buffer 4 New England Biolabs Search Results


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  • 95
    New England Biolabs new england biolabs streptavidin beads
    New England Biolabs Streptavidin Beads, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs new england biolabs buffer 4
    New England Biolabs Buffer 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 103 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs 1x new england biolabs
    1x New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs new england buffer 4 neb4
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    New England Buffer 4 Neb4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs 1x new england digestion buffer 4
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    1x New England Digestion Buffer 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rnl2 new england biolabs buffer
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    Rnl2 New England Biolabs Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs new england biolabs ligation buffer
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    New England Biolabs Ligation Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs new england biolabs nebuffer 3
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    New England Biolabs Nebuffer 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs new england biolabs nebuffer 1
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    New England Biolabs Nebuffer 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs new england biolabs ligase buffer
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    New England Biolabs Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 polynucleotide kinase pnk buffer buffer new england biolabs
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    T4 Polynucleotide Kinase Pnk Buffer Buffer New England Biolabs, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebuffer 4
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    Nebuffer 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 529 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs cutsmart buffer 4
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    Cutsmart Buffer 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 114 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs alui
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    Alui, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 882 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nlaiii restriction enzyme
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    Nlaiii Restriction Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 106 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs crimson taq buffer
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    Crimson Taq Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 58 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs phusion hf buffer
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    Phusion Hf Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 846 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs q5 reaction buffer
    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in <t>NEB</t> <t>buffer</t> 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.
    Q5 Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 404 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs buffer 4
    Strand selection by wt FokI. ( A ) The reactions, in <t>Buffer</t> 4 at 20°C, contained 5 nM wt FokI and 1 nM immobilized BIO-42, 32 P-labelled in either top or bottom strand. At various times after adding the enzyme, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown: left, top-strand label; right, bottom-strand label. Time ranges are indicated above each gel and the electrophoretic mobilities of the intact (S t or S b ) and cleaved strands (P 16 or P 12 ) marked on the left. ( B ) The amounts of the intact and the cleaved forms of the labelled strands were measured and the amounts of intact DNA are shown as a fraction of the total; top strand, black circles; bottom strand, white circles. ( C ) The reaction was identical to that in (B) except that it also contained 100 nM N13Y-D450A. In both (B) and (C), error bars denote standard deviations from ≥3 independent repeats and the lines drawn through each data set are best fits to single exponentials: top strand, solid line; bottom strand, dashed line. The best fits were obtained with: in (B), wt FokI, 0.4 min −1 and 0.3 min −1 for top and bottom strands, respectively; in (C), wt FokI and N13Y-D450A, 0.1 min −1 and 0.5 min −1 for top and bottom strands, respectively.
    Buffer 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 332 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs thermopol reaction buffer
    The temperatures of <t>ThermoPol</t> Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    Thermopol Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 927 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nebnext fragmentation buffer
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    Nebnext Fragmentation Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 82/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs gc buffer
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    Gc Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 830 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs onetaq hot start buffer
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    Onetaq Hot Start Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs restriction enzyme buffer 4
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    Restriction Enzyme Buffer 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 83/100, based on 38 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs 10x neb buffer 4
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    10x Neb Buffer 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 76/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs reaction buffer 4
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    Reaction Buffer 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 113 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ecori
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    Ecori, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 8899 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs restriction buffer 4
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    Restriction Buffer 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 kinase buffer
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    New England Biolabs 1x buffer 4
    The temperatures of <t>ThermoPol</t> Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
    1x Buffer 4, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 77/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs 1×neb buffer 4
    The temperatures of <t>ThermoPol</t> Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.
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    Image Search Results


    BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in NEB buffer 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.

    Journal: Journal of Bacteriology

    Article Title: Characterization of Type II and III Restriction-Modification Systems from Bacillus cereus Strains ATCC 10987 and ATCC 14579

    doi: 10.1128/JB.06248-11

    Figure Lengend Snippet: BceSI gene organization and characterization of BceSI restriction enzyme. (A) Gene organization of the BceSI R-M system. Mod, modification subunit; Res, restriction subunit. (B) SDS-PAGE analysis of BceSI after two-column purification (nickel agarose and Hi-Trap heparin Sepharose). (C) BceSI digestion of phage λ DNA. One μg of λ DNA was digested by various amounts of BceSI endonuclease (0.5 μg/μl) in NEB buffer 4 at 37°C for 1 h. (D) BceSI digestion pattern generated by NEB cutter. The real digestion and hypothetical digestion patterns do not completely match, presumably due to BceSI requiring two sites in a head-to-head or tail-to-tail orientation for efficient cleavage. unmeth., unmethylated; CpG, CpG dinucleotide.

    Article Snippet: DNA cleavage assays by BceSIV or Bce14579I were carried out using 1× NEB buffer 4 supplemented with ATP or GTP at 37°C for 1 h. One μg of DNA and various amounts of the indicated crude lysate or purified protein was used.

    Techniques: Modification, SDS Page, Purification, Generated

    Strand selection by wt FokI. ( A ) The reactions, in Buffer 4 at 20°C, contained 5 nM wt FokI and 1 nM immobilized BIO-42, 32 P-labelled in either top or bottom strand. At various times after adding the enzyme, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown: left, top-strand label; right, bottom-strand label. Time ranges are indicated above each gel and the electrophoretic mobilities of the intact (S t or S b ) and cleaved strands (P 16 or P 12 ) marked on the left. ( B ) The amounts of the intact and the cleaved forms of the labelled strands were measured and the amounts of intact DNA are shown as a fraction of the total; top strand, black circles; bottom strand, white circles. ( C ) The reaction was identical to that in (B) except that it also contained 100 nM N13Y-D450A. In both (B) and (C), error bars denote standard deviations from ≥3 independent repeats and the lines drawn through each data set are best fits to single exponentials: top strand, solid line; bottom strand, dashed line. The best fits were obtained with: in (B), wt FokI, 0.4 min −1 and 0.3 min −1 for top and bottom strands, respectively; in (C), wt FokI and N13Y-D450A, 0.1 min −1 and 0.5 min −1 for top and bottom strands, respectively.

    Journal: Nucleic Acids Research

    Article Title: Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

    doi: 10.1093/nar/gkp046

    Figure Lengend Snippet: Strand selection by wt FokI. ( A ) The reactions, in Buffer 4 at 20°C, contained 5 nM wt FokI and 1 nM immobilized BIO-42, 32 P-labelled in either top or bottom strand. At various times after adding the enzyme, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown: left, top-strand label; right, bottom-strand label. Time ranges are indicated above each gel and the electrophoretic mobilities of the intact (S t or S b ) and cleaved strands (P 16 or P 12 ) marked on the left. ( B ) The amounts of the intact and the cleaved forms of the labelled strands were measured and the amounts of intact DNA are shown as a fraction of the total; top strand, black circles; bottom strand, white circles. ( C ) The reaction was identical to that in (B) except that it also contained 100 nM N13Y-D450A. In both (B) and (C), error bars denote standard deviations from ≥3 independent repeats and the lines drawn through each data set are best fits to single exponentials: top strand, solid line; bottom strand, dashed line. The best fits were obtained with: in (B), wt FokI, 0.4 min −1 and 0.3 min −1 for top and bottom strands, respectively; in (C), wt FokI and N13Y-D450A, 0.1 min −1 and 0.5 min −1 for top and bottom strands, respectively.

    Article Snippet: After 5 min at 25°C, the beads were washed three times in 300 µl SSC and then resuspended in Buffer 4 (NEB).

    Techniques: Selection, Polyacrylamide Gel Electrophoresis

    Reaction rates. The reactions, in Buffer 4 at 37°C, contained 5 nM 3 H-labelled DNA and FokI protein at either 1 nM or 10 nM, as indicated below. At timed intervals after adding the protein(s) to the DNA, samples were removed, quenched and analysed as described in Materials and methods section to obtain the concentrations of the SC, OC and LIN DNA. The residual concentration of SC DNA at each time point is given as a percentage of the total DNA in that sample. ( A ) Reactions of 1 nM wt FokI on: pSKFokI (one recognition site), black circles; pIF190 (two FokI sites), white circles. ( B ) Reactions with a mixture of N13Y and D450A, both at 1 nM, on pSKFokI (black circles) and on pIF190 (white circles). Also shown in (B) are the reactions with the mix of N13Y and D450A, both at 10 nM, on pSKFokI (black squares) and on pIF190 (white squares), both with dashed lines.

    Journal: Nucleic Acids Research

    Article Title: Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

    doi: 10.1093/nar/gkp046

    Figure Lengend Snippet: Reaction rates. The reactions, in Buffer 4 at 37°C, contained 5 nM 3 H-labelled DNA and FokI protein at either 1 nM or 10 nM, as indicated below. At timed intervals after adding the protein(s) to the DNA, samples were removed, quenched and analysed as described in Materials and methods section to obtain the concentrations of the SC, OC and LIN DNA. The residual concentration of SC DNA at each time point is given as a percentage of the total DNA in that sample. ( A ) Reactions of 1 nM wt FokI on: pSKFokI (one recognition site), black circles; pIF190 (two FokI sites), white circles. ( B ) Reactions with a mixture of N13Y and D450A, both at 1 nM, on pSKFokI (black circles) and on pIF190 (white circles). Also shown in (B) are the reactions with the mix of N13Y and D450A, both at 10 nM, on pSKFokI (black squares) and on pIF190 (white squares), both with dashed lines.

    Article Snippet: After 5 min at 25°C, the beads were washed three times in 300 µl SSC and then resuspended in Buffer 4 (NEB).

    Techniques: Concentration Assay

    Strand-specific nicking. ( A ) A mixture of the N13Y and D450A mutants of the FokI endonuclease was added to immobilized BIO-42 to give a reaction with 10 nM N13Y, 10 nM D450A and 1 nM DNA in Buffer 4 at 20°C. The BIO-42 was 32 P-labelled in either the top (left-hand gel) or the bottom strand (right-hand). At various times after adding the mixture, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown. The electrophoretic mobilities of the intact strands (S t and S b , from top- and bottom-strand labelled BIO-42, respectively) are noted on the left of the gels and the lanes marked Wt show the products from reactions of wt FokI on the same DNA species (P 16 from the top strand, P 12 from the bottom). ( B ) The fraction of the total amount of radiolabel in each lane still present as the intact DNA were measured and these values plotted as a function of reaction time: top strand, black circles; bottom strand, white circles. Error bars denote standard deviations from ≥3 independent repeats. The line drawn through the data from the top strand (solid line) is the best fit to a single exponential, which gave a rate constant of 0.3 min −1 . Data points for the bottom strand are connected by a dashed line.

    Journal: Nucleic Acids Research

    Article Title: Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

    doi: 10.1093/nar/gkp046

    Figure Lengend Snippet: Strand-specific nicking. ( A ) A mixture of the N13Y and D450A mutants of the FokI endonuclease was added to immobilized BIO-42 to give a reaction with 10 nM N13Y, 10 nM D450A and 1 nM DNA in Buffer 4 at 20°C. The BIO-42 was 32 P-labelled in either the top (left-hand gel) or the bottom strand (right-hand). At various times after adding the mixture, samples were removed from the reactions, quenched and subjected to denaturing PAGE. Phosphorimager records of the gels are shown. The electrophoretic mobilities of the intact strands (S t and S b , from top- and bottom-strand labelled BIO-42, respectively) are noted on the left of the gels and the lanes marked Wt show the products from reactions of wt FokI on the same DNA species (P 16 from the top strand, P 12 from the bottom). ( B ) The fraction of the total amount of radiolabel in each lane still present as the intact DNA were measured and these values plotted as a function of reaction time: top strand, black circles; bottom strand, white circles. Error bars denote standard deviations from ≥3 independent repeats. The line drawn through the data from the top strand (solid line) is the best fit to a single exponential, which gave a rate constant of 0.3 min −1 . Data points for the bottom strand are connected by a dashed line.

    Article Snippet: After 5 min at 25°C, the beads were washed three times in 300 µl SSC and then resuspended in Buffer 4 (NEB).

    Techniques: Polyacrylamide Gel Electrophoresis

    Specificity of nicking. The reactions, in Buffer 4, contained 5 nM SC pSKFokI (a plasmid with one recognition site for FokI) and FokI protein as indicated below. Reactions were stopped after 1 h at 37°C and the samples analysed by electrophoresis through agarose. The symbols SC, OC and LIN on the right of each gel mark the electrophoretic mobilities of the intact SC DNA, the nicked OC form cut in one strand and the LIN form cut in both strands at one site. The lanes marked M contain 1 kb electrophoresis markers (NEB), and the lanes marked Wt are from equivalent 1 h reactions of 10 nM wt FokI on pSKFokI. ( A ) Left-hand gel: the reactions contained D450A at the concentrations indicated above each lane (0 → 200 nM). In the right-hand gel, the reactions with 10 → 200 nM D450A also contained 10 nM N13Y. ( B ) As (A) except that the protein whose concentration was varied was N13Y and that, in the right-hand gel, the samples with varied N13Y also contained 10 nM D450A.

    Journal: Nucleic Acids Research

    Article Title: Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands

    doi: 10.1093/nar/gkp046

    Figure Lengend Snippet: Specificity of nicking. The reactions, in Buffer 4, contained 5 nM SC pSKFokI (a plasmid with one recognition site for FokI) and FokI protein as indicated below. Reactions were stopped after 1 h at 37°C and the samples analysed by electrophoresis through agarose. The symbols SC, OC and LIN on the right of each gel mark the electrophoretic mobilities of the intact SC DNA, the nicked OC form cut in one strand and the LIN form cut in both strands at one site. The lanes marked M contain 1 kb electrophoresis markers (NEB), and the lanes marked Wt are from equivalent 1 h reactions of 10 nM wt FokI on pSKFokI. ( A ) Left-hand gel: the reactions contained D450A at the concentrations indicated above each lane (0 → 200 nM). In the right-hand gel, the reactions with 10 → 200 nM D450A also contained 10 nM N13Y. ( B ) As (A) except that the protein whose concentration was varied was N13Y and that, in the right-hand gel, the samples with varied N13Y also contained 10 nM D450A.

    Article Snippet: After 5 min at 25°C, the beads were washed three times in 300 µl SSC and then resuspended in Buffer 4 (NEB).

    Techniques: Plasmid Preparation, Electrophoresis, Concentration Assay

    The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Journal: PLoS ONE

    Article Title: Controlled Microwave Heating Accelerates Rolling Circle Amplification

    doi: 10.1371/journal.pone.0136532

    Figure Lengend Snippet: The temperatures of ThermoPol Buffer individual components at (a) 1-fold (b) and 4-fold higher concentrations heated from 13°C to 60°C by microwave heating.

    Article Snippet: The pH of fictitious RCA mixtures of one 4-fold constituent of Thermopol-buffer were measured (4-fold Tris–HCl: 8.73, 4-fold KCl; 8.69, 4-fold (NH4)2 SO4 : 8.37, 4-fold MgSO4 : 8.67).

    Techniques: