), were verified by distinct PCR reactions and subsequent nucleotide sequence analyses, as well as restriction fragment length polymorphism analysis (RFLP, using restriction endonuclease EcoRV-HF, NEB). .. To generate double-tagged TB40/E-UL78-EYFP/Flag-US27 (UL78-EYFP/Flag-US27), the parental BAC TB40/E-Flag-US27 (Flag-US27) was used to transform E.coli GS1783.

    Sequencing:

    Article Title: Intracellular Trafficking of the Human Cytomegalovirus-Encoded 7-trans-Membrane Protein Homologs pUS27 and pUL78 during Viral Infection: A Comparative Analysis
    Article Snippet: .. Resulting BAC clones, termed TB40/E-US27-EYFP (US27-EYFP), TB40/E-Flag-US27 (Flag-US27), or TB40/E-UL78-EYFP (UL78-EYFP), were verified by distinct PCR reactions and subsequent nucleotide sequence analyses, as well as restriction fragment length polymorphism analysis (RFLP, using restriction endonuclease EcoRV-HF, NEB). .. To generate double-tagged TB40/E-UL78-EYFP/Flag-US27 (UL78-EYFP/Flag-US27), the parental BAC TB40/E-Flag-US27 (Flag-US27) was used to transform E.coli GS1783.

    Ligation:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Through these studies, the relationship between tight nonspecific DNA-binding and end-joining activity was explored, and insight gained into how catalytic domains and DNA-binding domains are involved in DNA ligase in vitro substrate specificity. .. 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). .. Tris-HCl (1 M pH 7.5 @ 25°C) was obtained from Amresco (Solon, OH).

    Incubation:

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA
    Article Snippet: .. For topology analysis, 1-μg aliquots of mitochondrial nucleic acid were incubated with the following enzymes, in 30 μl of manufacturer-supplied reaction buffer at 37 °C except where stated, and conditions as follows: topoisomerase I (New England Biolabs), 2 units, 30 min; gyrase (Topogen), 2 units, 60 min; restriction endonucleases MbiI, XhoI, EcoRV, NdeI, and Bsp1407I (Thermo Fisher Scientific), 4 units, 4 h; RNase H (Thermo Fisher Scientific), 0.5 unit, 60 min; S1 nuclease (Thermo Fisher Scientific), 2 units, 2 min at room temperature; RusA as described previously ( ); and exonuclease I (Thermo Fisher Scientific), 10 units, 60 min. .. Reactions were deproteinized by phenol-chloroform extraction, and 2 μl of the aqueous phase was analyzed by 0.5% agarose gel electrophoresis (30-cm gels run for 40 h at 1.7 V/cm in Tris/Borate/EDTA).

    Isolation:

    Article Title: Molecular Epidemiology, Sequence Types, and Plasmid Analyses of KPC-Producing Klebsiella pneumoniae Strains in Israel ▿
    Article Snippet: Plasmid DNA was purified using a NucleoBond PC 100 plasmid midi-kit (Macherey-Nagel GmbH, Duren, Germany). .. Plasmids isolated from clinical isolates of K. pneumoniae and E. coli strains and their transformants were digested with different restriction endonucleases, such as BglII, SmaI, and EcoRV (New England Biolabs, Boston, MA), and their restriction patterns were compared. .. Plasmids were transformed by electroporation into the E. coli GeneHogs strain.

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    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: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/restriction endonucleases ecorv/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    New England Biolabs ecorv restriction endonuclease
    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 <t>pBSK/EcoRV.</t> 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.
    Ecorv Restriction Endonuclease, 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
    https://www.bioz.com/result/ecorv restriction endonuclease/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    New England Biolabs ecorv hf restriction endonuclease
    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 <t>pBSK/EcoRV.</t> 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.
    Ecorv Hf Restriction Endonuclease, 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
    https://www.bioz.com/result/ecorv hf restriction endonuclease/product/New England Biolabs
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    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

    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

    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