e coli dna ligase New England Biolabs Search Results


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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.
    N Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs dna polymerase
    Overview of the nonhomologous random recombination (NRR) method. (A) Starting <t>DNA</t> sequences are randomly digested with DNase I, blunt-ended with <t>T4</t> DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 3856 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    New England Biolabs neb5α competent escherichia coli
    Overview of the nonhomologous random recombination (NRR) method. (A) Starting <t>DNA</t> sequences are randomly digested with DNase I, blunt-ended with <t>T4</t> DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    Neb5α Competent Escherichia Coli, 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 escherichia coli dna polymerase
    Overview of the nonhomologous random recombination (NRR) method. (A) Starting <t>DNA</t> sequences are randomly digested with DNase I, blunt-ended with <t>T4</t> DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    Escherichia Coli Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    New England Biolabs 1x e coli dna ligase reaction buffer
    Overview of the nonhomologous random recombination (NRR) method. (A) Starting <t>DNA</t> sequences are randomly digested with DNase I, blunt-ended with <t>T4</t> DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    1x E Coli Dna Ligase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 80/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs exonuclease i e coli
    Overview of the nonhomologous random recombination (NRR) method. (A) Starting <t>DNA</t> sequences are randomly digested with DNase I, blunt-ended with <t>T4</t> DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    Exonuclease I E Coli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs 5 alpha high efficiency competent e coli cells
    Overview of the nonhomologous random recombination (NRR) method. (A) Starting <t>DNA</t> sequences are randomly digested with DNase I, blunt-ended with <t>T4</t> DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.
    5 Alpha High Efficiency Competent E Coli Cells, 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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    95
    New England Biolabs e coli uracil dna glycosylase
    Verification of each step during second strand <t>DNA</t> synthesis and <t>glycosylase/nuclease</t> treatments. Samples were separated on 1% agarose gel containing 2 μg/ml ethidium bromide. Lane M, DNA size markers. Lane 1, 200ng of sspAVR88. Lane 2, product after annealing of the 2 nd strand primer. Lane 3, product after second strand synthesis and ligation. Lane 4, product after glycosylase and nuclease treatment. Lane 5 and 6 are different amounts of purified pSOcpd. Equal portions corresponding to 200ng of sspAVR88 were loaded in lanes 1-5.
    E Coli Uracil Dna Glycosylase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs exonuclease iii
    Verification of each step during second strand <t>DNA</t> synthesis and <t>glycosylase/nuclease</t> treatments. Samples were separated on 1% agarose gel containing 2 μg/ml ethidium bromide. Lane M, DNA size markers. Lane 1, 200ng of sspAVR88. Lane 2, product after annealing of the 2 nd strand primer. Lane 3, product after second strand synthesis and ligation. Lane 4, product after glycosylase and nuclease treatment. Lane 5 and 6 are different amounts of purified pSOcpd. Equal portions corresponding to 200ng of sspAVR88 were loaded in lanes 1-5.
    Exonuclease Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 517 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs restriction enzymes ligase agarose gels competent e coli cells dh5α
    Verification of each step during second strand <t>DNA</t> synthesis and <t>glycosylase/nuclease</t> treatments. Samples were separated on 1% agarose gel containing 2 μg/ml ethidium bromide. Lane M, DNA size markers. Lane 1, 200ng of sspAVR88. Lane 2, product after annealing of the 2 nd strand primer. Lane 3, product after second strand synthesis and ligation. Lane 4, product after glycosylase and nuclease treatment. Lane 5 and 6 are different amounts of purified pSOcpd. Equal portions corresponding to 200ng of sspAVR88 were loaded in lanes 1-5.
    Restriction Enzymes Ligase Agarose Gels Competent E Coli Cells Dh5α, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs ptxb1 vector
    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the <t>pTXB1</t> vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Ptxb1 Vector, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 411 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    New England Biolabs t4 dna ligase
    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the <t>pTXB1</t> vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 38631 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 ligase buffer
    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the <t>pTXB1</t> vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    T4 Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 968 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs xhoi
    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the <t>pTXB1</t> vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Xhoi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 7470 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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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

    Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.

    Journal: Nature biotechnology

    Article Title: Nucleic acid evolution and minimization by nonhomologous random recombination

    doi: 10.1038/nbt736

    Figure Lengend Snippet: Overview of the nonhomologous random recombination (NRR) method. (A) Starting DNA sequences are randomly digested with DNase I, blunt-ended with T4 DNA polymerase, and recombined with T4 DNA ligase under conditions that strongly favor intermolecular ligation over intramolecular circularization. (B) A defined stoichiometry of hairpin DNA added to the ligation reaction controls the average length of the recombined products. The completed ligation reaction is digested with a restriction endonuclease to provide a library of double-stranded recombined DNA flanked by defined primer-binding sequences.

    Article Snippet: Restriction endonucleases, T4 DNA ligase, Vent DNA polymerase, T4 polynucleotide kinase, and T4 DNA polymerase were obtained from New England Biolabs (Beverly, MA).

    Techniques: Ligation, Binding Assay

    Verification of each step during second strand DNA synthesis and glycosylase/nuclease treatments. Samples were separated on 1% agarose gel containing 2 μg/ml ethidium bromide. Lane M, DNA size markers. Lane 1, 200ng of sspAVR88. Lane 2, product after annealing of the 2 nd strand primer. Lane 3, product after second strand synthesis and ligation. Lane 4, product after glycosylase and nuclease treatment. Lane 5 and 6 are different amounts of purified pSOcpd. Equal portions corresponding to 200ng of sspAVR88 were loaded in lanes 1-5.

    Journal: DNA repair

    Article Title: Construction of a circular single-stranded DNA template containing a defined lesion

    doi: 10.1016/j.dnarep.2009.03.006

    Figure Lengend Snippet: Verification of each step during second strand DNA synthesis and glycosylase/nuclease treatments. Samples were separated on 1% agarose gel containing 2 μg/ml ethidium bromide. Lane M, DNA size markers. Lane 1, 200ng of sspAVR88. Lane 2, product after annealing of the 2 nd strand primer. Lane 3, product after second strand synthesis and ligation. Lane 4, product after glycosylase and nuclease treatment. Lane 5 and 6 are different amounts of purified pSOcpd. Equal portions corresponding to 200ng of sspAVR88 were loaded in lanes 1-5.

    Article Snippet: E.coli DNA polymerase I (Klenow Fragment) [pol I (Kf)], E.coli Exonuclease III, E.coli Exonuclease I, E.coli Uracil DNA glycosylase, E.coli RecA, T7 DNA polymerase, T4 Polynucleotide kinase, and M13KO7 helper phage were all purchased from New England Biolabs (Ipswich, MA).

    Techniques: DNA Synthesis, Agarose Gel Electrophoresis, Ligation, Purification

    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Journal: Biopolymers

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides

    doi: 10.1002/bip.21391

    Figure Lengend Snippet: Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Article Snippet: As described above, a miniprep of DNA for the pTXB1 vector containing Aβ1-29 was used as a template for synthesis of the mutated strand by PCR.

    Techniques: Sequencing, Plasmid Preparation, Expressing, Binding Assay, Affinity Purification, Recombinant, Ligation