e coli dna ligase New England Biolabs Search Results


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  • 99
    New England Biolabs dna ligase
    Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease <t>III</t> in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid <t>DNA</t> which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.
    Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1350 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 5169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs e coli dna polymerase i
    A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli <t>DNA</t> polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.
    E Coli Dna Polymerase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 554 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 dna ligase
    A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli <t>DNA</t> polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.
    T4 Dna Ligase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 49235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 ligase buffer
    A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli <t>DNA</t> polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.
    T4 Ligase Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1325 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs exonuclease i e coli
    A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli <t>DNA</t> polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.
    Exonuclease I E Coli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1055 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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: 97/100, based on 490 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs 5 alpha high efficiency competent e coli cells
    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 .
    5 Alpha High Efficiency Competent E Coli Cells, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Thermo Fisher ambion rnase h from e coli
    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 .
    Ambion Rnase H From E Coli, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs biotinylated rna polymerase gabizon
    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 .
    Biotinylated Rna Polymerase Gabizon, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Journal: The Journal of General Virology

    Article Title: Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2

    doi: 10.1099/vir.0.013946-0

    Figure Lengend Snippet: Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Article Snippet: Restriction enzymes, reverse transcriptase SuperScript III, high-fidelity Taq DNA polymerase, Pfu polymerase, Phusion DNA polymerase, Klenow fragment of DNA polymerase, exonuclease III and DNA ligase were obtained from New England Biolabs (NEB) or Invitrogen and used according to the manufacturers' instructions.

    Techniques: Ligation, Clone Assay, Plasmid Preparation, Sequencing, Mutagenesis, In Vitro, Methylation

    The large terminase gp17 is a weak endonuclease. ( A ) Time course of cleavage of circular pET28b plasmid (100 ng) by gp17 (1.5 µM). ( B ) Cleavage of topoisomerase 1 relaxed DNA by gp17. ( C ) Increasing concentrations of gp17 (9–900 nM) were incubated with circular pAd10 DNA (400 ng, 0.9 nM) and the amount of undigested circular DNA in each lane was quantified by laser densitometry. ( D ) Comparison of the nuclease activity of T4 gp17 with DNase I (non-specific nickase) and Sau3A1 (frequent cutting restriction endonuclease). Increasing concentrations of each enzyme were incubated with circular pAd10 DNA (400 ng, 0.9 nM) with the enzyme (monomer) to DNA ratio (number of molecules of each) varied over a range of 10–1000:1. The enzyme:DNA ratio at 50% cleavage was determined by quantifying the amount of undigested circular DNA in each lane. Values represent average of duplicates from two independent experiments. The ‘C’ lanes represent untreated DNA. See ‘Materials and Methods’ section for additional details.

    Journal: Nucleic Acids Research

    Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    doi: 10.1093/nar/gkq1191

    Figure Lengend Snippet: The large terminase gp17 is a weak endonuclease. ( A ) Time course of cleavage of circular pET28b plasmid (100 ng) by gp17 (1.5 µM). ( B ) Cleavage of topoisomerase 1 relaxed DNA by gp17. ( C ) Increasing concentrations of gp17 (9–900 nM) were incubated with circular pAd10 DNA (400 ng, 0.9 nM) and the amount of undigested circular DNA in each lane was quantified by laser densitometry. ( D ) Comparison of the nuclease activity of T4 gp17 with DNase I (non-specific nickase) and Sau3A1 (frequent cutting restriction endonuclease). Increasing concentrations of each enzyme were incubated with circular pAd10 DNA (400 ng, 0.9 nM) with the enzyme (monomer) to DNA ratio (number of molecules of each) varied over a range of 10–1000:1. The enzyme:DNA ratio at 50% cleavage was determined by quantifying the amount of undigested circular DNA in each lane. Values represent average of duplicates from two independent experiments. The ‘C’ lanes represent untreated DNA. See ‘Materials and Methods’ section for additional details.

    Article Snippet: The digested DNA was precipitated by 8 M ammonium acetate and resuspended in water, followed by the addition of T4 DNA ligase (Fermentas) or E. coli DNA ligase (New England Biolabs) in a 25 µl reaction mixture.

    Techniques: Plasmid Preparation, Incubation, Activity Assay

    ATP stimulates gp17 nuclease. ( A ) Nuclease activity of gp17 is stimulated in presence of ATP. gp17 (1 µM) was incubated with linear pAd10 DNA (100 ng) in the presence of increasing concentrations of ATP (0.05–5 mM) . Note that the DNA is degraded to small fragments in some of the lanes. Therefore, very little DNA smear is seen in these lanes. ( B ) Nuclease activity of gp17 in the presence of ATP analogs. gp17 (1.2 µM) was incubated with circular pAd10 DNA (200 ng) in the presence of ATP, ADP, ATP-γS or AMP-PNP (1 mM). ( C ) The T4 nuclease domain (C360, amino acids 360–577) (left panel; lanes 1–7) or the RB49 nuclease domain (C360, amino acids 358–607) (right panel; lanes 8–14) is not stimulated by ATP. T4 C360 (4 µM) or RB49 C360 (1 µM), either alone (lanes 2 and 9) or in the presence of ATP (lanes 3–7 and 10–14) was incubated with linear pAd10 DNA (100 ng) for 15 min and the samples were analyzed by 0.8% (w/v) agarose gel electrophoresis. Lanes 1 and 8 labeled as ‘C’ are control lanes having untreated DNA.

    Journal: Nucleic Acids Research

    Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    doi: 10.1093/nar/gkq1191

    Figure Lengend Snippet: ATP stimulates gp17 nuclease. ( A ) Nuclease activity of gp17 is stimulated in presence of ATP. gp17 (1 µM) was incubated with linear pAd10 DNA (100 ng) in the presence of increasing concentrations of ATP (0.05–5 mM) . Note that the DNA is degraded to small fragments in some of the lanes. Therefore, very little DNA smear is seen in these lanes. ( B ) Nuclease activity of gp17 in the presence of ATP analogs. gp17 (1.2 µM) was incubated with circular pAd10 DNA (200 ng) in the presence of ATP, ADP, ATP-γS or AMP-PNP (1 mM). ( C ) The T4 nuclease domain (C360, amino acids 360–577) (left panel; lanes 1–7) or the RB49 nuclease domain (C360, amino acids 358–607) (right panel; lanes 8–14) is not stimulated by ATP. T4 C360 (4 µM) or RB49 C360 (1 µM), either alone (lanes 2 and 9) or in the presence of ATP (lanes 3–7 and 10–14) was incubated with linear pAd10 DNA (100 ng) for 15 min and the samples were analyzed by 0.8% (w/v) agarose gel electrophoresis. Lanes 1 and 8 labeled as ‘C’ are control lanes having untreated DNA.

    Article Snippet: The digested DNA was precipitated by 8 M ammonium acetate and resuspended in water, followed by the addition of T4 DNA ligase (Fermentas) or E. coli DNA ligase (New England Biolabs) in a 25 µl reaction mixture.

    Techniques: Activity Assay, Incubation, Agarose Gel Electrophoresis, Labeling

    gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.

    Journal: Nucleic Acids Research

    Article Title: Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4

    doi: 10.1093/nar/gkq1191

    Figure Lengend Snippet: gp17 nuclease prefers long DNA substrates and cleaves at the ends of linear DNA. ( A ) Increasing concentrations of gp17 were incubated with 0.9 nM each of 29 kb pAd10 plasmid DNA or 2.6 kb pUC19 plasmid DNA. The undigested circular DNA was quantified and used to determine the percent of cleaved DNA at different gp17:DNA ratios. Values represent the average of duplicates from two independent experiments. ( B ) gp17 preference for longer DNA molecules was seen by incubating gp17 (3 µM, lanes 2–7) with a 2-log DNA ladder (400 ng, 0.1–10 kb, New England Biolabs) for 2–30 min. ( C ) Autoradiogram showing the cleavage of γ 32 P end-labeled λ-HindIII DNA fragments (0.5 pmol, 125–23 130 bp, Promega) by gp17 (1.2 µM) (lanes 2–6) or DNase I (0.0024 µM, 500-fold less than gp17) (lanes 7–11). Lane 1 has untreated DNA. ( D ) gp17 nuclease generates blunt ends. Circular pUC19 DNA (40 ng) was cleaved by gp17 (lanes 2–4) or BamH1 (lanes 5–7). The cleaved DNA was then treated with E. coli DNA ligase (lanes 3 and 6) or T4 DNA ligase (lanes 4 and 7). Lanes labeled as ‘C’ are control untreated lanes. See ‘Materials and Methods’ section for additional details.

    Article Snippet: The digested DNA was precipitated by 8 M ammonium acetate and resuspended in water, followed by the addition of T4 DNA ligase (Fermentas) or E. coli DNA ligase (New England Biolabs) in a 25 µl reaction mixture.

    Techniques: Incubation, Plasmid Preparation, Labeling

    Analysis of supercoiling of relaxed pHOT1 by E. coli gyrase in the presence of DMA. Lane 1, relaxed DNA as control; lane 2, supercoiling of relaxed DNA by E. coli DNA gyrase; lane 3; relaxed DNA in the presence of 100 μM DMA; lanes 4–10,

    Journal: Journal of Antimicrobial Chemotherapy

    Article Title: 3,4-Dimethoxyphenyl bis-benzimidazole, a novel DNA topoisomerase inhibitor that preferentially targets Escherichia coli topoisomerase I

    doi: 10.1093/jac/dks322

    Figure Lengend Snippet: Analysis of supercoiling of relaxed pHOT1 by E. coli gyrase in the presence of DMA. Lane 1, relaxed DNA as control; lane 2, supercoiling of relaxed DNA by E. coli DNA gyrase; lane 3; relaxed DNA in the presence of 100 μM DMA; lanes 4–10,

    Article Snippet: E. coli DNA gyrase and its relaxed substrate were purchased from New England Biolabs (GmBH, Germany).

    Techniques:

    Optimization of primers for real-time DOP-PCR. Amplification profiles of real-time DOP-PCR were obtained by using various degenerate primers. Serially diluted human placental DNA samples ranging from 80 fg to 80 ng and a no-template control (NTC) were amplified. The primers used were Tag-N6- ATGTGG (A), Tag-N6- CCGCCC (B), Tag-N6- ATTTCG (C), Tag-N4- ATGTGG (D), Tag-N8- ATGTGG (E), and a combination of Tag-N6- ATGTGG and Tag-N6- TGTTGC (F).

    Journal: PLoS ONE

    Article Title: Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

    doi: 10.1371/journal.pone.0028661

    Figure Lengend Snippet: Optimization of primers for real-time DOP-PCR. Amplification profiles of real-time DOP-PCR were obtained by using various degenerate primers. Serially diluted human placental DNA samples ranging from 80 fg to 80 ng and a no-template control (NTC) were amplified. The primers used were Tag-N6- ATGTGG (A), Tag-N6- CCGCCC (B), Tag-N6- ATTTCG (C), Tag-N4- ATGTGG (D), Tag-N8- ATGTGG (E), and a combination of Tag-N6- ATGTGG and Tag-N6- TGTTGC (F).

    Article Snippet: DNA samples Genomic DNAs from four different species were used as templates for PCR analysis: human placental DNA (HPD, Sigma), calf thymus DNA (CTD, Invitrogen), E. coli DNA (extracted from BL21 strain), and lambda phage DNA (NEB).

    Techniques: Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Amplification

    Application of the real-time DOP-PCR to diverse DNA species. Amplification profiles and their standard curves were obtained from human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and lambda phage DNA (D). Standard DNA samples from 80 fg to 80 ng and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.

    Journal: PLoS ONE

    Article Title: Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification

    doi: 10.1371/journal.pone.0028661

    Figure Lengend Snippet: Application of the real-time DOP-PCR to diverse DNA species. Amplification profiles and their standard curves were obtained from human placental DNA (HPD; A), calf thymus DNA (CTD; B), E. coli DNA (C), and lambda phage DNA (D). Standard DNA samples from 80 fg to 80 ng and a no-template control were amplified. Six independent experiments each comprising triplicate reactions were performed, and typical results of one experiment are presented. Data for 80 ng and NTC were omitted for the plotting of standard curves.

    Article Snippet: DNA samples Genomic DNAs from four different species were used as templates for PCR analysis: human placental DNA (HPD, Sigma), calf thymus DNA (CTD, Invitrogen), E. coli DNA (extracted from BL21 strain), and lambda phage DNA (NEB).

    Techniques: Degenerate Oligonucleotide–primed Polymerase Chain Reaction, Amplification

    β-clamp inhibits the contact between the 5’end of nicked DNA and Pol-I. (A) Structural model of E . coli Klenow /DNA complex shows that finger domain of Klenow makes a contact with downstream nick site. The model shows that the conserved F771 (in blue surface representation), which participates in the strand displacement, is positioned between the downstream nicked DNA strands. ( B) A fraction of BrdU base containing radiolabelled 28 bases oligonucleotide shows a gel shift under the near UV light, suggesting that the oligonucleotide makes cross-linked adduct with Klenow during strand displacement (lane 2). The presence of β-clamp and γ-complex in the absence of ATP (lane 3), or ATP alone (lane 4) also exhibited similar crosslinking adduct. However, β-clamp, γ-complex, and ATP together suppressed the formation of crosslinking product (lane 6), suggesting that loading of the β-clamp on the template blocks the crosslinking. * Represents a minor contaminated band with the custom synthesized 28 bases oligonucleotide.

    Journal: PLoS ONE

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation

    doi: 10.1371/journal.pone.0199559

    Figure Lengend Snippet: β-clamp inhibits the contact between the 5’end of nicked DNA and Pol-I. (A) Structural model of E . coli Klenow /DNA complex shows that finger domain of Klenow makes a contact with downstream nick site. The model shows that the conserved F771 (in blue surface representation), which participates in the strand displacement, is positioned between the downstream nicked DNA strands. ( B) A fraction of BrdU base containing radiolabelled 28 bases oligonucleotide shows a gel shift under the near UV light, suggesting that the oligonucleotide makes cross-linked adduct with Klenow during strand displacement (lane 2). The presence of β-clamp and γ-complex in the absence of ATP (lane 3), or ATP alone (lane 4) also exhibited similar crosslinking adduct. However, β-clamp, γ-complex, and ATP together suppressed the formation of crosslinking product (lane 6), suggesting that loading of the β-clamp on the template blocks the crosslinking. * Represents a minor contaminated band with the custom synthesized 28 bases oligonucleotide.

    Article Snippet: Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB).

    Techniques: Electrophoretic Mobility Shift Assay, Synthesized

    β-clamp inhibits strand displacement activity of exo - Klenow and exo - Pol I. (A) The urea PAGE shows that exo - Klenow exhibits a slower speed of strand displacement. Presence of the template-loaded β-clamp stalls the exo - Klenow pauses mostly at the 39 th nucleotide position. The exo - Klenow-mediated strand displacement coupled with ligation represents that the DNA ligase fails to ligate nicks. However, the presence of template-loaded β-clamp increases the ligation frequency. ( B) exo - Pol I exhibits similar functional consequences on the strand displacement and ligation as observed in case of exo - Klenow (panel A). The values representing mean ± sd are calculated from three different experiments.

    Journal: PLoS ONE

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation

    doi: 10.1371/journal.pone.0199559

    Figure Lengend Snippet: β-clamp inhibits strand displacement activity of exo - Klenow and exo - Pol I. (A) The urea PAGE shows that exo - Klenow exhibits a slower speed of strand displacement. Presence of the template-loaded β-clamp stalls the exo - Klenow pauses mostly at the 39 th nucleotide position. The exo - Klenow-mediated strand displacement coupled with ligation represents that the DNA ligase fails to ligate nicks. However, the presence of template-loaded β-clamp increases the ligation frequency. ( B) exo - Pol I exhibits similar functional consequences on the strand displacement and ligation as observed in case of exo - Klenow (panel A). The values representing mean ± sd are calculated from three different experiments.

    Article Snippet: Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB).

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Ligation, Functional Assay

    β-clamp inhibits Pol I-mediated nick translation. (A ) The template used in the assay was prepared by assembling 67 bases, 5’-phosphorylated 28 bases and 5’-radiolabelled (asterisk) 19 bases oligonucleotides. ( B) ) indicate that the presence of β-clamp nominally affected nick translation traversing 28 bases downstream RNA. ( C) The urea-denaturing gel shows that Pol I efficiently translated the nick, degrading 28 bases downstream DNA, but the presence of β-clamp dramatically reduced the speed of nick translation. The presence of ligase allowed early ligation when β-clamp slowed down Pol I-mediated nick translation. ( D) The urea PAGE represents that the 3’-exo - Pol I-mediated nick translation was slowed down in the presence of template-loaded β-clamp, in a manner similar to the nick translation with β-clamp and Pol I combinations, as shown in panel C. Moreover, the presence of template-loaded β-clamp increases the ligation efficiency during the 3’-exo - Pol I-mediated nick translation. ( E) The urea denaturing gel represents that the 5’-exo - Pol I intensely paused at the 39 th nucleotide and downstream positions, affecting the ligation process. The presence of template-loaded β-clamp moderately enhances these pauses, but facilitates ligation to some extent. The # indicates the position of truncated by-products that originated from the 67-nucleotide long product by the 3’ exonuclease function of Pol I and 5’-exo - Pol I (panel B, C, E). This truncated product is missing in the assay with 3’-exo - Pol I (panel D). All the experiments were performed at least three times. The values represent mean ± standard deviation (sd).

    Journal: PLoS ONE

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation

    doi: 10.1371/journal.pone.0199559

    Figure Lengend Snippet: β-clamp inhibits Pol I-mediated nick translation. (A ) The template used in the assay was prepared by assembling 67 bases, 5’-phosphorylated 28 bases and 5’-radiolabelled (asterisk) 19 bases oligonucleotides. ( B) ) indicate that the presence of β-clamp nominally affected nick translation traversing 28 bases downstream RNA. ( C) The urea-denaturing gel shows that Pol I efficiently translated the nick, degrading 28 bases downstream DNA, but the presence of β-clamp dramatically reduced the speed of nick translation. The presence of ligase allowed early ligation when β-clamp slowed down Pol I-mediated nick translation. ( D) The urea PAGE represents that the 3’-exo - Pol I-mediated nick translation was slowed down in the presence of template-loaded β-clamp, in a manner similar to the nick translation with β-clamp and Pol I combinations, as shown in panel C. Moreover, the presence of template-loaded β-clamp increases the ligation efficiency during the 3’-exo - Pol I-mediated nick translation. ( E) The urea denaturing gel represents that the 5’-exo - Pol I intensely paused at the 39 th nucleotide and downstream positions, affecting the ligation process. The presence of template-loaded β-clamp moderately enhances these pauses, but facilitates ligation to some extent. The # indicates the position of truncated by-products that originated from the 67-nucleotide long product by the 3’ exonuclease function of Pol I and 5’-exo - Pol I (panel B, C, E). This truncated product is missing in the assay with 3’-exo - Pol I (panel D). All the experiments were performed at least three times. The values represent mean ± standard deviation (sd).

    Article Snippet: Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB).

    Techniques: Nick Translation, Ligation, Polyacrylamide Gel Electrophoresis, Standard Deviation

    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

    Amplification of the HCV 3′UTR by ligation-mediated RT-PCR. Three linkers were estimated for their efficiency in the ligation and subsequent RT-PCR. Lanes 1, 2, 3, 4 represent samples #069, #0273, #1099 and #1564, respectively; Lane 5, negative control. M1, 100 bp DNA ladder (Invitrogen). M2, 123 bp DNA ladder (Invitrogen).

    Journal: Journal of virological methods

    Article Title: Enhanced Protocol for Determining the 3? Terminus of Hepatitis C Virus

    doi: 10.1016/j.jviromet.2010.03.030

    Figure Lengend Snippet: Amplification of the HCV 3′UTR by ligation-mediated RT-PCR. Three linkers were estimated for their efficiency in the ligation and subsequent RT-PCR. Lanes 1, 2, 3, 4 represent samples #069, #0273, #1099 and #1564, respectively; Lane 5, negative control. M1, 100 bp DNA ladder (Invitrogen). M2, 123 bp DNA ladder (Invitrogen).

    Article Snippet: Fifth, the first round of PCR amplification was tried with different DNA polymerases, including rTth XL, DyNAzyme EXT, Vent (New England Biolabs), DeepVent (New England Biolabs) and Phusion High-Fidelity (New England Biolabs).

    Techniques: Amplification, Ligation, Reverse Transcription Polymerase Chain Reaction, Negative Control

    Estimation of processing activity and fidelity of 5 DNA polymerases in the amplification of ~130 bp poly(T/TG) domain. The plasmid p90H/FL pol- consisting of the full-length HCV genome was used as the PCR template. A series of 1:10 dilution of the plasmid template, starting at 4 ng, was applied and matched to lanes 1, 2, 3 and 4, respectively. Lane 5, negative control. M, 123 DNA ladder (Invitrogen).

    Journal: Journal of virological methods

    Article Title: Enhanced Protocol for Determining the 3? Terminus of Hepatitis C Virus

    doi: 10.1016/j.jviromet.2010.03.030

    Figure Lengend Snippet: Estimation of processing activity and fidelity of 5 DNA polymerases in the amplification of ~130 bp poly(T/TG) domain. The plasmid p90H/FL pol- consisting of the full-length HCV genome was used as the PCR template. A series of 1:10 dilution of the plasmid template, starting at 4 ng, was applied and matched to lanes 1, 2, 3 and 4, respectively. Lane 5, negative control. M, 123 DNA ladder (Invitrogen).

    Article Snippet: Fifth, the first round of PCR amplification was tried with different DNA polymerases, including rTth XL, DyNAzyme EXT, Vent (New England Biolabs), DeepVent (New England Biolabs) and Phusion High-Fidelity (New England Biolabs).

    Techniques: Activity Assay, Amplification, Plasmid Preparation, Polymerase Chain Reaction, Negative Control

    Amplification of the HCV poly(U/UC) tract and 3′ X tail. Three reverse primers, located at different sites of 3′ X tail, were used for RT and the first round PCR. While the RT-PCR with either 3HBVR1UTR2 or 3HBVR1UTR3 generated DNA bands with expected sizes, ~300 and 324 bp, respectively, the primer 3HBVR1UTR1, located on the uttermost part of 3′ X tail, gave a much smaller product (~123 bp), indicating the existence of potential replication slippage. Lanes 1, 2, 3, 4 represent samples #069, #0273, #1099 and #1564, respectively; Lane 5, negative control. M, 123 bp DNA ladder (Invitrogen).

    Journal: Journal of virological methods

    Article Title: Enhanced Protocol for Determining the 3? Terminus of Hepatitis C Virus

    doi: 10.1016/j.jviromet.2010.03.030

    Figure Lengend Snippet: Amplification of the HCV poly(U/UC) tract and 3′ X tail. Three reverse primers, located at different sites of 3′ X tail, were used for RT and the first round PCR. While the RT-PCR with either 3HBVR1UTR2 or 3HBVR1UTR3 generated DNA bands with expected sizes, ~300 and 324 bp, respectively, the primer 3HBVR1UTR1, located on the uttermost part of 3′ X tail, gave a much smaller product (~123 bp), indicating the existence of potential replication slippage. Lanes 1, 2, 3, 4 represent samples #069, #0273, #1099 and #1564, respectively; Lane 5, negative control. M, 123 bp DNA ladder (Invitrogen).

    Article Snippet: Fifth, the first round of PCR amplification was tried with different DNA polymerases, including rTth XL, DyNAzyme EXT, Vent (New England Biolabs), DeepVent (New England Biolabs) and Phusion High-Fidelity (New England Biolabs).

    Techniques: Amplification, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Generated, Negative Control

    A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli DNA polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.

    Journal: Biochemistry

    Article Title: Cross-Link Structure Affects Replication-Independent DNA Interstrand Cross-Link Repair in Mammalian Cells †

    doi: 10.1021/bi902169q

    Figure Lengend Snippet: A) Scheme of cross-linked plasmid preparation. First, p3CMV was double digested with Van91I and BstXI to create the linear duplex, 2A.1 . Next, an adaptor duplex was ligated onto the BstXI compatible end followed by ligation with the cross-linked duplex to form 2A.2 . Blocking one end of the duplex prevented multiple ligations of the cross-linked duplex. Digestion by BstXI released the adaptor duplex to produce the cross-linked linear duplex, 2A.3 . Finally, the linear cross-linked duplex was circularized under dilute conditions to form the single site-specific interstrand cross-linked plasmid, p3CMV-X . B) Characterization of interstrand cross-linked plasmids. Plasmids were digested with a restriction enzyme to release a 150bp fragment and the fragments were radiolabeled using the Klenow fragment of E. coli DNA polymerase I. The labeled fragments were then analyzed on a 6% gel under denaturing conditions.

    Article Snippet: Protected deoxyribonucleoside 3′-O-phosphoramidites and oligonucleotide synthesis reagents were obtained from Glen Research, Inc. Polynucleotide kinase, T4 DNA ligase, E. coli DNA polymerase I (Klenow fragment), EcoRI, and BstXI were obtained from New England Biolabs, Inc. Van91I was obtained from Fermantas, Inc.

    Techniques: Plasmid Preparation, Ligation, Blocking Assay, Labeling

    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