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    New England Biolabs new england biolabs buffer 2
    New England Biolabs Buffer 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 192 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs new england biolabs buffer 2 1
    Determination of divalent cation cofactor requirement and DNA substrate preference for GmrSD digestion. (A). Metal ion cofactor requirement for GmrSD digestion. Divalent cations or EDTA are indicated on top of each lane. (B). Substrate preference and optimal substrate size for GmrSD digestion. PCR DNA substrates containing 5hmC or regular dC were generated by PCR using pBR322 template and digested by GmrSD endonuclease in the presence of 1 mM ATP in <t>NEB</t> buffer 2. The same DNA substrates were also digested by HpaII (CCGG) in NEB buffer 4 (to confirm modified DNA).
    New England Biolabs Buffer 2 1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 39 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs r0137l ne buffer2
    Determination of divalent cation cofactor requirement and DNA substrate preference for GmrSD digestion. (A). Metal ion cofactor requirement for GmrSD digestion. Divalent cations or EDTA are indicated on top of each lane. (B). Substrate preference and optimal substrate size for GmrSD digestion. PCR DNA substrates containing 5hmC or regular dC were generated by PCR using pBR322 template and digested by GmrSD endonuclease in the presence of 1 mM ATP in <t>NEB</t> buffer 2. The same DNA substrates were also digested by HpaII (CCGG) in NEB buffer 4 (to confirm modified DNA).
    R0137l Ne Buffer2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs new england biolabs buffer 3
    Determination of divalent cation cofactor requirement and DNA substrate preference for GmrSD digestion. (A). Metal ion cofactor requirement for GmrSD digestion. Divalent cations or EDTA are indicated on top of each lane. (B). Substrate preference and optimal substrate size for GmrSD digestion. PCR DNA substrates containing 5hmC or regular dC were generated by PCR using pBR322 template and digested by GmrSD endonuclease in the presence of 1 mM ATP in <t>NEB</t> buffer 2. The same DNA substrates were also digested by HpaII (CCGG) in NEB buffer 4 (to confirm modified DNA).
    New England Biolabs Buffer 3, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs reaction buffer 2
    Determination of divalent cation cofactor requirement and DNA substrate preference for GmrSD digestion. (A). Metal ion cofactor requirement for GmrSD digestion. Divalent cations or EDTA are indicated on top of each lane. (B). Substrate preference and optimal substrate size for GmrSD digestion. PCR DNA substrates containing 5hmC or regular dC were generated by PCR using pBR322 template and digested by GmrSD endonuclease in the presence of 1 mM ATP in <t>NEB</t> buffer 2. The same DNA substrates were also digested by HpaII (CCGG) in NEB buffer 4 (to confirm modified DNA).
    Reaction Buffer 2, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 287 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs reaction buffer
    Determination of divalent cation cofactor requirement and DNA substrate preference for GmrSD digestion. (A). Metal ion cofactor requirement for GmrSD digestion. Divalent cations or EDTA are indicated on top of each lane. (B). Substrate preference and optimal substrate size for GmrSD digestion. PCR DNA substrates containing 5hmC or regular dC were generated by PCR using pBR322 template and digested by GmrSD endonuclease in the presence of 1 mM ATP in <t>NEB</t> buffer 2. The same DNA substrates were also digested by HpaII (CCGG) in NEB buffer 4 (to confirm modified DNA).
    Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2709 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs digestion mixture
    Determination of divalent cation cofactor requirement and DNA substrate preference for GmrSD digestion. (A). Metal ion cofactor requirement for GmrSD digestion. Divalent cations or EDTA are indicated on top of each lane. (B). Substrate preference and optimal substrate size for GmrSD digestion. PCR DNA substrates containing 5hmC or regular dC were generated by PCR using pBR322 template and digested by GmrSD endonuclease in the presence of 1 mM ATP in <t>NEB</t> buffer 2. The same DNA substrates were also digested by HpaII (CCGG) in NEB buffer 4 (to confirm modified DNA).
    Digestion Mixture, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Determination of divalent cation cofactor requirement and DNA substrate preference for GmrSD digestion. (A). Metal ion cofactor requirement for GmrSD digestion. Divalent cations or EDTA are indicated on top of each lane. (B). Substrate preference and optimal substrate size for GmrSD digestion. PCR DNA substrates containing 5hmC or regular dC were generated by PCR using pBR322 template and digested by GmrSD endonuclease in the presence of 1 mM ATP in NEB buffer 2. The same DNA substrates were also digested by HpaII (CCGG) in NEB buffer 4 (to confirm modified DNA).

    Journal: Scientific Reports

    Article Title: Expression and purification of a single-chain Type IV restriction enzyme Eco94GmrSD and determination of its substrate preference

    doi: 10.1038/srep09747

    Figure Lengend Snippet: Determination of divalent cation cofactor requirement and DNA substrate preference for GmrSD digestion. (A). Metal ion cofactor requirement for GmrSD digestion. Divalent cations or EDTA are indicated on top of each lane. (B). Substrate preference and optimal substrate size for GmrSD digestion. PCR DNA substrates containing 5hmC or regular dC were generated by PCR using pBR322 template and digested by GmrSD endonuclease in the presence of 1 mM ATP in NEB buffer 2. The same DNA substrates were also digested by HpaII (CCGG) in NEB buffer 4 (to confirm modified DNA).

    Article Snippet: GmrSD enzyme activity assay T4 (glc-5hmC), T4gt (5hmC), and λ DNA (Dam+ Dcm+ ) or 5hmC-modified PCR DNA were digested with purified GmrSD enzyme in NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTT) supplemented with 1 mM ATP at 37°C for 1 h unless specified otherwise.

    Techniques: Polymerase Chain Reaction, Generated, Modification

    TagI activity assays on (A) 5hm C and 5m C containing PCR products and (B,C) modified phage DNA in vitro and in vivo . ( A ) One μg of mixed PCR DNA (∼12 nM) made from modified dNTP mixtures was digested at 37°C for 1 h with 1 μg TagI (∼0.3 μM) in 2-fold serial dilutions in NEB buffer 2.1. The substrates for TagI digestion in 10 mM Mg 2+ contained C (3 kb) and 5hm C or 5m C (2.2 kb). The assay in 1 mM Mn 2+ was performed on C (3 kb), 5hm C (2.2 kb) and 5m C (1.2 kb) containing PCR DNA. The amount of TagI (μg) shown on top of each lane corresponds to 295, 147, 74, 37, and 18 nM of protein dimer, respectively. ( B ) Modified DNA from phage T4gt ( 5hm C, ∼0.2 nM), T4 ( g5hm C, ∼0.2 nM) or XP12 ( 5m C, 0.5 nM) was digested by TagI (∼0.3 μM) and control enzymes: tolerant to the presence of modified cytosines (MluCI (/AATT), 10 U), inhibited by cytosine modifications (HpaII (C/CGG), 10 U) and affected only by the presence of g5hm C (MspJI, 5U). ( C ) Late-log phase host cells were plated on soft agar to form a cell lawn, and diluted phages (Lambda, T4gt or T4) were spotted onto the cell lawns. Cell lysis and plaque formation indicated susceptibility to phage infection. No plaque formation indicated the restriction of T4gt phage by TagI expressing cells.

    Journal: Nucleic Acids Research

    Article Title: Crystal structure of the modification-dependent SRA-HNH endonuclease TagI

    doi: 10.1093/nar/gky781

    Figure Lengend Snippet: TagI activity assays on (A) 5hm C and 5m C containing PCR products and (B,C) modified phage DNA in vitro and in vivo . ( A ) One μg of mixed PCR DNA (∼12 nM) made from modified dNTP mixtures was digested at 37°C for 1 h with 1 μg TagI (∼0.3 μM) in 2-fold serial dilutions in NEB buffer 2.1. The substrates for TagI digestion in 10 mM Mg 2+ contained C (3 kb) and 5hm C or 5m C (2.2 kb). The assay in 1 mM Mn 2+ was performed on C (3 kb), 5hm C (2.2 kb) and 5m C (1.2 kb) containing PCR DNA. The amount of TagI (μg) shown on top of each lane corresponds to 295, 147, 74, 37, and 18 nM of protein dimer, respectively. ( B ) Modified DNA from phage T4gt ( 5hm C, ∼0.2 nM), T4 ( g5hm C, ∼0.2 nM) or XP12 ( 5m C, 0.5 nM) was digested by TagI (∼0.3 μM) and control enzymes: tolerant to the presence of modified cytosines (MluCI (/AATT), 10 U), inhibited by cytosine modifications (HpaII (C/CGG), 10 U) and affected only by the presence of g5hm C (MspJI, 5U). ( C ) Late-log phase host cells were plated on soft agar to form a cell lawn, and diluted phages (Lambda, T4gt or T4) were spotted onto the cell lawns. Cell lysis and plaque formation indicated susceptibility to phage infection. No plaque formation indicated the restriction of T4gt phage by TagI expressing cells.

    Article Snippet: Plasmid based assays TagI restriction activity was first assessed by digestion of 0.5–1 μg of pBR322 (Dcm+ or Dcm− ), pBRFM+ (Dcm+ ) or pACYC-HpyCH4IVM+ plasmid in NEB buffer 2.1 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9) at 37°C for 1 h. After the reaction Protease K (1.6 U) was added at 37°C for 15 min.

    Techniques: Activity Assay, Polymerase Chain Reaction, Modification, In Vitro, In Vivo, Lysis, Infection, Expressing

    Efficacy of DNA TagI cleavage of substrates containing none to four 5m C bases. 20 ng (∼32 nM) of annealed 48-mer oligonucleotides were digested by TagI (0.125 μg, ∼92 nM of TagI dimer) in NEB buffer 2.1 for 30 min. Cleavage products were resolved in 15% urea-PAGE, stained by SYBR Gold and imaged on Typhoon imager. The predicted TagI cleavage site GCS/GC at the center of the duplexes conforms to the GC/NGC consensus for cleavage by Fnu4HI (20 U), which was used as a positive control. 5m Cs in oligoduplexes are represented by small black dots in the diagrams above each lane.

    Journal: Nucleic Acids Research

    Article Title: Crystal structure of the modification-dependent SRA-HNH endonuclease TagI

    doi: 10.1093/nar/gky781

    Figure Lengend Snippet: Efficacy of DNA TagI cleavage of substrates containing none to four 5m C bases. 20 ng (∼32 nM) of annealed 48-mer oligonucleotides were digested by TagI (0.125 μg, ∼92 nM of TagI dimer) in NEB buffer 2.1 for 30 min. Cleavage products were resolved in 15% urea-PAGE, stained by SYBR Gold and imaged on Typhoon imager. The predicted TagI cleavage site GCS/GC at the center of the duplexes conforms to the GC/NGC consensus for cleavage by Fnu4HI (20 U), which was used as a positive control. 5m Cs in oligoduplexes are represented by small black dots in the diagrams above each lane.

    Article Snippet: Plasmid based assays TagI restriction activity was first assessed by digestion of 0.5–1 μg of pBR322 (Dcm+ or Dcm− ), pBRFM+ (Dcm+ ) or pACYC-HpyCH4IVM+ plasmid in NEB buffer 2.1 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9) at 37°C for 1 h. After the reaction Protease K (1.6 U) was added at 37°C for 15 min.

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, Positive Control