e coli dna gyrase  (New England Biolabs)


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    Name:
    E coli DNA Ligase
    Description:
    E coli DNA Ligase 1 000 units
    Catalog Number:
    m0205l
    Price:
    248
    Size:
    1 000 units
    Category:
    DNA Ligases
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    Structured Review

    New England Biolabs e coli dna gyrase
    E coli DNA Ligase
    E coli DNA Ligase 1 000 units
    https://www.bioz.com/result/e coli dna gyrase/product/New England Biolabs
    Average 95 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    e coli dna gyrase - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases"

    Article Title: Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    Journal: Scientific Reports

    doi: 10.1038/srep36006

    An experimental strategy to construct relaxed (rx) or supercoiled (sc) pAB1_FL905. ( A ) Oligomer FL905 that contains the 42 nt. AT sequence is ligated between the two Nt.BbvCI sites of plasmid pAB1 to yield rx pAB1_FL905. ( B ) Sc pAB1_FL905 can be generated through the treatment of rx pAB1_FL905 by E. coli DNA gyrase. The fluorescence intensity of fluorescein is dependent on the supercoiling status of pAB1_FL905.
    Figure Legend Snippet: An experimental strategy to construct relaxed (rx) or supercoiled (sc) pAB1_FL905. ( A ) Oligomer FL905 that contains the 42 nt. AT sequence is ligated between the two Nt.BbvCI sites of plasmid pAB1 to yield rx pAB1_FL905. ( B ) Sc pAB1_FL905 can be generated through the treatment of rx pAB1_FL905 by E. coli DNA gyrase. The fluorescence intensity of fluorescein is dependent on the supercoiling status of pAB1_FL905.

    Techniques Used: Construct, Sequencing, Plasmid Preparation, Generated, Fluorescence

    ( A ) Fluorescence spectra of sc (red line), rx (black line), and nk (blue line) pAB1_FL509. λex = 470 nm. ( B ) Kinetics of the nicking reaction by Nt.BbvCI. Briefly, 60 μL of 1 × CutSmart buffer containing 500 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of Nt.BbvCI were added to initiate the nicking reaction. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( C ) Kinetics of the relaxation reaction by E. coli DNA topoisomerase I. For the relaxation reaction, 90 μL of 1 × NEBuffer 4 (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(AC) 2 , 1 mM DTT, pH 7.9) containing 270 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 0.67 μM of E. coli DNA topoisomerase I was used to relax the sc pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( D ) Kinetics of the supercoiling reaction by E. coli DNA gyrase. For the supercoiling reaction, 90 μL of 1 × gyrase buffer containing 1 μg of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 30 units of E. coli DNA gyrase was used to supercoil the rx pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm.
    Figure Legend Snippet: ( A ) Fluorescence spectra of sc (red line), rx (black line), and nk (blue line) pAB1_FL509. λex = 470 nm. ( B ) Kinetics of the nicking reaction by Nt.BbvCI. Briefly, 60 μL of 1 × CutSmart buffer containing 500 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of Nt.BbvCI were added to initiate the nicking reaction. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( C ) Kinetics of the relaxation reaction by E. coli DNA topoisomerase I. For the relaxation reaction, 90 μL of 1 × NEBuffer 4 (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(AC) 2 , 1 mM DTT, pH 7.9) containing 270 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 0.67 μM of E. coli DNA topoisomerase I was used to relax the sc pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( D ) Kinetics of the supercoiling reaction by E. coli DNA gyrase. For the supercoiling reaction, 90 μL of 1 × gyrase buffer containing 1 μg of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 30 units of E. coli DNA gyrase was used to supercoil the rx pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm.

    Techniques Used: Fluorescence

    DNA gyrase was potently inhibited by novobiocin ( A ) and ciprofloxacin ( B ). For DNA supercoiling reactions, 60 μL μL of 1 × gyrase buffer containing 670 ng of of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of DNA gyrase was used to supercoil the rx pAB1_FL905 in the presence of different concentrations of novobiocin and ciprofloxacin. The fluorescence intensity at λem = 521 nm was monitor with λex = 494 nm. The inhibition IC50 was estimated to be 0.48 ± 0.14 and 2.57 ± 1.b μM for novobiocin and ciprofloxacin, respectively.
    Figure Legend Snippet: DNA gyrase was potently inhibited by novobiocin ( A ) and ciprofloxacin ( B ). For DNA supercoiling reactions, 60 μL μL of 1 × gyrase buffer containing 670 ng of of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of DNA gyrase was used to supercoil the rx pAB1_FL905 in the presence of different concentrations of novobiocin and ciprofloxacin. The fluorescence intensity at λem = 521 nm was monitor with λex = 494 nm. The inhibition IC50 was estimated to be 0.48 ± 0.14 and 2.57 ± 1.b μM for novobiocin and ciprofloxacin, respectively.

    Techniques Used: Fluorescence, Inhibition

    2) Product Images from "Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A"

    Article Title: Partial Reconstitution of Human DNA Mismatch Repair In Vitro: Characterization of the Role of Human Replication Protein A

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.7.2037-2046.2002

    Reconstitution of MMR in vitro. (A) Fractionation of a HeLa nuclear extract into three components required for MMR. (B) Reconstitution of MMR requires SS1, SS2, and FII. The DNA substrate (100 ng of the 5′ G-T heteroduplex) was incubated for 15 min at 37°C in the reaction buffer with fractions as indicated. Amounts of protein used were 15 μg of SS1, 1.5 μg of SS2, or 30 μg of FII. DNA was extracted, treated with Hin dIII and Bsp 106, electrophoresed on an agarose gel, and visualized by ethidium bromide staining under UV illumination. ND, not detectable.
    Figure Legend Snippet: Reconstitution of MMR in vitro. (A) Fractionation of a HeLa nuclear extract into three components required for MMR. (B) Reconstitution of MMR requires SS1, SS2, and FII. The DNA substrate (100 ng of the 5′ G-T heteroduplex) was incubated for 15 min at 37°C in the reaction buffer with fractions as indicated. Amounts of protein used were 15 μg of SS1, 1.5 μg of SS2, or 30 μg of FII. DNA was extracted, treated with Hin dIII and Bsp 106, electrophoresed on an agarose gel, and visualized by ethidium bromide staining under UV illumination. ND, not detectable.

    Techniques Used: In Vitro, Fractionation, Incubation, Agarose Gel Electrophoresis, Staining

    3) Product Images from "Independent and Stochastic Action of DNA Polymerases in the Replisome"

    Article Title: Independent and Stochastic Action of DNA Polymerases in the Replisome

    Journal: Cell

    doi: 10.1016/j.cell.2017.05.041

    Individual replication fork progression is independent of primase A Micrographs showing replication products at 10 min where: i , all components present; or a component omitted: ii, DnaB and DnaC810; iii, Pol III*; iv, β; v, SSB; vi, primase. Composite, false-colored fields show anchor points for molecules that contain ssDNA, except i or v , where only long products were seen. In vi , surfaces were sparsely populated with DNA to avoid any ambiguity in molecule identification. Cyan, fields with flow off; magenta, same field with flow on showing fully-extended molecules. Molecules are bracketed for clarity. Scale bar: 10 μm, equal to 33.9 kb dsDNA or 80.3 knt SSB-bound ssDNA at 4,000 μl/h, without Mg 2+ ). B. Cartoon showing leading strand only product in a reaction lacking primase. C. Composite, false-colored image showing leading strand only replication without primase. Three replicating molecules ( 1, 2, 3 ). Molecules a, b and c are referred to later. D. Time-lapse, at 50-second intervals, of Molecules 1, 2 and 3 identified in C , colored by time-point as per C . E. Kymographs of molecules, numbered per C and D , showing fork progression without primase. Dashed grey line: position of anchor. Linear fits are from initiation to termination, yielding average fork rates. Pauses are included in the average here. F. Histograms of fork progression rates in the presence (grey) and absence of primase (light blue). Histograms fit to single Gaussians ( R 2 : with primase, 0.80; without primase, 0.94); no outliers were rejected. n , molecules. G. Processivities of single replisomes from live imaging experiments. Whisker plots of molecule lengths, with (320 nM) or without primase and/or β in flow. Data from 2 (primase, no β) or 3 (others) experiments. Horizontal bars, median; vertical bars, interquartile range. ***, significantly different pairs of populations (Kruskal-Wallis; P
    Figure Legend Snippet: Individual replication fork progression is independent of primase A Micrographs showing replication products at 10 min where: i , all components present; or a component omitted: ii, DnaB and DnaC810; iii, Pol III*; iv, β; v, SSB; vi, primase. Composite, false-colored fields show anchor points for molecules that contain ssDNA, except i or v , where only long products were seen. In vi , surfaces were sparsely populated with DNA to avoid any ambiguity in molecule identification. Cyan, fields with flow off; magenta, same field with flow on showing fully-extended molecules. Molecules are bracketed for clarity. Scale bar: 10 μm, equal to 33.9 kb dsDNA or 80.3 knt SSB-bound ssDNA at 4,000 μl/h, without Mg 2+ ). B. Cartoon showing leading strand only product in a reaction lacking primase. C. Composite, false-colored image showing leading strand only replication without primase. Three replicating molecules ( 1, 2, 3 ). Molecules a, b and c are referred to later. D. Time-lapse, at 50-second intervals, of Molecules 1, 2 and 3 identified in C , colored by time-point as per C . E. Kymographs of molecules, numbered per C and D , showing fork progression without primase. Dashed grey line: position of anchor. Linear fits are from initiation to termination, yielding average fork rates. Pauses are included in the average here. F. Histograms of fork progression rates in the presence (grey) and absence of primase (light blue). Histograms fit to single Gaussians ( R 2 : with primase, 0.80; without primase, 0.94); no outliers were rejected. n , molecules. G. Processivities of single replisomes from live imaging experiments. Whisker plots of molecule lengths, with (320 nM) or without primase and/or β in flow. Data from 2 (primase, no β) or 3 (others) experiments. Horizontal bars, median; vertical bars, interquartile range. ***, significantly different pairs of populations (Kruskal-Wallis; P

    Techniques Used: Flow Cytometry, Imaging, Whisker Assay

    4) Product Images from "Regulation by interdomain communication of a headful packaging nuclease from bacteriophage T4"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1191

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Incubation, Plasmid Preparation, Labeling

    5) Product Images from "Deoxyinosine repair in nuclear extracts of human cells"

    Article Title: Deoxyinosine repair in nuclear extracts of human cells

    Journal: Cell & Bioscience

    doi: 10.1186/s13578-015-0044-8

    Map of M13mp18 and f1PM based heteroduplex substrates. a The map of bacteriophage M13mp18 replicative form (RF) DNA shows restriction enzyme sites relevant to this study with derivatives M13LR1 and M13LR3 containing 22-bp insertions at the unique HindIII restriction site, and phage M13WX1 and M13X22 containing 26-bp and 22-bp insertions at Xba I site respectively. b The map of bacteriophage f1PM RF DNA with its derivative f1PMA with a 27-bp insertion at Xba I. ‘V’, phage viral strand. ‘C’, phage complementary strand. Underlines beneath each viral strand are the original insertion sequences. The C-strand from parental phage RF DNA was paired with viral strand of its insertion derivative to produce gapped duplex DNA, and the gap was sealed with dI or deoxyuridine containing synthetic oligodeoxyribonucleotide. A-I, C-I, G-I, T-I, and G-U are the resulting substrates and DNA sequence shown on each C-strand of the the synthetic linker used. In the presence of dI, the substrates were refractory to the restriction endonuclease scoring. After the repair, DNA products become sensitive to restriction endonuclease cleavage. The recognition sequence of corresponding restriction endonuclease markers for repair products are shown in bold on V-strands
    Figure Legend Snippet: Map of M13mp18 and f1PM based heteroduplex substrates. a The map of bacteriophage M13mp18 replicative form (RF) DNA shows restriction enzyme sites relevant to this study with derivatives M13LR1 and M13LR3 containing 22-bp insertions at the unique HindIII restriction site, and phage M13WX1 and M13X22 containing 26-bp and 22-bp insertions at Xba I site respectively. b The map of bacteriophage f1PM RF DNA with its derivative f1PMA with a 27-bp insertion at Xba I. ‘V’, phage viral strand. ‘C’, phage complementary strand. Underlines beneath each viral strand are the original insertion sequences. The C-strand from parental phage RF DNA was paired with viral strand of its insertion derivative to produce gapped duplex DNA, and the gap was sealed with dI or deoxyuridine containing synthetic oligodeoxyribonucleotide. A-I, C-I, G-I, T-I, and G-U are the resulting substrates and DNA sequence shown on each C-strand of the the synthetic linker used. In the presence of dI, the substrates were refractory to the restriction endonuclease scoring. After the repair, DNA products become sensitive to restriction endonuclease cleavage. The recognition sequence of corresponding restriction endonuclease markers for repair products are shown in bold on V-strands

    Techniques Used: Sequencing

    Related Articles

    Clone Assay:

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: The PCR products were cloned at the Nde I and Eco RI sites of pET21a (+) vector and expressed as tag-less native proteins. .. Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB).

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: The PCR products were cloned at the Nde I and Eco RI sites of pET21a (+) vector and expressed as tag-less native proteins. .. Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB).

    Article Title: A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli
    Article Snippet: Paragraph title: 4.2. Gene Construction and Cloning ... The ligation mixture was prepared by adding digested vector and digested ADP fragment with DNA ligase and its suitable ligation buffer (New England Biolabs, UK).

    Amplification:

    Article Title: Direct Selection of IS903 Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of Actinobacillus actinomycetemcomitans
    Article Snippet: A 1-μg portion of Hin dIII-digested genomic DNA was ligated with 10 U of DNA ligase (New England Biolabs) in a 50-μl reaction mixture at 14°C for 16 h. The large reaction volume was used to facilitate intramolecular ligation. .. The A. actinomycetemcomitans genomic DNA adjacent to the transposon was amplified by PCR with primers which hybridize within the transposon and are oriented outward: kan Start (5′-GTTTCCCGTTGAATATGGCTGGG-3′) and kan Stop (5′-GCAGTTTCATTTGATGCTCGA-3′).

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB). .. Furthermore, to get full length hexa-histidine-tagged Pol I, PCR amplified E . coli polA gene was cloned at the NdeI and HindIII sites of pET28a (+) vector.

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB). .. Furthermore, to get full length hexa-histidine-tagged Pol I, PCR amplified E . coli polA gene was cloned at the NdeI and HindIII sites of pET28a (+) vector.

    Blocking Assay:

    Article Title: The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *
    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). .. A DNA extension primer (5′-TAM-CGC CAG GGT TTT CCC AGT CAC GAC), labeled with 5-carboxytetramethylrhodamine (TAM) and a RNA-DNA blocking oligonucleotide (5′-rGrCrC rArArG rCrUrU rGCA TGC CTG CAG GTC GAC TCT AGA GGA TCC CCG GGT ACC GAG CTC GAA TT-FAM-3′) 3′-labeled with 5-carboxyfluorescein (FAM) were purchased from Integrated DNA Technologies (IDT, Coralville, IA).

    Real-time Polymerase Chain Reaction:

    Article Title: Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification
    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). .. Calibration standards for real-time PCR were prepared by gravimetric ten-fold serial dilutions of HPD in TE buffer.

    Incubation:

    Article Title: GyrA Interacts with MarR To Reduce Repression of the marRAB Operon in Escherichia coli ▿
    Article Snippet: One unit (120 pmol) of E. coli DNA gyrase and 0.5 μg of pUC19 DNA relaxed by topoisomerase I (both from New England Biolabs) were mixed with or without 250 pmol MarR in 30 μl reaction buffer containing 35 mM Tris-HCl, 24 mM KCl, 4 mM MgCl2, 2 mM dithiothreitol (DTT), 1.75 mM ATP, 5 mM spermidine, 0.1 mg/ml bovine serum albumin, 6.5% glycerol at pH 7.5. .. After incubation for 30 min at 37°C, the degree of DNA supercoiling, with or without GyrA and/or MarR, was assessed by agarose gel electrophoresis with 0.8% separation at 30 V, stained by 0.25 mg·liter−1 ethidium bromide without SDS and EDTA.

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA
    Article Snippet: .. E. coli DNA gyrase (NEB) at varying concentrations (184 – 920 nM) was incubated with 40 nM of Cy5-labeled, 160 bp double stranded DNA for 30 min. in 35 mM Tris-HCl pH 7.5, 5 mM MgCl2 , 24 mM KCl, 2mM DTT and 10% glycerol at room temperature. ..

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Paragraph title: Supporting information Protein gels to evaluate ligase purity. Incubation of ligases with ssDNA to test for contaminating nuclease activity. Reaction endpoint and dsDNA nuclease contamination assessment for wild type ligases. Reaction endpoint and dsDNA nuclease contamination assessment for fusion ligases. Effect of monovalent cations on active blunt/cohesive-end ligases. Separate addition of DNA binding domains to PBCV1 DNA ligase. Fluorescence anisotropy DNA binding curves. Capillary gel electrophoresis panel raw data. ... In this case the buffer used was E. Coli DNA Ligase reaction buffer from NEB: 30 mM Tris-HCl, pH 8 @ 25°C,4 mM MgCl2 , 26 μM NAD, 1 mM DTT, 50 μg/ml BSA.

    Direct Fluorescent Antibody Test:

    Article Title: 3,4-Dimethoxyphenyl bis-benzimidazole, a novel DNA topoisomerase inhibitor that preferentially targets Escherichia coli topoisomerase I
    Article Snippet: Synthesis of the bis-benzimidazole derivatives DCA, DFA and DMA has been described previously. .. E. coli DNA gyrase and its relaxed substrate were purchased from New England Biolabs (GmBH, Germany).

    Activity Assay:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Paragraph title: Supporting information Protein gels to evaluate ligase purity. Incubation of ligases with ssDNA to test for contaminating nuclease activity. Reaction endpoint and dsDNA nuclease contamination assessment for wild type ligases. Reaction endpoint and dsDNA nuclease contamination assessment for fusion ligases. Effect of monovalent cations on active blunt/cohesive-end ligases. Separate addition of DNA binding domains to PBCV1 DNA ligase. Fluorescence anisotropy DNA binding curves. Capillary gel electrophoresis panel raw data. ... In this case the buffer used was E. Coli DNA Ligase reaction buffer from NEB: 30 mM Tris-HCl, pH 8 @ 25°C,4 mM MgCl2 , 26 μM NAD, 1 mM DTT, 50 μg/ml BSA.

    Countercurrent Chromatography:

    Article Title: The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *
    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). .. A DNA extension primer (5′-TAM-CGC CAG GGT TTT CCC AGT CAC GAC), labeled with 5-carboxytetramethylrhodamine (TAM) and a RNA-DNA blocking oligonucleotide (5′-rGrCrC rArArG rCrUrU rGCA TGC CTG CAG GTC GAC TCT AGA GGA TCC CCG GGT ACC GAG CTC GAA TT-FAM-3′) 3′-labeled with 5-carboxyfluorescein (FAM) were purchased from Integrated DNA Technologies (IDT, Coralville, IA).

    Flow Cytometry:

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA
    Article Snippet: E. coli DNA gyrase (NEB) was used at a final concentration of about 9.2 nM (20 units/ml). .. The flow chamber was flushed with Topo II buffer before adding TOP2A or gyrase.

    Inverse PCR:

    Article Title: Direct Selection of IS903 Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of Actinobacillus actinomycetemcomitans
    Article Snippet: Paragraph title: Inverse PCR. ... A 1-μg portion of Hin dIII-digested genomic DNA was ligated with 10 U of DNA ligase (New England Biolabs) in a 50-μl reaction mixture at 14°C for 16 h. The large reaction volume was used to facilitate intramolecular ligation.

    Ligation:

    Article Title: Direct Selection of IS903 Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of Actinobacillus actinomycetemcomitans
    Article Snippet: .. A 1-μg portion of Hin dIII-digested genomic DNA was ligated with 10 U of DNA ligase (New England Biolabs) in a 50-μl reaction mixture at 14°C for 16 h. The large reaction volume was used to facilitate intramolecular ligation. .. The A. actinomycetemcomitans genomic DNA adjacent to the transposon was amplified by PCR with primers which hybridize within the transposon and are oriented outward: kan Start (5′-GTTTCCCGTTGAATATGGCTGGG-3′) and kan Stop (5′-GCAGTTTCATTTGATGCTCGA-3′).

    Article Title: A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli
    Article Snippet: .. The ligation mixture was prepared by adding digested vector and digested ADP fragment with DNA ligase and its suitable ligation buffer (New England Biolabs, UK). .. Adiponectin fragment was cloned in pGEM® -T cloning vector (Promega, USA).

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: In this case the buffer used was E. Coli DNA Ligase reaction buffer from NEB: 30 mM Tris-HCl, pH 8 @ 25°C,4 mM MgCl2 , 26 μM NAD, 1 mM DTT, 50 μg/ml BSA. .. QL = Quick Ligation.

    other:

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

    Polymerase Chain Reaction:

    Article Title: Direct Selection of IS903 Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of Actinobacillus actinomycetemcomitans
    Article Snippet: A 1-μg portion of Hin dIII-digested genomic DNA was ligated with 10 U of DNA ligase (New England Biolabs) in a 50-μl reaction mixture at 14°C for 16 h. The large reaction volume was used to facilitate intramolecular ligation. .. The A. actinomycetemcomitans genomic DNA adjacent to the transposon was amplified by PCR with primers which hybridize within the transposon and are oriented outward: kan Start (5′-GTTTCCCGTTGAATATGGCTGGG-3′) and kan Stop (5′-GCAGTTTCATTTGATGCTCGA-3′).

    Article Title: Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification
    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). .. HPD was fragmented by sonication to an average size of 3,000 bp and absolutely quantified by ICP-OES .

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: The PCR products were cloned at the Nde I and Eco RI sites of pET21a (+) vector and expressed as tag-less native proteins. .. Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB).

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: The PCR products were cloned at the Nde I and Eco RI sites of pET21a (+) vector and expressed as tag-less native proteins. .. Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB).

    Article Title: A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli
    Article Snippet: The reaction conditions of an overlap-extension PCR through 10 cycles were denaturing step at 95 °C for 45 s, annealing step at 60 °C for 45 s and elongating step at 72 °C for 1 min. .. The ligation mixture was prepared by adding digested vector and digested ADP fragment with DNA ligase and its suitable ligation buffer (New England Biolabs, UK).

    Sonication:

    Article Title: Quantification of Trace-Level DNA by Real-Time Whole Genome Amplification
    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). .. HPD was fragmented by sonication to an average size of 3,000 bp and absolutely quantified by ICP-OES .

    Recombinant:

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA
    Article Snippet: Recombinant human topoisomerase II α (USB) was used at a final concentration of about 0.6 nM. .. E. coli DNA gyrase (NEB) was used at a final concentration of about 9.2 nM (20 units/ml).

    Article Title: A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli
    Article Snippet: The ligation mixture was prepared by adding digested vector and digested ADP fragment with DNA ligase and its suitable ligation buffer (New England Biolabs, UK). .. After sequence verification, the recombinant plasmid was double digested with Eco RI and Not I restriction enzymes to generate fragments with cohesive termini.

    High Performance Liquid Chromatography:

    Article Title: Molecular Anatomy of the Human Excision Nuclease Assembled at Sites of DNA Damage
    Article Snippet: T4 polynucleotide kinase and DNA ligase were purchased from New England BioLabs, DNase I was from Invitrogen, and the TnT Quick Coupled Transcription/Translation system was from Promega. .. The dodecamer containing a furan-side psoralen-thymine adduct was prepared by reacting a synthetic oligomer with 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) followed by high-performance liquid chromatography purification and was a generous gift from J. E. Hearst (University of California, Berkeley).

    Nucleic Acid Electrophoresis:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Paragraph title: Supporting information Protein gels to evaluate ligase purity. Incubation of ligases with ssDNA to test for contaminating nuclease activity. Reaction endpoint and dsDNA nuclease contamination assessment for wild type ligases. Reaction endpoint and dsDNA nuclease contamination assessment for fusion ligases. Effect of monovalent cations on active blunt/cohesive-end ligases. Separate addition of DNA binding domains to PBCV1 DNA ligase. Fluorescence anisotropy DNA binding curves. Capillary gel electrophoresis panel raw data. ... In this case the buffer used was E. Coli DNA Ligase reaction buffer from NEB: 30 mM Tris-HCl, pH 8 @ 25°C,4 mM MgCl2 , 26 μM NAD, 1 mM DTT, 50 μg/ml BSA.

    Fluorescence:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Paragraph title: Supporting information Protein gels to evaluate ligase purity. Incubation of ligases with ssDNA to test for contaminating nuclease activity. Reaction endpoint and dsDNA nuclease contamination assessment for wild type ligases. Reaction endpoint and dsDNA nuclease contamination assessment for fusion ligases. Effect of monovalent cations on active blunt/cohesive-end ligases. Separate addition of DNA binding domains to PBCV1 DNA ligase. Fluorescence anisotropy DNA binding curves. Capillary gel electrophoresis panel raw data. ... In this case the buffer used was E. Coli DNA Ligase reaction buffer from NEB: 30 mM Tris-HCl, pH 8 @ 25°C,4 mM MgCl2 , 26 μM NAD, 1 mM DTT, 50 μg/ml BSA.

    Magnetic Beads:

    Article Title: Rho-dependent transcription termination in bacteria recycles RNA polymerases stalled at DNA lesions
    Article Snippet: The streptavidin-coated magnetic beads were from Promega. .. The restriction enzymes, the DNA ligase and the RNA polymerase were purchased from NEB, USA.

    Mutagenesis:

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB). .. Site directed mutagenesis was performed on the pET28a-polA vector using Q5 Site-Directed Mutagenesis Kit from NEB to get 3’-exo- Pol I (D355 A, E357 A), 5’-exo- Pol I (D13 N, D138 N), and exo- Pol I (D13 N, D138 N, D355 A, E357 A) [ , ].

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB). .. Site directed mutagenesis was performed on the pET28a- polA vector using Q5 Site-Directed Mutagenesis Kit from NEB to get 3’-exo- Pol I (D355 A, E357 A), 5’-exo- Pol I (D13 N, D138 N), and exo- Pol I (D13 N, D138 N, D355 A, E357 A) [ , ].

    Labeling:

    Article Title: The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *
    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). .. A DNA extension primer (5′-TAM-CGC CAG GGT TTT CCC AGT CAC GAC), labeled with 5-carboxytetramethylrhodamine (TAM) and a RNA-DNA blocking oligonucleotide (5′-rGrCrC rArArG rCrUrU rGCA TGC CTG CAG GTC GAC TCT AGA GGA TCC CCG GGT ACC GAG CTC GAA TT-FAM-3′) 3′-labeled with 5-carboxyfluorescein (FAM) were purchased from Integrated DNA Technologies (IDT, Coralville, IA).

    Purification:

    Article Title: Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases
    Article Snippet: Materials Restriction enzymes Nt.BbvCI, SphI, BamHI, E. coli DNA gyrase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA). .. E. coli DNA topoisomerase I was purified as described previously .

    Article Title: The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *
    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). .. 9°N polD was purified as previously described ( ).

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: The overexpressed proteins were purified using standard protocols [ , ]. .. Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB).

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: The overexpressed proteins were purified using standard protocols [ , ]. .. Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB).

    Article Title: A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli
    Article Snippet: The ligation mixture was prepared by adding digested vector and digested ADP fragment with DNA ligase and its suitable ligation buffer (New England Biolabs, UK). .. Digested fragments were purified using the QIAquick Gel Extraction kit (Qiagen, USA), as described in the manufacturer’s protocol.

    Article Title: Molecular Anatomy of the Human Excision Nuclease Assembled at Sites of DNA Damage
    Article Snippet: T4 polynucleotide kinase and DNA ligase were purchased from New England BioLabs, DNase I was from Invitrogen, and the TnT Quick Coupled Transcription/Translation system was from Promega. .. The dodecamer containing a furan-side psoralen-thymine adduct was prepared by reacting a synthetic oligomer with 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) followed by high-performance liquid chromatography purification and was a generous gift from J. E. Hearst (University of California, Berkeley).

    Sequencing:

    Article Title: A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli
    Article Snippet: The ligation mixture was prepared by adding digested vector and digested ADP fragment with DNA ligase and its suitable ligation buffer (New England Biolabs, UK). .. After sequence verification, the recombinant plasmid was double digested with Eco RI and Not I restriction enzymes to generate fragments with cohesive termini.

    Affinity Chromatography:

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB). .. The hexa-histidine-tagged Pol I and its mutated versions were overexpressed and were purified by nickel-NTA affinity chromatography.

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB). .. The hexa-histidine-tagged Pol I and its mutated versions were overexpressed and were purified by nickel-NTA affinity chromatography.

    Electrophoretic Mobility Shift Assay:

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA
    Article Snippet: Paragraph title: Electrophoretic Mobility shift Assay ... E. coli DNA gyrase (NEB) at varying concentrations (184 – 920 nM) was incubated with 40 nM of Cy5-labeled, 160 bp double stranded DNA for 30 min. in 35 mM Tris-HCl pH 7.5, 5 mM MgCl2 , 24 mM KCl, 2mM DTT and 10% glycerol at room temperature.

    Gel Extraction:

    Article Title: Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases
    Article Snippet: Materials Restriction enzymes Nt.BbvCI, SphI, BamHI, E. coli DNA gyrase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA). .. QIAquick Nucleotide Removal Kit and QIAquick Gel Extraction Kit were obtained from Qiagen, Inc (Valencia, CA).

    Article Title: A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli
    Article Snippet: The ligation mixture was prepared by adding digested vector and digested ADP fragment with DNA ligase and its suitable ligation buffer (New England Biolabs, UK). .. Digested fragments were purified using the QIAquick Gel Extraction kit (Qiagen, USA), as described in the manufacturer’s protocol.

    IA:

    Article Title: The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *
    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). .. A DNA extension primer (5′-TAM-CGC CAG GGT TTT CCC AGT CAC GAC), labeled with 5-carboxytetramethylrhodamine (TAM) and a RNA-DNA blocking oligonucleotide (5′-rGrCrC rArArG rCrUrU rGCA TGC CTG CAG GTC GAC TCT AGA GGA TCC CCG GGT ACC GAG CTC GAA TT-FAM-3′) 3′-labeled with 5-carboxyfluorescein (FAM) were purchased from Integrated DNA Technologies (IDT, Coralville, IA).

    Plasmid Preparation:

    Article Title: 3,4-Dimethoxyphenyl bis-benzimidazole, a novel DNA topoisomerase inhibitor that preferentially targets Escherichia coli topoisomerase I
    Article Snippet: Catenated kinetoplast DNA (kDNA) and pBAD/Thio and pHOT1 plasmid DNA were purchased from TopoGen Inc. (Port Orange, FL, USA). .. E. coli DNA gyrase and its relaxed substrate were purchased from New England Biolabs (GmBH, Germany).

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: The PCR products were cloned at the Nde I and Eco RI sites of pET21a (+) vector and expressed as tag-less native proteins. .. Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB).

    Article Title: Escherichia coli β-clamp slows down DNA polymerase I dependent nick translation while accelerating ligation
    Article Snippet: The PCR products were cloned at the Nde I and Eco RI sites of pET21a (+) vector and expressed as tag-less native proteins. .. Pol I, exo- Klenow, E . coli DNA ligase, and ultrapure nucleotides were purchased from New England Biolabs (NEB).

    Article Title: A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli
    Article Snippet: .. The ligation mixture was prepared by adding digested vector and digested ADP fragment with DNA ligase and its suitable ligation buffer (New England Biolabs, UK). .. Adiponectin fragment was cloned in pGEM® -T cloning vector (Promega, USA).

    Software:

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA
    Article Snippet: E. coli DNA gyrase (NEB) at varying concentrations (184 – 920 nM) was incubated with 40 nM of Cy5-labeled, 160 bp double stranded DNA for 30 min. in 35 mM Tris-HCl pH 7.5, 5 mM MgCl2 , 24 mM KCl, 2mM DTT and 10% glycerol at room temperature. .. The free and gyrase-complexed DNA bands were quantified using ImageQuant software (GE Healthcare).

    Electrophoresis:

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA
    Article Snippet: E. coli DNA gyrase (NEB) at varying concentrations (184 – 920 nM) was incubated with 40 nM of Cy5-labeled, 160 bp double stranded DNA for 30 min. in 35 mM Tris-HCl pH 7.5, 5 mM MgCl2 , 24 mM KCl, 2mM DTT and 10% glycerol at room temperature. .. After electrophoresis on ice for about 2 hours at 8V/cm, the DNA was visualized using a Typhoon Trio fluorescent scanner (GE Healthcare) with detection at 670 nm.

    Binding Assay:

    Article Title: Comparative analysis of the end-joining activity of several DNA ligases
    Article Snippet: Paragraph title: Supporting information Protein gels to evaluate ligase purity. Incubation of ligases with ssDNA to test for contaminating nuclease activity. Reaction endpoint and dsDNA nuclease contamination assessment for wild type ligases. Reaction endpoint and dsDNA nuclease contamination assessment for fusion ligases. Effect of monovalent cations on active blunt/cohesive-end ligases. Separate addition of DNA binding domains to PBCV1 DNA ligase. Fluorescence anisotropy DNA binding curves. Capillary gel electrophoresis panel raw data. ... In this case the buffer used was E. Coli DNA Ligase reaction buffer from NEB: 30 mM Tris-HCl, pH 8 @ 25°C,4 mM MgCl2 , 26 μM NAD, 1 mM DTT, 50 μg/ml BSA.

    Agarose Gel Electrophoresis:

    Article Title: GyrA Interacts with MarR To Reduce Repression of the marRAB Operon in Escherichia coli ▿
    Article Snippet: One unit (120 pmol) of E. coli DNA gyrase and 0.5 μg of pUC19 DNA relaxed by topoisomerase I (both from New England Biolabs) were mixed with or without 250 pmol MarR in 30 μl reaction buffer containing 35 mM Tris-HCl, 24 mM KCl, 4 mM MgCl2, 2 mM dithiothreitol (DTT), 1.75 mM ATP, 5 mM spermidine, 0.1 mg/ml bovine serum albumin, 6.5% glycerol at pH 7.5. .. After incubation for 30 min at 37°C, the degree of DNA supercoiling, with or without GyrA and/or MarR, was assessed by agarose gel electrophoresis with 0.8% separation at 30 V, stained by 0.25 mg·liter−1 ethidium bromide without SDS and EDTA.

    Concentration Assay:

    Article Title: E. coli gyrase fails to negatively supercoil diaminopurine-substituted DNA
    Article Snippet: .. E. coli DNA gyrase (NEB) was used at a final concentration of about 9.2 nM (20 units/ml). ..

    CTG Assay:

    Article Title: The Roles of Family B and D DNA Polymerases in Thermococcus Species 9°N Okazaki Fragment Maturation *
    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). .. A DNA extension primer (5′-TAM-CGC CAG GGT TTT CCC AGT CAC GAC), labeled with 5-carboxytetramethylrhodamine (TAM) and a RNA-DNA blocking oligonucleotide (5′-rGrCrC rArArG rCrUrU rGCA TGC CTG CAG GTC GAC TCT AGA GGA TCC CCG GGT ACC GAG CTC GAA TT-FAM-3′) 3′-labeled with 5-carboxyfluorescein (FAM) were purchased from Integrated DNA Technologies (IDT, Coralville, IA).

    Staining:

    Article Title: GyrA Interacts with MarR To Reduce Repression of the marRAB Operon in Escherichia coli ▿
    Article Snippet: One unit (120 pmol) of E. coli DNA gyrase and 0.5 μg of pUC19 DNA relaxed by topoisomerase I (both from New England Biolabs) were mixed with or without 250 pmol MarR in 30 μl reaction buffer containing 35 mM Tris-HCl, 24 mM KCl, 4 mM MgCl2, 2 mM dithiothreitol (DTT), 1.75 mM ATP, 5 mM spermidine, 0.1 mg/ml bovine serum albumin, 6.5% glycerol at pH 7.5. .. After incubation for 30 min at 37°C, the degree of DNA supercoiling, with or without GyrA and/or MarR, was assessed by agarose gel electrophoresis with 0.8% separation at 30 V, stained by 0.25 mg·liter−1 ethidium bromide without SDS and EDTA.

    HMT Assay:

    Article Title: Molecular Anatomy of the Human Excision Nuclease Assembled at Sites of DNA Damage
    Article Snippet: T4 polynucleotide kinase and DNA ligase were purchased from New England BioLabs, DNase I was from Invitrogen, and the TnT Quick Coupled Transcription/Translation system was from Promega. .. The dodecamer containing a furan-side psoralen-thymine adduct was prepared by reacting a synthetic oligomer with 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) followed by high-performance liquid chromatography purification and was a generous gift from J. E. Hearst (University of California, Berkeley).

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    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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    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

    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

    In vitro construction of ADP fragment using overlap-extension PCR. ADP fragment was amplified by PCR through 32 cycles include denaturing step (95 °C for 45 s), annealing step (60 °C for 45 s) and elongation step (72 °C for 1 min). The two fragments were then purified and joined through 10 cycles of overlap-extension PCR include denaturing step at 95 °C for 45 s, annealing step at 60 °C for 45 s and elongating step at 72 °C for 1 min. L1 : PCR product of exon 2 (204 bp). L2 : PCR product of exon 3 (531 bp). L3 : the full length of ADP fragment (734 bp). M : 100 bp DNA marker.

    Journal: International Journal of Molecular Sciences

    Article Title: A Comparative Study on the Expression, Purification and Functional Characterization of Human Adiponectin in Pichia pastoris and Escherichia coli

    doi: 10.3390/ijms13033549

    Figure Lengend Snippet: In vitro construction of ADP fragment using overlap-extension PCR. ADP fragment was amplified by PCR through 32 cycles include denaturing step (95 °C for 45 s), annealing step (60 °C for 45 s) and elongation step (72 °C for 1 min). The two fragments were then purified and joined through 10 cycles of overlap-extension PCR include denaturing step at 95 °C for 45 s, annealing step at 60 °C for 45 s and elongating step at 72 °C for 1 min. L1 : PCR product of exon 2 (204 bp). L2 : PCR product of exon 3 (531 bp). L3 : the full length of ADP fragment (734 bp). M : 100 bp DNA marker.

    Article Snippet: The ligation mixture was prepared by adding digested vector and digested ADP fragment with DNA ligase and its suitable ligation buffer (New England Biolabs, UK).

    Techniques: In Vitro, Polymerase Chain Reaction, Amplification, Purification, Marker

    An experimental strategy to construct relaxed (rx) or supercoiled (sc) pAB1_FL905. ( A ) Oligomer FL905 that contains the 42 nt. AT sequence is ligated between the two Nt.BbvCI sites of plasmid pAB1 to yield rx pAB1_FL905. ( B ) Sc pAB1_FL905 can be generated through the treatment of rx pAB1_FL905 by E. coli DNA gyrase. The fluorescence intensity of fluorescein is dependent on the supercoiling status of pAB1_FL905.

    Journal: Scientific Reports

    Article Title: Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    doi: 10.1038/srep36006

    Figure Lengend Snippet: An experimental strategy to construct relaxed (rx) or supercoiled (sc) pAB1_FL905. ( A ) Oligomer FL905 that contains the 42 nt. AT sequence is ligated between the two Nt.BbvCI sites of plasmid pAB1 to yield rx pAB1_FL905. ( B ) Sc pAB1_FL905 can be generated through the treatment of rx pAB1_FL905 by E. coli DNA gyrase. The fluorescence intensity of fluorescein is dependent on the supercoiling status of pAB1_FL905.

    Article Snippet: Materials Restriction enzymes Nt.BbvCI, SphI, BamHI, E. coli DNA gyrase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA).

    Techniques: Construct, Sequencing, Plasmid Preparation, Generated, Fluorescence

    ( A ) Fluorescence spectra of sc (red line), rx (black line), and nk (blue line) pAB1_FL509. λex = 470 nm. ( B ) Kinetics of the nicking reaction by Nt.BbvCI. Briefly, 60 μL of 1 × CutSmart buffer containing 500 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of Nt.BbvCI were added to initiate the nicking reaction. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( C ) Kinetics of the relaxation reaction by E. coli DNA topoisomerase I. For the relaxation reaction, 90 μL of 1 × NEBuffer 4 (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(AC) 2 , 1 mM DTT, pH 7.9) containing 270 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 0.67 μM of E. coli DNA topoisomerase I was used to relax the sc pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( D ) Kinetics of the supercoiling reaction by E. coli DNA gyrase. For the supercoiling reaction, 90 μL of 1 × gyrase buffer containing 1 μg of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 30 units of E. coli DNA gyrase was used to supercoil the rx pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm.

    Journal: Scientific Reports

    Article Title: Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    doi: 10.1038/srep36006

    Figure Lengend Snippet: ( A ) Fluorescence spectra of sc (red line), rx (black line), and nk (blue line) pAB1_FL509. λex = 470 nm. ( B ) Kinetics of the nicking reaction by Nt.BbvCI. Briefly, 60 μL of 1 × CutSmart buffer containing 500 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of Nt.BbvCI were added to initiate the nicking reaction. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( C ) Kinetics of the relaxation reaction by E. coli DNA topoisomerase I. For the relaxation reaction, 90 μL of 1 × NEBuffer 4 (50 mM KAc, 20 mM Tris-Ac, 10 mM Mg(AC) 2 , 1 mM DTT, pH 7.9) containing 270 ng of sc pAB1_FL905 was prepared and equilibrated to 37 °C. 0.67 μM of E. coli DNA topoisomerase I was used to relax the sc pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm. ( D ) Kinetics of the supercoiling reaction by E. coli DNA gyrase. For the supercoiling reaction, 90 μL of 1 × gyrase buffer containing 1 μg of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 30 units of E. coli DNA gyrase was used to supercoil the rx pAB1_FL905. The fluorescence intensity at λem = 521 nm was monitor with λex = 470 nm.

    Article Snippet: Materials Restriction enzymes Nt.BbvCI, SphI, BamHI, E. coli DNA gyrase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA).

    Techniques: Fluorescence

    DNA gyrase was potently inhibited by novobiocin ( A ) and ciprofloxacin ( B ). For DNA supercoiling reactions, 60 μL μL of 1 × gyrase buffer containing 670 ng of of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of DNA gyrase was used to supercoil the rx pAB1_FL905 in the presence of different concentrations of novobiocin and ciprofloxacin. The fluorescence intensity at λem = 521 nm was monitor with λex = 494 nm. The inhibition IC50 was estimated to be 0.48 ± 0.14 and 2.57 ± 1.b μM for novobiocin and ciprofloxacin, respectively.

    Journal: Scientific Reports

    Article Title: Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    doi: 10.1038/srep36006

    Figure Lengend Snippet: DNA gyrase was potently inhibited by novobiocin ( A ) and ciprofloxacin ( B ). For DNA supercoiling reactions, 60 μL μL of 1 × gyrase buffer containing 670 ng of of rx pAB1_FL905 was prepared and equilibrated to 37 °C. 20 units of DNA gyrase was used to supercoil the rx pAB1_FL905 in the presence of different concentrations of novobiocin and ciprofloxacin. The fluorescence intensity at λem = 521 nm was monitor with λex = 494 nm. The inhibition IC50 was estimated to be 0.48 ± 0.14 and 2.57 ± 1.b μM for novobiocin and ciprofloxacin, respectively.

    Article Snippet: Materials Restriction enzymes Nt.BbvCI, SphI, BamHI, E. coli DNA gyrase, and T4 DNA ligase were purchased from New England Biolabs (Beverly, MA, USA).

    Techniques: Fluorescence, Inhibition