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  • 95
    New England Biolabs n dna ligase
    Determinants of Thermococcus Okazaki fragment maturation. Panel I , a schematic of the Okazaki fragment maturation experiment is shown. The processing substrate was prepared by annealing a 5′-TAM-labeled extension primer (shaded black ) and 3′-FAM-labeled blocking oligonucleotide (shaded blue ) to ssM13 as described in the text. <t>9°N</t> polB, polD, PCNA/RFC, Fen1, and <t>DNA</t> ligase were incubated with the processing substrate at 60 °C for 15 min. Processed products (103 nt) result from complete Okazaki fragment maturation. B , Okazaki fragment maturation assays were performed without any proteins added, all proteins added (9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase), or in reactions that omit one protein as noted in the figure. Reaction products were analyzed by capillary electrophoresis. 5′-TAM-labeled extension primer is shaded black and 3′-FAM-labeled blocking oligonucleotide is shaded blue . Processed products (103 nt) result from a complete Okazaki fragment maturation assay and are labeled with both 5′-TAM ( black ) and 3′-FAM ( blue ). C, processed products from reactions omitting various replication proteins in panel B were quantitated and plotted. The data shown are averages with standard deviations from three independent experiments.
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    New England Biolabs t4 dna ligase
    Determinants of Thermococcus Okazaki fragment maturation. Panel I , a schematic of the Okazaki fragment maturation experiment is shown. The processing substrate was prepared by annealing a 5′-TAM-labeled extension primer (shaded black ) and 3′-FAM-labeled blocking oligonucleotide (shaded blue ) to ssM13 as described in the text. <t>9°N</t> polB, polD, PCNA/RFC, Fen1, and <t>DNA</t> ligase were incubated with the processing substrate at 60 °C for 15 min. Processed products (103 nt) result from complete Okazaki fragment maturation. B , Okazaki fragment maturation assays were performed without any proteins added, all proteins added (9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase), or in reactions that omit one protein as noted in the figure. Reaction products were analyzed by capillary electrophoresis. 5′-TAM-labeled extension primer is shaded black and 3′-FAM-labeled blocking oligonucleotide is shaded blue . Processed products (103 nt) result from a complete Okazaki fragment maturation assay and are labeled with both 5′-TAM ( black ) and 3′-FAM ( blue ). C, processed products from reactions omitting various replication proteins in panel B were quantitated and plotted. The data shown are averages with standard deviations from three independent experiments.
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    New England Biolabs dna polymerases
    Determinants of Thermococcus Okazaki fragment maturation. Panel I , a schematic of the Okazaki fragment maturation experiment is shown. The processing substrate was prepared by annealing a 5′-TAM-labeled extension primer (shaded black ) and 3′-FAM-labeled blocking oligonucleotide (shaded blue ) to ssM13 as described in the text. <t>9°N</t> polB, polD, PCNA/RFC, Fen1, and <t>DNA</t> ligase were incubated with the processing substrate at 60 °C for 15 min. Processed products (103 nt) result from complete Okazaki fragment maturation. B , Okazaki fragment maturation assays were performed without any proteins added, all proteins added (9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase), or in reactions that omit one protein as noted in the figure. Reaction products were analyzed by capillary electrophoresis. 5′-TAM-labeled extension primer is shaded black and 3′-FAM-labeled blocking oligonucleotide is shaded blue . Processed products (103 nt) result from a complete Okazaki fragment maturation assay and are labeled with both 5′-TAM ( black ) and 3′-FAM ( blue ). C, processed products from reactions omitting various replication proteins in panel B were quantitated and plotted. The data shown are averages with standard deviations from three independent experiments.
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    New England Biolabs 1x e coli dna ligase reaction buffer
    Determinants of Thermococcus Okazaki fragment maturation. Panel I , a schematic of the Okazaki fragment maturation experiment is shown. The processing substrate was prepared by annealing a 5′-TAM-labeled extension primer (shaded black ) and 3′-FAM-labeled blocking oligonucleotide (shaded blue ) to ssM13 as described in the text. <t>9°N</t> polB, polD, PCNA/RFC, Fen1, and <t>DNA</t> ligase were incubated with the processing substrate at 60 °C for 15 min. Processed products (103 nt) result from complete Okazaki fragment maturation. B , Okazaki fragment maturation assays were performed without any proteins added, all proteins added (9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase), or in reactions that omit one protein as noted in the figure. Reaction products were analyzed by capillary electrophoresis. 5′-TAM-labeled extension primer is shaded black and 3′-FAM-labeled blocking oligonucleotide is shaded blue . Processed products (103 nt) result from a complete Okazaki fragment maturation assay and are labeled with both 5′-TAM ( black ) and 3′-FAM ( blue ). C, processed products from reactions omitting various replication proteins in panel B were quantitated and plotted. The data shown are averages with standard deviations from three independent experiments.
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    New England Biolabs restriction enzymes ligase agarose gels competent e coli cells dh5α
    Determinants of Thermococcus Okazaki fragment maturation. Panel I , a schematic of the Okazaki fragment maturation experiment is shown. The processing substrate was prepared by annealing a 5′-TAM-labeled extension primer (shaded black ) and 3′-FAM-labeled blocking oligonucleotide (shaded blue ) to ssM13 as described in the text. <t>9°N</t> polB, polD, PCNA/RFC, Fen1, and <t>DNA</t> ligase were incubated with the processing substrate at 60 °C for 15 min. Processed products (103 nt) result from complete Okazaki fragment maturation. B , Okazaki fragment maturation assays were performed without any proteins added, all proteins added (9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase), or in reactions that omit one protein as noted in the figure. Reaction products were analyzed by capillary electrophoresis. 5′-TAM-labeled extension primer is shaded black and 3′-FAM-labeled blocking oligonucleotide is shaded blue . Processed products (103 nt) result from a complete Okazaki fragment maturation assay and are labeled with both 5′-TAM ( black ) and 3′-FAM ( blue ). C, processed products from reactions omitting various replication proteins in panel B were quantitated and plotted. The data shown are averages with standard deviations from three independent experiments.
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    Image Search Results


    Determinants of Thermococcus Okazaki fragment maturation. Panel I , a schematic of the Okazaki fragment maturation experiment is shown. The processing substrate was prepared by annealing a 5′-TAM-labeled extension primer (shaded black ) and 3′-FAM-labeled blocking oligonucleotide (shaded blue ) to ssM13 as described in the text. 9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase were incubated with the processing substrate at 60 °C for 15 min. Processed products (103 nt) result from complete Okazaki fragment maturation. B , Okazaki fragment maturation assays were performed without any proteins added, all proteins added (9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase), or in reactions that omit one protein as noted in the figure. Reaction products were analyzed by capillary electrophoresis. 5′-TAM-labeled extension primer is shaded black and 3′-FAM-labeled blocking oligonucleotide is shaded blue . Processed products (103 nt) result from a complete Okazaki fragment maturation assay and are labeled with both 5′-TAM ( black ) and 3′-FAM ( blue ). C, processed products from reactions omitting various replication proteins in panel B were quantitated and plotted. The data shown are averages with standard deviations from three independent experiments.

    Journal: The Journal of Biological Chemistry

    Article Title: The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *

    doi: 10.1074/jbc.M115.638130

    Figure Lengend Snippet: Determinants of Thermococcus Okazaki fragment maturation. Panel I , a schematic of the Okazaki fragment maturation experiment is shown. The processing substrate was prepared by annealing a 5′-TAM-labeled extension primer (shaded black ) and 3′-FAM-labeled blocking oligonucleotide (shaded blue ) to ssM13 as described in the text. 9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase were incubated with the processing substrate at 60 °C for 15 min. Processed products (103 nt) result from complete Okazaki fragment maturation. B , Okazaki fragment maturation assays were performed without any proteins added, all proteins added (9°N polB, polD, PCNA/RFC, Fen1, and DNA ligase), or in reactions that omit one protein as noted in the figure. Reaction products were analyzed by capillary electrophoresis. 5′-TAM-labeled extension primer is shaded black and 3′-FAM-labeled blocking oligonucleotide is shaded blue . Processed products (103 nt) result from a complete Okazaki fragment maturation assay and are labeled with both 5′-TAM ( black ) and 3′-FAM ( blue ). C, processed products from reactions omitting various replication proteins in panel B were quantitated and plotted. The data shown are averages with standard deviations from three independent experiments.

    Article Snippet: Enzymes and Reagents All restriction endonucleases, modifying enzymes, polB (9°Nm DNA polymerase; 9°N/E143D), 9°N DNA ligase, T4 DNA ligase, nucleotides, and single-stranded M13mp18 DNA (ssM13) were from New England Biolabs, Inc. (Ipswich, MA).

    Techniques: Labeling, Blocking Assay, Incubation, Electrophoresis

    Simplified models of Okazaki fragment maturation in bacteria ( A ), Eukarya ( B ), and Thermococcus species 9°N ( C ). A, in bacteria, pol III synthesizes the lagging strand. pol I replaces pol III to complete Okazaki fragment maturation. pol I 5′-3′ exonuclease removes the RNA primer as its DNA polymerase activity fills the gap. DNA ligase seals the Okazaki fragments. B, the eukaryal lagging strand DNA polymerase, pol δ, strand displacement activity generates a flap for Fen1 cleavage. DNA ligase seals the Okazaki fragments. C, polD synthesizes the lagging strand and stops at a downstream Okazaki fragment. polB replaces polD and its strand displacement activity generates a flap for Fen1 cleavage. DNA ligase seals the Okazaki fragments. Further details are described in the text.

    Journal: The Journal of Biological Chemistry

    Article Title: The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *

    doi: 10.1074/jbc.M115.638130

    Figure Lengend Snippet: Simplified models of Okazaki fragment maturation in bacteria ( A ), Eukarya ( B ), and Thermococcus species 9°N ( C ). A, in bacteria, pol III synthesizes the lagging strand. pol I replaces pol III to complete Okazaki fragment maturation. pol I 5′-3′ exonuclease removes the RNA primer as its DNA polymerase activity fills the gap. DNA ligase seals the Okazaki fragments. B, the eukaryal lagging strand DNA polymerase, pol δ, strand displacement activity generates a flap for Fen1 cleavage. DNA ligase seals the Okazaki fragments. C, polD synthesizes the lagging strand and stops at a downstream Okazaki fragment. polB replaces polD and its strand displacement activity generates a flap for Fen1 cleavage. DNA ligase seals the Okazaki fragments. Further details are described in the text.

    Article Snippet: Enzymes and Reagents All restriction endonucleases, modifying enzymes, polB (9°Nm DNA polymerase; 9°N/E143D), 9°N DNA ligase, T4 DNA ligase, nucleotides, and single-stranded M13mp18 DNA (ssM13) were from New England Biolabs, Inc. (Ipswich, MA).

    Techniques: Activity Assay