β nicotinamide adenine dinucleotide  (New England Biolabs)


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    Name:
    beta Nicotinamide adenine dinucleotide NAD
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    beta Nicotinamide adenine dinucleotide NAD 0 2 ml
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    b9007s
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    New England Biolabs β nicotinamide adenine dinucleotide
    beta Nicotinamide adenine dinucleotide NAD
    beta Nicotinamide adenine dinucleotide NAD 0 2 ml
    https://www.bioz.com/result/β nicotinamide adenine dinucleotide/product/New England Biolabs
    Average 80 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    β nicotinamide adenine dinucleotide - by Bioz Stars, 2020-01
    80/100 stars

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    1) Product Images from "Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions"

    Article Title: Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw998

    ATP-hydrolysis by RecA under the conditions for the ligation enhancement. Radioactive [α- 33 P]ATP (1.3 mM) was incubated with the indicated amounts of RecA for 30 min with RecA in the complete system, except for the amounts of DNA and the absence of DNA ligase and NAD. After the reaction, the amounts of hydrolyzed ATP were analyzed. The averages of more than three independent experiments were plotted with standard deviations. The small extent of ATP-hydrolysis observed in the absence of DNA was probably caused by an impurity in the RecA preparations used in these experiments, since the values varied from 0 to a few nmol among RecA preparations. Closed symbols, reactions in the basic reaction mixture (with 3.8% (w/v) PEG), open symbols, omit PEG. •, ∘ (circles), linear dsDNA with 5′ four nucleotide overhangs generated by Hin dIII; ▪, □ (squares), linear dsDNA with 3′ four nucleotide overhangs generated by Pst I; ▴, Δ (triangles), linear dsDNA with blunt ends generated by Hin cII; ♦, ◊ (diamonds), ssDNA; ▿ (reversed triangles), ssDNA without PEG and in the presence of 13 mM MgCl 2 (the standard reaction mixtures for homologous joint formation by RecA: ( 31 )); ▹ (sideways triangles), without DNA (with 1.3 mM MgCl 2 , without PEG).
    Figure Legend Snippet: ATP-hydrolysis by RecA under the conditions for the ligation enhancement. Radioactive [α- 33 P]ATP (1.3 mM) was incubated with the indicated amounts of RecA for 30 min with RecA in the complete system, except for the amounts of DNA and the absence of DNA ligase and NAD. After the reaction, the amounts of hydrolyzed ATP were analyzed. The averages of more than three independent experiments were plotted with standard deviations. The small extent of ATP-hydrolysis observed in the absence of DNA was probably caused by an impurity in the RecA preparations used in these experiments, since the values varied from 0 to a few nmol among RecA preparations. Closed symbols, reactions in the basic reaction mixture (with 3.8% (w/v) PEG), open symbols, omit PEG. •, ∘ (circles), linear dsDNA with 5′ four nucleotide overhangs generated by Hin dIII; ▪, □ (squares), linear dsDNA with 3′ four nucleotide overhangs generated by Pst I; ▴, Δ (triangles), linear dsDNA with blunt ends generated by Hin cII; ♦, ◊ (diamonds), ssDNA; ▿ (reversed triangles), ssDNA without PEG and in the presence of 13 mM MgCl 2 (the standard reaction mixtures for homologous joint formation by RecA: ( 31 )); ▹ (sideways triangles), without DNA (with 1.3 mM MgCl 2 , without PEG).

    Techniques Used: Ligation, Incubation, Generated

    Related Articles

    Electroporation:

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: For bacmid recombineering, the bacmid construct was moved via electroporation into SW106 (generous gift from Don Court, NCI), an E. coli strain which has a temperature-inducible lambda prophage integrated into the chromosome. .. To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O .

    Amplification:

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O . .. The cassette was amplified using 1ul of the isothermal assembly reaction, with primers 15165 and 15169 (0.4 μM) in a PCR reaction with 2× Phusion HF mastermix, (100 ul, 25 cycle, 60 °C melting temperature, 6 minute extension) and purified using the Qiagen Qiaquick PCR purification kit.

    In Vitro:

    Article Title: A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro
    Article Snippet: Paragraph title: In vitro mariner mutagenesis ... Finally 2μl of 1mM NAD+ (β-Nicotinamide adenine dinucleotide, NEB) and 4μl E .coli DNA ligase (NEB) was added, incubated at 16°C overnight and then stored at 4°C until transformation.

    Polymerase Chain Reaction:

    Article Title: A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro
    Article Snippet: The reaction was cleaned using the Wizard SV gel and PCR clean up system (Promega) as per manufacturers guide and heated for 10 minutes at 75°C to inactivate the transposase. .. Finally 2μl of 1mM NAD+ (β-Nicotinamide adenine dinucleotide, NEB) and 4μl E .coli DNA ligase (NEB) was added, incubated at 16°C overnight and then stored at 4°C until transformation.

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O . .. The cassette was amplified using 1ul of the isothermal assembly reaction, with primers 15165 and 15169 (0.4 μM) in a PCR reaction with 2× Phusion HF mastermix, (100 ul, 25 cycle, 60 °C melting temperature, 6 minute extension) and purified using the Qiagen Qiaquick PCR purification kit.

    Mutagenesis:

    Article Title: A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro
    Article Snippet: Paragraph title: In vitro mariner mutagenesis ... Finally 2μl of 1mM NAD+ (β-Nicotinamide adenine dinucleotide, NEB) and 4μl E .coli DNA ligase (NEB) was added, incubated at 16°C overnight and then stored at 4°C until transformation.

    Incubation:

    Article Title: A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro
    Article Snippet: .. Finally 2μl of 1mM NAD+ (β-Nicotinamide adenine dinucleotide, NEB) and 4μl E .coli DNA ligase (NEB) was added, incubated at 16°C overnight and then stored at 4°C until transformation. ..

    Construct:

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: Paragraph title: Preparation of constructs for recombineering ... To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O .

    Purification:

    Article Title: A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro
    Article Snippet: In vitro mariner mutagenesis MarC9 transposase was purified as previously described [ , ]. .. Finally 2μl of 1mM NAD+ (β-Nicotinamide adenine dinucleotide, NEB) and 4μl E .coli DNA ligase (NEB) was added, incubated at 16°C overnight and then stored at 4°C until transformation.

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O . .. The cassette was amplified using 1ul of the isothermal assembly reaction, with primers 15165 and 15169 (0.4 μM) in a PCR reaction with 2× Phusion HF mastermix, (100 ul, 25 cycle, 60 °C melting temperature, 6 minute extension) and purified using the Qiagen Qiaquick PCR purification kit.

    Knock-In:

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: A linear knock-in cassette was constructed with the isothermal assembly of 5 amplicons including two flanks representing ≈300bp of homology to the regions upstream and downstream from the chitinase-cathepsin deletion, the counter-selectable marker cat-SacB, and both FNT subunits generated from the Gateway expression clone mentioned above ( ) . .. To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O .

    Generated:

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: A linear knock-in cassette was constructed with the isothermal assembly of 5 amplicons including two flanks representing ≈300bp of homology to the regions upstream and downstream from the chitinase-cathepsin deletion, the counter-selectable marker cat-SacB, and both FNT subunits generated from the Gateway expression clone mentioned above ( ) . .. To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O .

    other:

    Article Title: Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions
    Article Snippet: Basic reaction mixture The basic reaction mixture contained 30 mM Tris-HCl (pH 7.5), 7 mM MgCl2 , 3.8% (w/v) polyethylene glycol 6000 (PEG), 0.1 mM nicotinamide adenine dinucleotide (NAD), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1.8 mM dithiothreitol (DTT) and 88 μg/ml bovine serum albumin ((BSA); New England Biolabs and Roche, molecular biology grade).

    Introduce:

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: To introduce the genes into the bacmid, we utilized a region of the bacmid which formerly contained the genes for chitinase and cathepsin; this region was previously shown to tolerate insertions . .. To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O .

    Expressing:

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: A linear knock-in cassette was constructed with the isothermal assembly of 5 amplicons including two flanks representing ≈300bp of homology to the regions upstream and downstream from the chitinase-cathepsin deletion, the counter-selectable marker cat-SacB, and both FNT subunits generated from the Gateway expression clone mentioned above ( ) . .. To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O .

    Marker:

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: A linear knock-in cassette was constructed with the isothermal assembly of 5 amplicons including two flanks representing ≈300bp of homology to the regions upstream and downstream from the chitinase-cathepsin deletion, the counter-selectable marker cat-SacB, and both FNT subunits generated from the Gateway expression clone mentioned above ( ) . .. To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O .

    Transformation Assay:

    Article Title: A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro
    Article Snippet: .. Finally 2μl of 1mM NAD+ (β-Nicotinamide adenine dinucleotide, NEB) and 4μl E .coli DNA ligase (NEB) was added, incubated at 16°C overnight and then stored at 4°C until transformation. ..

    Crocin Bleaching Assay:

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: .. To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O . .. The cassette was amplified using 1ul of the isothermal assembly reaction, with primers 15165 and 15169 (0.4 μM) in a PCR reaction with 2× Phusion HF mastermix, (100 ul, 25 cycle, 60 °C melting temperature, 6 minute extension) and purified using the Qiagen Qiaquick PCR purification kit.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: A linear knock-in cassette was constructed with the isothermal assembly of 5 amplicons including two flanks representing ≈300bp of homology to the regions upstream and downstream from the chitinase-cathepsin deletion, the counter-selectable marker cat-SacB, and both FNT subunits generated from the Gateway expression clone mentioned above ( ) . .. To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O .

    Plasmid Preparation:

    Article Title: A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro
    Article Snippet: The reaction mixture consisted of 1μg of stkP PCR product, 10μl 2x transposition buffer (10% glycerol, 2mM DTT, 25mM Hepes pH 7.9, 250μg/ml BSA, 100mM NaCl, 10mM MgCl2 ), 1μg pR412 plasmid DNA, 0.5μl transposase enzyme and made to 20μl with PCR grade water. .. Finally 2μl of 1mM NAD+ (β-Nicotinamide adenine dinucleotide, NEB) and 4μl E .coli DNA ligase (NEB) was added, incubated at 16°C overnight and then stored at 4°C until transformation.

    Article Title: Farnesylated and methylated KRAS4b: high yield production of protein suitable for biophysical studies of prenylated protein-lipid interactions
    Article Snippet: To make the linear cassette, 25 fmoles of each fragment were combined in a 20 ul isothermal assembly reaction with 2× CBA isothermal assembly mastermix (320 μl of 5 × ISO Reaction Buffer [25% polyethylene glycol 8000, 500 mM tris-HCl (pH 8.0), 50 mM MgCl2 , 50 mM dithiothreitol (DTT), 1.0 μM each dNTPs, 5 μM nicotinamide adenine dinucleotide, 6.4 μl of T5 Exonuclease (1 U/ml) (Epicentre), 20 μl of Phusion polymerase (2 U/ml) [New England Biolabs (NEB)], 80 μl of Taq ligase (40 U/ml) (NEB), and 374 μl of deionized H2 O . .. The cat-SacB cassette was amplified from the plasmid template pELO4 (generous gift from Don Court, NCI) in a PCR reaction with 2× Phusion HF mastermix, 0.9 ng template plasmid DNA, 0.4 mM MgCl2 , and 0.4 μM primers 15173 and 15174 (100 μl, 25 cycle, 62 °C melting temperature, 2 minute extension).

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    New England Biolabs β nicotinamide adenine dinucleotide
    ATP-hydrolysis by RecA under the conditions for the ligation enhancement. Radioactive [α- 33 P]ATP (1.3 mM) was incubated with the indicated amounts of RecA for 30 min with RecA in the complete system, except for the amounts of DNA and the absence of DNA ligase and <t>NAD.</t> After the reaction, the amounts of hydrolyzed ATP were analyzed. The averages of more than three independent experiments were plotted with standard deviations. The small extent of ATP-hydrolysis observed in the absence of DNA was probably caused by an impurity in the RecA preparations used in these experiments, since the values varied from 0 to a few nmol among RecA preparations. Closed symbols, reactions in the basic reaction mixture (with 3.8% (w/v) <t>PEG),</t> open symbols, omit PEG. •, ∘ (circles), linear dsDNA with 5′ four nucleotide overhangs generated by Hin dIII; ▪, □ (squares), linear dsDNA with 3′ four nucleotide overhangs generated by Pst I; ▴, Δ (triangles), linear dsDNA with blunt ends generated by Hin cII; ♦, ◊ (diamonds), ssDNA; ▿ (reversed triangles), ssDNA without PEG and in the presence of 13 mM MgCl 2 (the standard reaction mixtures for homologous joint formation by RecA: ( 31 )); ▹ (sideways triangles), without DNA (with 1.3 mM MgCl 2 , without PEG).
    β Nicotinamide Adenine Dinucleotide, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/β nicotinamide adenine dinucleotide/product/New England Biolabs
    Average 80 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    β nicotinamide adenine dinucleotide - by Bioz Stars, 2020-01
    80/100 stars
      Buy from Supplier

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    ATP-hydrolysis by RecA under the conditions for the ligation enhancement. Radioactive [α- 33 P]ATP (1.3 mM) was incubated with the indicated amounts of RecA for 30 min with RecA in the complete system, except for the amounts of DNA and the absence of DNA ligase and NAD. After the reaction, the amounts of hydrolyzed ATP were analyzed. The averages of more than three independent experiments were plotted with standard deviations. The small extent of ATP-hydrolysis observed in the absence of DNA was probably caused by an impurity in the RecA preparations used in these experiments, since the values varied from 0 to a few nmol among RecA preparations. Closed symbols, reactions in the basic reaction mixture (with 3.8% (w/v) PEG), open symbols, omit PEG. •, ∘ (circles), linear dsDNA with 5′ four nucleotide overhangs generated by Hin dIII; ▪, □ (squares), linear dsDNA with 3′ four nucleotide overhangs generated by Pst I; ▴, Δ (triangles), linear dsDNA with blunt ends generated by Hin cII; ♦, ◊ (diamonds), ssDNA; ▿ (reversed triangles), ssDNA without PEG and in the presence of 13 mM MgCl 2 (the standard reaction mixtures for homologous joint formation by RecA: ( 31 )); ▹ (sideways triangles), without DNA (with 1.3 mM MgCl 2 , without PEG).

    Journal: Nucleic Acids Research

    Article Title: Rad51 and RecA juxtapose dsDNA ends ready for DNA ligase-catalyzed end-joining under recombinase-suppressive conditions

    doi: 10.1093/nar/gkw998

    Figure Lengend Snippet: ATP-hydrolysis by RecA under the conditions for the ligation enhancement. Radioactive [α- 33 P]ATP (1.3 mM) was incubated with the indicated amounts of RecA for 30 min with RecA in the complete system, except for the amounts of DNA and the absence of DNA ligase and NAD. After the reaction, the amounts of hydrolyzed ATP were analyzed. The averages of more than three independent experiments were plotted with standard deviations. The small extent of ATP-hydrolysis observed in the absence of DNA was probably caused by an impurity in the RecA preparations used in these experiments, since the values varied from 0 to a few nmol among RecA preparations. Closed symbols, reactions in the basic reaction mixture (with 3.8% (w/v) PEG), open symbols, omit PEG. •, ∘ (circles), linear dsDNA with 5′ four nucleotide overhangs generated by Hin dIII; ▪, □ (squares), linear dsDNA with 3′ four nucleotide overhangs generated by Pst I; ▴, Δ (triangles), linear dsDNA with blunt ends generated by Hin cII; ♦, ◊ (diamonds), ssDNA; ▿ (reversed triangles), ssDNA without PEG and in the presence of 13 mM MgCl 2 (the standard reaction mixtures for homologous joint formation by RecA: ( 31 )); ▹ (sideways triangles), without DNA (with 1.3 mM MgCl 2 , without PEG).

    Article Snippet: Basic reaction mixture The basic reaction mixture contained 30 mM Tris-HCl (pH 7.5), 7 mM MgCl2 , 3.8% (w/v) polyethylene glycol 6000 (PEG), 0.1 mM nicotinamide adenine dinucleotide (NAD), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1.8 mM dithiothreitol (DTT) and 88 μg/ml bovine serum albumin ((BSA); New England Biolabs and Roche, molecular biology grade).

    Techniques: Ligation, Incubation, Generated