pbr322  (New England Biolabs)


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    Name:
    pBR322 Vector
    Description:
    pBR322 Vector 250 ug
    Catalog Number:
    n3033l
    Price:
    302
    Size:
    250 ug
    Category:
    Vectors Plasmids
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    New England Biolabs pbr322
    pBR322 Vector
    pBR322 Vector 250 ug
    https://www.bioz.com/result/pbr322/product/New England Biolabs
    Average 95 stars, based on 70 article reviews
    Price from $9.99 to $1999.99
    pbr322 - by Bioz Stars, 2021-01
    95/100 stars

    Images

    1) Product Images from "A robust assay to measure DNA topology-dependent protein binding affinity"

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1381

    Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).
    Figure Legend Snippet: Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).

    Techniques Used: Binding Assay, SYBR Green Assay, Staining, Electrophoresis, Agarose Gel Electrophoresis, Plasmid Preparation, Mutagenesis

    (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).
    Figure Legend Snippet: (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).

    Techniques Used: Binding Assay

    2) Product Images from "Inverse transposition by the RAG1 and RAG2 proteins: role reversal of donor and target DNA"

    Article Title: Inverse transposition by the RAG1 and RAG2 proteins: role reversal of donor and target DNA

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdf630

    Fig. 2. Transposition of non-RSS sequence mediated by RAG1 and RAG2 proteins. ( A ) Schematic drawings of oligonucleotides used in the plasmid assay for inverse transposition. The position of radioactive label on the bottom strand of an RSS-containing oligonucleotide is indicated by an asterisk. The top and bottom strands are defined by the orientation of the RSS sequence. Either intact (substrates 2 and 4) or pre-nicked dideoxy (substrates 1 and 3) oligonucleotides were used. The labeled oligonucleotide was incubated with supercoiled pBR322 DNA in the presence of RAG1/2, HMG1 and Mg 2+ at 37°C for 2 h. Reaction samples were deproteinized and analyzed by agarose gel electrophoresis. ( B ) Strand transfer of the coding sequence to a non-RSS plasmid requires both RAG1 and RAG2 proteins. In addition to 3′- 32 P-labeled intact substrate (substrate 4), pBR322 plasmid and reaction buffer, lanes 1, 2 and 3 (all from the same experiment) contain purified RAG1, purified RAG2 and co-expressed RAG1/2 proteins, respectively. The reactions were analyzed on native agarose gels. N, nicked circular plasmid; L, linear plasmid; M, molecular marker of λ Hin dIII ladder. ( C ) Covalent strand connection of the inverse- transposition products was analyzed by comparing the deproteinized inverse-transposition products on a native gel (1.2%, left panel) and an alkaline denaturing gel (0.8%, right panel). The substrate for each reaction is indicated. 1× and 2× indicate the products with a single and double length of the linear single-stranded plasmid, respectively.
    Figure Legend Snippet: Fig. 2. Transposition of non-RSS sequence mediated by RAG1 and RAG2 proteins. ( A ) Schematic drawings of oligonucleotides used in the plasmid assay for inverse transposition. The position of radioactive label on the bottom strand of an RSS-containing oligonucleotide is indicated by an asterisk. The top and bottom strands are defined by the orientation of the RSS sequence. Either intact (substrates 2 and 4) or pre-nicked dideoxy (substrates 1 and 3) oligonucleotides were used. The labeled oligonucleotide was incubated with supercoiled pBR322 DNA in the presence of RAG1/2, HMG1 and Mg 2+ at 37°C for 2 h. Reaction samples were deproteinized and analyzed by agarose gel electrophoresis. ( B ) Strand transfer of the coding sequence to a non-RSS plasmid requires both RAG1 and RAG2 proteins. In addition to 3′- 32 P-labeled intact substrate (substrate 4), pBR322 plasmid and reaction buffer, lanes 1, 2 and 3 (all from the same experiment) contain purified RAG1, purified RAG2 and co-expressed RAG1/2 proteins, respectively. The reactions were analyzed on native agarose gels. N, nicked circular plasmid; L, linear plasmid; M, molecular marker of λ Hin dIII ladder. ( C ) Covalent strand connection of the inverse- transposition products was analyzed by comparing the deproteinized inverse-transposition products on a native gel (1.2%, left panel) and an alkaline denaturing gel (0.8%, right panel). The substrate for each reaction is indicated. 1× and 2× indicate the products with a single and double length of the linear single-stranded plasmid, respectively.

    Techniques Used: Sequencing, Plasmid Preparation, Labeling, Incubation, Agarose Gel Electrophoresis, Purification, Marker

    Fig. 3. Hairpin structure on the plasmid DNA resulting from inverse transposition. ( A ) Inverse-transposition reactions with an intact 3′- 32 P-12 RSS oligonucleotide (*) and supercoiled pBR322 were carried out in the presence of RAG1/2, HMG1 and 5 mM Mg 2+ /0.5 mM Mn 2+ (Mn 2+ conditions) at 37°C for 2 h, as described in Materials and methods. Predicted configurations of the inverse-transposition products are shown. ( B ) Two-dimensional gel analysis of the inverse-transposition product. The reaction product was treated with mung bean nuclease or with mock digestion before deproteinization, and analyzed by two-dimensional gel electrophoresis. The first dimension was fractionated on a 1.2% native agarose gel, and the second dimension on a 1% alkaline denaturing gel. Three species, 1L, 2L and 1N, were detected. The numbers on the left indicate the positions of molecular markers.
    Figure Legend Snippet: Fig. 3. Hairpin structure on the plasmid DNA resulting from inverse transposition. ( A ) Inverse-transposition reactions with an intact 3′- 32 P-12 RSS oligonucleotide (*) and supercoiled pBR322 were carried out in the presence of RAG1/2, HMG1 and 5 mM Mg 2+ /0.5 mM Mn 2+ (Mn 2+ conditions) at 37°C for 2 h, as described in Materials and methods. Predicted configurations of the inverse-transposition products are shown. ( B ) Two-dimensional gel analysis of the inverse-transposition product. The reaction product was treated with mung bean nuclease or with mock digestion before deproteinization, and analyzed by two-dimensional gel electrophoresis. The first dimension was fractionated on a 1.2% native agarose gel, and the second dimension on a 1% alkaline denaturing gel. Three species, 1L, 2L and 1N, were detected. The numbers on the left indicate the positions of molecular markers.

    Techniques Used: Plasmid Preparation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Agarose Gel Electrophoresis

    3) Product Images from "The Fitness Landscapes of cis-Acting Binding Sites in Different Promoter and Environmental Contexts"

    Article Title: The Fitness Landscapes of cis-Acting Binding Sites in Different Promoter and Environmental Contexts

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001042

    Schematic Diagram of selection promoter. Sequences of the four randomized promoter libraries (top), and a diagram mapping the promoter components (bottom). The MarA or sites were varied (blue boxes). Spacing between the binding sites may affect transcriptional output [5] . We used the same sequence between the and found in the tet promoter of pBR322 in our selection system because it has the optimal spacing [11] . We used a slight variation of the spacer between the MarA binding site and the from the mar gene [22] . Spacer sequences are shown in gray. Restriction sites used to clone synthesized libraries into the selection plasmid are marked in orange. All libraries have 6 randomized bases at the hexamer (green box).
    Figure Legend Snippet: Schematic Diagram of selection promoter. Sequences of the four randomized promoter libraries (top), and a diagram mapping the promoter components (bottom). The MarA or sites were varied (blue boxes). Spacing between the binding sites may affect transcriptional output [5] . We used the same sequence between the and found in the tet promoter of pBR322 in our selection system because it has the optimal spacing [11] . We used a slight variation of the spacer between the MarA binding site and the from the mar gene [22] . Spacer sequences are shown in gray. Restriction sites used to clone synthesized libraries into the selection plasmid are marked in orange. All libraries have 6 randomized bases at the hexamer (green box).

    Techniques Used: Selection, Binding Assay, Sequencing, Synthesized, Plasmid Preparation

    4) Product Images from "Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV "

    Article Title: Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00113-09

    Assay for DNA unwinding of closed circular DNA by PD 0305970 and ciprofloxacin. Relaxed pBR322 DNA (0.8 μg) was preincubated with calf thymus DNA topo I (10 U) at room temperature for 10 min in the presence of MgCl 2 at the concentrations (mM)
    Figure Legend Snippet: Assay for DNA unwinding of closed circular DNA by PD 0305970 and ciprofloxacin. Relaxed pBR322 DNA (0.8 μg) was preincubated with calf thymus DNA topo I (10 U) at room temperature for 10 min in the presence of MgCl 2 at the concentrations (mM)

    Techniques Used:

    Reversal of drug-stabilized DNA cleavage in the absence (top) or presence (bottom) of ATP. (Top) Supercoiled plasmid pBR322 (0.45 μg) was incubated with S. pneumoniae GyrA (0.45 μg) and GyrB (1.7 μg) in a DNA cleavage assay in
    Figure Legend Snippet: Reversal of drug-stabilized DNA cleavage in the absence (top) or presence (bottom) of ATP. (Top) Supercoiled plasmid pBR322 (0.45 μg) was incubated with S. pneumoniae GyrA (0.45 μg) and GyrB (1.7 μg) in a DNA cleavage assay in

    Techniques Used: Plasmid Preparation, Incubation, DNA Cleavage Assay

    Inhibitory activity of PD 0305970 against wild-type and mutant S. pneumoniae type II topoisomerases. Inhibition of DNA supercoiling by DNA gyrase. Relaxed pBR322 plasmid DNA (0.4 μg) was incubated with wild-type (wt) gyrase activity (1 U) or mutant
    Figure Legend Snippet: Inhibitory activity of PD 0305970 against wild-type and mutant S. pneumoniae type II topoisomerases. Inhibition of DNA supercoiling by DNA gyrase. Relaxed pBR322 plasmid DNA (0.4 μg) was incubated with wild-type (wt) gyrase activity (1 U) or mutant

    Techniques Used: Activity Assay, Mutagenesis, Inhibition, Plasmid Preparation, Incubation

    PD 0305970 induces site-specific DNA cleavage by gyrase. (A) PD 0305970 promotes gyrase-mediated double-stranded DNA cleavage. pBR322 DNA linearized with EcoRI was employed as a substrate in a cleavage assay with wild-type S. pneumoniae gyrase (as described
    Figure Legend Snippet: PD 0305970 induces site-specific DNA cleavage by gyrase. (A) PD 0305970 promotes gyrase-mediated double-stranded DNA cleavage. pBR322 DNA linearized with EcoRI was employed as a substrate in a cleavage assay with wild-type S. pneumoniae gyrase (as described

    Techniques Used: Cleavage Assay

    5) Product Images from "Investigation into the antimicrobial action and mechanism of a novel endogenous peptide β-casein 197 from human milk"

    Article Title: Investigation into the antimicrobial action and mechanism of a novel endogenous peptide β-casein 197 from human milk

    Journal: AMB Express

    doi: 10.1186/s13568-017-0409-y

    The DNA-binding properties of β-Casein 197. a ProtParam analysis of predicted binding residues of β-casein 197, predicted binding residues was labeled with a red “+”. b Gel retardation assays. Binding was assayed by the migration of pDNA. Various concentrations of peptides were incubated with 5 µL of 25 µg mL −1 pBR322 vector from E. coli at 37 °C for 1 h, and then the reaction mixtures were applied to 0.7% agarose gel electrophoresis. Lane M DNA marker DL 10,000; Lane 1 5 µL pDNA as control; Lanes 2–6 mixture of pDNA and various concentrations of peptides (12.5, 25, 50, 100 and 500 µg mL −1 )
    Figure Legend Snippet: The DNA-binding properties of β-Casein 197. a ProtParam analysis of predicted binding residues of β-casein 197, predicted binding residues was labeled with a red “+”. b Gel retardation assays. Binding was assayed by the migration of pDNA. Various concentrations of peptides were incubated with 5 µL of 25 µg mL −1 pBR322 vector from E. coli at 37 °C for 1 h, and then the reaction mixtures were applied to 0.7% agarose gel electrophoresis. Lane M DNA marker DL 10,000; Lane 1 5 µL pDNA as control; Lanes 2–6 mixture of pDNA and various concentrations of peptides (12.5, 25, 50, 100 and 500 µg mL −1 )

    Techniques Used: Binding Assay, Labeling, Electrophoretic Mobility Shift Assay, Migration, Incubation, Plasmid Preparation, Agarose Gel Electrophoresis, Marker

    6) Product Images from "Pyridine and p-Nitrophenyl Oxime Esters with Possible Photochemotherapeutic Activity: Synthesis, DNA Photocleavage and DNA Binding Studies"

    Article Title: Pyridine and p-Nitrophenyl Oxime Esters with Possible Photochemotherapeutic Activity: Synthesis, DNA Photocleavage and DNA Binding Studies

    Journal: Molecules

    doi: 10.3390/molecules21070864

    DNA photo-cleavage by various oxime ester conjugates at concentration of 500 μM with plasmid DNA pBR322, at 365 nm for 120 min. Top: Gel electrophoresis picture: Lane 1: DNA without UV irradiation; Lane 2: DNA with UV irradiation; Lanes 3–9: DNA + oxime derivatives ( 1 , 2 , 3 , 5 , 10 , 21 , and 19 , respectively) + UV irradiation; Bottom: Calculation of the % conversion to ss and ds damage.
    Figure Legend Snippet: DNA photo-cleavage by various oxime ester conjugates at concentration of 500 μM with plasmid DNA pBR322, at 365 nm for 120 min. Top: Gel electrophoresis picture: Lane 1: DNA without UV irradiation; Lane 2: DNA with UV irradiation; Lanes 3–9: DNA + oxime derivatives ( 1 , 2 , 3 , 5 , 10 , 21 , and 19 , respectively) + UV irradiation; Bottom: Calculation of the % conversion to ss and ds damage.

    Techniques Used: Concentration Assay, Plasmid Preparation, Nucleic Acid Electrophoresis, Irradiation

    7) Product Images from "Diabetes and aging alter bone marrow contributions to tissue maintenance"

    Article Title: Diabetes and aging alter bone marrow contributions to tissue maintenance

    Journal: International Journal of Physiology, Pathophysiology and Pharmacology

    doi:

    Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and
    Figure Legend Snippet: Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and

    Techniques Used: Derivative Assay, In Situ Hybridization, Labeling, Mouse Assay

    Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young
    Figure Legend Snippet: Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young

    Techniques Used: Quantitation Assay, Derivative Assay, Mouse Assay, Generated

    8) Product Images from "Diabetes and aging alter bone marrow contributions to tissue maintenance"

    Article Title: Diabetes and aging alter bone marrow contributions to tissue maintenance

    Journal: International Journal of Physiology, Pathophysiology and Pharmacology

    doi:

    Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and
    Figure Legend Snippet: Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and

    Techniques Used: Derivative Assay, In Situ Hybridization, Labeling, Mouse Assay

    Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young
    Figure Legend Snippet: Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young

    Techniques Used: Quantitation Assay, Derivative Assay, Mouse Assay, Generated

    9) Product Images from "Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB"

    Article Title: Perturbed structural dynamics underlie inhibition and altered efflux of the multidrug resistance pump AcrB

    Journal: Nature Communications

    doi: 10.1038/s41467-020-19397-2

    AcrB G288D is inhibited by the EPI PAβN. a Molecular docking and multi-copy μs-long MD simulations reveal stable interactions of CIP (orange) and PAβN (cyan) to AcrB G288D T-state monomer and show their likely binding locations. The pose and its orientation are the same as shown for AcrB WT in Fig. 3c . EG = exit channel gate (blue spheres), SL = switch-loop (yellow), and HT = hydrophobic trap (purple). All computational data, including binding free energies can be found in Supplementary Table 5 and Supplementary Figs. 8 , 14 – 16 . b Binding of CIP by AcrB G288D in the presence of 150 μM of PAβN as determined by a fluorescence polarization assay performed by Su et al. 39 . All data are fit as in Fig. 3a ( R 2 = 0.99). Data are the average and standard deviation from independent measurements ( n = 3). c Binding competition assay between PAβN and CIP for AcrB WT . Data are the average and standard deviation from independent measurements ( n = 3). d MIC assays of Escherichia coli containing AcrB WT or AcrB G288D in the presence of PAβN and antibiotics. AcrB was overexpressed in MG1655 ∆acrB from a pBR322 plasmid containing its corresponding acrAB genes, natural promoter and “marbox” sequence. Minocycline = MIN, ciprofloxacin = CIP, and phenylalanine-arginine-β-naphthylamide = PAβN. † PAβN was added at a concentration of 50 μg/ml.
    Figure Legend Snippet: AcrB G288D is inhibited by the EPI PAβN. a Molecular docking and multi-copy μs-long MD simulations reveal stable interactions of CIP (orange) and PAβN (cyan) to AcrB G288D T-state monomer and show their likely binding locations. The pose and its orientation are the same as shown for AcrB WT in Fig. 3c . EG = exit channel gate (blue spheres), SL = switch-loop (yellow), and HT = hydrophobic trap (purple). All computational data, including binding free energies can be found in Supplementary Table 5 and Supplementary Figs. 8 , 14 – 16 . b Binding of CIP by AcrB G288D in the presence of 150 μM of PAβN as determined by a fluorescence polarization assay performed by Su et al. 39 . All data are fit as in Fig. 3a ( R 2 = 0.99). Data are the average and standard deviation from independent measurements ( n = 3). c Binding competition assay between PAβN and CIP for AcrB WT . Data are the average and standard deviation from independent measurements ( n = 3). d MIC assays of Escherichia coli containing AcrB WT or AcrB G288D in the presence of PAβN and antibiotics. AcrB was overexpressed in MG1655 ∆acrB from a pBR322 plasmid containing its corresponding acrAB genes, natural promoter and “marbox” sequence. Minocycline = MIN, ciprofloxacin = CIP, and phenylalanine-arginine-β-naphthylamide = PAβN. † PAβN was added at a concentration of 50 μg/ml.

    Techniques Used: Binding Assay, Fluorescence, Standard Deviation, Competitive Binding Assay, Plasmid Preparation, Sequencing, Concentration Assay

    10) Product Images from "Genetic Fusions of Heat-Labile Toxoid (LT) and Heat-Stable Toxin b (STb) of Porcine Enterotoxigenic Escherichia coli Elicit Protective Anti-LT and Anti-STb Antibodies ▿"

    Article Title: Genetic Fusions of Heat-Labile Toxoid (LT) and Heat-Stable Toxin b (STb) of Porcine Enterotoxigenic Escherichia coli Elicit Protective Anti-LT and Anti-STb Antibodies ▿

    Journal: Clinical and Vaccine Immunology : CVI

    doi: 10.1128/CVI.00095-10

    GM1-ELISA to measure binding of LT 192 and LT 192 -Gly:Pro-STb proteins to GM1. Total proteins extracted from equivalent amounts of cells (determined by OD readings) of each strain (3030-2 [K88ac + LT + STb + ], 8017 [1836-2/pBR322], 8035 [1836-2 LT], 8221 [1836-2 LT 192 ], 8145 [1836-2 LT-Gly:Pro-STb], and 8488 [1836-2 LT 192 -Gly:Pro-STb]) were tested in GM1 binding using anti-CT as the primary antibody (1:5,000) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000) as the secondary antibody. Optical densities, measured at 405 nm, showed significant differences for 3030-2, 8035, and 8221 strains ( P
    Figure Legend Snippet: GM1-ELISA to measure binding of LT 192 and LT 192 -Gly:Pro-STb proteins to GM1. Total proteins extracted from equivalent amounts of cells (determined by OD readings) of each strain (3030-2 [K88ac + LT + STb + ], 8017 [1836-2/pBR322], 8035 [1836-2 LT], 8221 [1836-2 LT 192 ], 8145 [1836-2 LT-Gly:Pro-STb], and 8488 [1836-2 LT 192 -Gly:Pro-STb]) were tested in GM1 binding using anti-CT as the primary antibody (1:5,000) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000) as the secondary antibody. Optical densities, measured at 405 nm, showed significant differences for 3030-2, 8035, and 8221 strains ( P

    Techniques Used: Enzyme-linked Immunosorbent Assay, Binding Assay

    Detection of toxic activity in a porcine ligated-gut-loop assay. Individual ligated intestinal loops from the ileum and jejunum sections were inoculated with 2 × 10 9 CFU of overnight culture from strain 3030-2 (K88 + LT + STb + , a positive control), 8488 (1836-2 LT 192 -Gly:Pro-STb), 8221(1836-2 LT 192 ), 8017 (1836-2/pBR322, a negative control), or 8816 (1836-2 STb). Fluid accumulation was measured after 8 h postinoculation; the data are presented in the inserted table. Statistical analysis indicated that the fluid accumulation in loops incubated with strains 8488 and 8221 was not significantly different from that in loops incubated with the negative control 8017 strain ( P > 0.05), whereas fluid accumulated in the loops incubated with 8816 and 3030-2 was significantly different ( P
    Figure Legend Snippet: Detection of toxic activity in a porcine ligated-gut-loop assay. Individual ligated intestinal loops from the ileum and jejunum sections were inoculated with 2 × 10 9 CFU of overnight culture from strain 3030-2 (K88 + LT + STb + , a positive control), 8488 (1836-2 LT 192 -Gly:Pro-STb), 8221(1836-2 LT 192 ), 8017 (1836-2/pBR322, a negative control), or 8816 (1836-2 STb). Fluid accumulation was measured after 8 h postinoculation; the data are presented in the inserted table. Statistical analysis indicated that the fluid accumulation in loops incubated with strains 8488 and 8221 was not significantly different from that in loops incubated with the negative control 8017 strain ( P > 0.05), whereas fluid accumulated in the loops incubated with 8816 and 3030-2 was significantly different ( P

    Techniques Used: Activity Assay, Positive Control, Negative Control, Incubation

    11) Product Images from "A robust assay to measure DNA topology-dependent protein binding affinity"

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1381

    Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).
    Figure Legend Snippet: Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).

    Techniques Used: Binding Assay, SYBR Green Assay, Staining, Electrophoresis, Agarose Gel Electrophoresis, Plasmid Preparation, Mutagenesis

    (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).
    Figure Legend Snippet: (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).

    Techniques Used: Binding Assay

    12) Product Images from "A robust assay to measure DNA topology-dependent protein binding affinity"

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1381

    Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).
    Figure Legend Snippet: Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).

    Techniques Used: Binding Assay, SYBR Green Assay, Staining, Electrophoresis, Agarose Gel Electrophoresis, Plasmid Preparation, Mutagenesis

    (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).
    Figure Legend Snippet: (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).

    Techniques Used: Binding Assay

    13) Product Images from "Single-molecule supercoil-relaxation assay as a screening tool to determine the mechanism and efficacy of human topoisomerase IB inhibitors"

    Article Title: Single-molecule supercoil-relaxation assay as a screening tool to determine the mechanism and efficacy of human topoisomerase IB inhibitors

    Journal: Molecular cancer therapeutics

    doi: 10.1158/1535-7163.MCT-15-0454

    DNA topology modulates the level of Top1cc formation induced by Top1 inhibitors. ( a) Visualization of Top1cc formation with increasing CPT concentration (0– 50 μM) with three different DNA substrates: negative supercoiled pBR322 (top), positive supercoiled pBR322 (middle), and relaxed pBR322 DNA (bottom). ( b) Fraction of nicked DNA as a function of drug concentration for three different DNA topological substrates. The amount of nicked DNA indicates the amount of Top1cc trapped by an inhibitor. Red dotted line indicates no difference in the fraction of nicked DNA relative to measurements without inhibitor.
    Figure Legend Snippet: DNA topology modulates the level of Top1cc formation induced by Top1 inhibitors. ( a) Visualization of Top1cc formation with increasing CPT concentration (0– 50 μM) with three different DNA substrates: negative supercoiled pBR322 (top), positive supercoiled pBR322 (middle), and relaxed pBR322 DNA (bottom). ( b) Fraction of nicked DNA as a function of drug concentration for three different DNA topological substrates. The amount of nicked DNA indicates the amount of Top1cc trapped by an inhibitor. Red dotted line indicates no difference in the fraction of nicked DNA relative to measurements without inhibitor.

    Techniques Used: Cycling Probe Technology, Concentration Assay

    14) Product Images from "Diabetes and aging alter bone marrow contributions to tissue maintenance"

    Article Title: Diabetes and aging alter bone marrow contributions to tissue maintenance

    Journal: International Journal of Physiology, Pathophysiology and Pharmacology

    doi:

    Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and
    Figure Legend Snippet: Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and

    Techniques Used: Derivative Assay, In Situ Hybridization, Labeling, Mouse Assay

    Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young
    Figure Legend Snippet: Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young

    Techniques Used: Quantitation Assay, Derivative Assay, Mouse Assay, Generated

    15) Product Images from "Construction of recB-recD genetic fusion and functional analysis of RecBDC fusion enzyme in Escherichia coli"

    Article Title: Construction of recB-recD genetic fusion and functional analysis of RecBDC fusion enzyme in Escherichia coli

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-9-27

    Both RecBD fusion mutants are resistant to Mitomycin C. Strains are transformants of V2831 containing rec alleles on a plasmid. Cultures were grown to mid log phase in LB broth, and plated on LB agar plates with or without Mitomycin C. Survival was determined by colony formation. a, b, c and d represent pBD2729, pDWS2, pBD2728 and pBR322, respectively.
    Figure Legend Snippet: Both RecBD fusion mutants are resistant to Mitomycin C. Strains are transformants of V2831 containing rec alleles on a plasmid. Cultures were grown to mid log phase in LB broth, and plated on LB agar plates with or without Mitomycin C. Survival was determined by colony formation. a, b, c and d represent pBD2729, pDWS2, pBD2728 and pBR322, respectively.

    Techniques Used: Plasmid Preparation

    RecBD fusion enzymes unwind dsDNA and make a cut after Chi. DNA unwinding and Chi cutting activities of the fusion enzymes were measured using pBR322 χ + DNA substrate labeled with 5'- 32 P. DNA substrates (4.5 nM) were incubated with crude extracts at 3.5 mM magnesium, monitored on 1% agarose gels and analyzed by autoradiography.
    Figure Legend Snippet: RecBD fusion enzymes unwind dsDNA and make a cut after Chi. DNA unwinding and Chi cutting activities of the fusion enzymes were measured using pBR322 χ + DNA substrate labeled with 5'- 32 P. DNA substrates (4.5 nM) were incubated with crude extracts at 3.5 mM magnesium, monitored on 1% agarose gels and analyzed by autoradiography.

    Techniques Used: Labeling, Incubation, Autoradiography

    16) Product Images from "A robust assay to measure DNA topology-dependent protein binding affinity"

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1381

    Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).
    Figure Legend Snippet: Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).

    Techniques Used: Binding Assay, SYBR Green Assay, Staining, Electrophoresis, Agarose Gel Electrophoresis, Plasmid Preparation, Mutagenesis

    (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).
    Figure Legend Snippet: (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).

    Techniques Used: Binding Assay

    17) Product Images from "High Performance DNA Purification using a Novel Ion Exchange Matrix"

    Article Title: High Performance DNA Purification using a Novel Ion Exchange Matrix

    Journal: Journal of Biomolecular Techniques : JBT

    doi:

    Isolation of plasmids with anion exchange membranes. A: Purification of high-copy plasmid pUC19 (lanes 1–4) and low-copy plasmid pBR322 (lanes 5–8) using IEXM. Fractions were precipitated by isopropanol to remove excess salts and redissolved
    Figure Legend Snippet: Isolation of plasmids with anion exchange membranes. A: Purification of high-copy plasmid pUC19 (lanes 1–4) and low-copy plasmid pBR322 (lanes 5–8) using IEXM. Fractions were precipitated by isopropanol to remove excess salts and redissolved

    Techniques Used: Isolation, Purification, Plasmid Preparation

    18) Product Images from "A robust assay to measure DNA topology-dependent protein binding affinity"

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1381

    Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).
    Figure Legend Snippet: Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).

    Techniques Used: Binding Assay, SYBR Green Assay, Staining, Electrophoresis, Agarose Gel Electrophoresis, Plasmid Preparation, Mutagenesis

    (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).
    Figure Legend Snippet: (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).

    Techniques Used: Binding Assay

    19) Product Images from "A robust assay to measure DNA topology-dependent protein binding affinity"

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1381

    Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).
    Figure Legend Snippet: Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).

    Techniques Used: Binding Assay, SYBR Green Assay, Staining, Electrophoresis, Agarose Gel Electrophoresis, Plasmid Preparation, Mutagenesis

    (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).
    Figure Legend Snippet: (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).

    Techniques Used: Binding Assay

    20) Product Images from "The GAAoTTC triplet repeat expanded in Friedreich's ataxia impedes transcription elongation by T7 RNA polymerase in a length and supercoil dependent manner"

    Article Title: The GAAoTTC triplet repeat expanded in Friedreich's ataxia impedes transcription elongation by T7 RNA polymerase in a length and supercoil dependent manner

    Journal: Nucleic Acids Research

    doi:

    Negative supercoils exacerbate transcription inhibition by a (GAA•TTC) 88 tract. ( A ) The templates used in these experiments contain the sequence for a self-cleaving ribozyme that cuts the transcript ∼270 bases 3′ to the end of the repeat tract, so the size of the full-length cleaved transcript (590 bases) is the same for both linear (L) and supercoiled (SC) templates. Linear templates were opened with restriction enzyme Ssp I and the primary transcript was 2698 bases. The templates produced RNA containing either (CUG) 88 or (GAA) 88 as indicated above the lanes. Templates were transcribed in the presence of [γ- 32 P]GTP (10 µCi per reaction). Bands immediately below the full-length transcripts extending to a length of ∼350 bases are due to deletions within the repeats in the templates. The numbers to the left indicate the size in bases of selected bands of the Msp I digest of pBR322 used as a marker. ( B ) A scan of lane 4 aligned to the gel highlights the distribution of truncation products within the (GAA) 88 tract. The bracket to the right of the scan labeled repeat tract indicates the location of the 88 triplets within the 5′ end-labeled transcripts in both the scan in (B) and the gel in (A).
    Figure Legend Snippet: Negative supercoils exacerbate transcription inhibition by a (GAA•TTC) 88 tract. ( A ) The templates used in these experiments contain the sequence for a self-cleaving ribozyme that cuts the transcript ∼270 bases 3′ to the end of the repeat tract, so the size of the full-length cleaved transcript (590 bases) is the same for both linear (L) and supercoiled (SC) templates. Linear templates were opened with restriction enzyme Ssp I and the primary transcript was 2698 bases. The templates produced RNA containing either (CUG) 88 or (GAA) 88 as indicated above the lanes. Templates were transcribed in the presence of [γ- 32 P]GTP (10 µCi per reaction). Bands immediately below the full-length transcripts extending to a length of ∼350 bases are due to deletions within the repeats in the templates. The numbers to the left indicate the size in bases of selected bands of the Msp I digest of pBR322 used as a marker. ( B ) A scan of lane 4 aligned to the gel highlights the distribution of truncation products within the (GAA) 88 tract. The bracket to the right of the scan labeled repeat tract indicates the location of the 88 triplets within the 5′ end-labeled transcripts in both the scan in (B) and the gel in (A).

    Techniques Used: Inhibition, Sequencing, Produced, Marker, Labeling

    21) Product Images from "The Fitness Landscapes of cis-Acting Binding Sites in Different Promoter and Environmental Contexts"

    Article Title: The Fitness Landscapes of cis-Acting Binding Sites in Different Promoter and Environmental Contexts

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1001042

    Schematic Diagram of selection promoter. Sequences of the four randomized promoter libraries (top), and a diagram mapping the promoter components (bottom). The MarA or sites were varied (blue boxes). Spacing between the binding sites may affect transcriptional output [5] . We used the same sequence between the and found in the tet promoter of pBR322 in our selection system because it has the optimal spacing [11] . We used a slight variation of the spacer between the MarA binding site and the from the mar gene [22] . Spacer sequences are shown in gray. Restriction sites used to clone synthesized libraries into the selection plasmid are marked in orange. All libraries have 6 randomized bases at the hexamer (green box).
    Figure Legend Snippet: Schematic Diagram of selection promoter. Sequences of the four randomized promoter libraries (top), and a diagram mapping the promoter components (bottom). The MarA or sites were varied (blue boxes). Spacing between the binding sites may affect transcriptional output [5] . We used the same sequence between the and found in the tet promoter of pBR322 in our selection system because it has the optimal spacing [11] . We used a slight variation of the spacer between the MarA binding site and the from the mar gene [22] . Spacer sequences are shown in gray. Restriction sites used to clone synthesized libraries into the selection plasmid are marked in orange. All libraries have 6 randomized bases at the hexamer (green box).

    Techniques Used: Selection, Binding Assay, Sequencing, Synthesized, Plasmid Preparation

    22) Product Images from "Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV "

    Article Title: Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00113-09

    Reversal of drug-stabilized DNA cleavage in the absence (top) or presence (bottom) of ATP. (Top) Supercoiled plasmid pBR322 (0.45 μg) was incubated with S. pneumoniae GyrA (0.45 μg) and GyrB (1.7 μg) in a DNA cleavage assay in
    Figure Legend Snippet: Reversal of drug-stabilized DNA cleavage in the absence (top) or presence (bottom) of ATP. (Top) Supercoiled plasmid pBR322 (0.45 μg) was incubated with S. pneumoniae GyrA (0.45 μg) and GyrB (1.7 μg) in a DNA cleavage assay in

    Techniques Used: Plasmid Preparation, Incubation, DNA Cleavage Assay

    23) Product Images from "Dataset on the effects of spermidine on linking number differences between histone H1-free and histone H1-bound circular polynucleosomes"

    Article Title: Dataset on the effects of spermidine on linking number differences between histone H1-free and histone H1-bound circular polynucleosomes

    Journal: Data in Brief

    doi: 10.1016/j.dib.2018.01.091

    (a) Illustration of our preparations of Structure 26 and Structure 28 in the presence of 5 mM spermidine. (b) Chloroquine-based agarose gel electrophoretic analysis on Structure 26 and Structure 28. Lane 1: molecular weight markers; Lane 2: relaxed forms of pBR322; Lane 3: Structure 26 and Lane 4: Structure 28. (c) Densitometry tracing of gel electrophoretic results in Lane 3. (d) Plot of Gauss fit on the data shown in Fig. 5 c, which gave rise to − 5.20 as its mean value of ΔLk (e) Densitometry tracing of gel electrophoretic results in Lane 4. (f) Plot of Gauss fit on the data shown in Fig. 5 e, which gave rise to − 5.32 as its mean value of ΔLk .
    Figure Legend Snippet: (a) Illustration of our preparations of Structure 26 and Structure 28 in the presence of 5 mM spermidine. (b) Chloroquine-based agarose gel electrophoretic analysis on Structure 26 and Structure 28. Lane 1: molecular weight markers; Lane 2: relaxed forms of pBR322; Lane 3: Structure 26 and Lane 4: Structure 28. (c) Densitometry tracing of gel electrophoretic results in Lane 3. (d) Plot of Gauss fit on the data shown in Fig. 5 c, which gave rise to − 5.20 as its mean value of ΔLk (e) Densitometry tracing of gel electrophoretic results in Lane 4. (f) Plot of Gauss fit on the data shown in Fig. 5 e, which gave rise to − 5.32 as its mean value of ΔLk .

    Techniques Used: Agarose Gel Electrophoresis, Molecular Weight

    (a) Illustration of our preparations of Structure 10 and Structure 12 in the presence of 0.75 mM spermidine. (b) Chloroquine-based agarose gel electrophoretic analysis on Structure 10 and Structure 12. Lane 1: molecular weight markers; Lane 2: relaxed forms of pBR322; Lane 3: Structure 10 and Lane 4: Structure 12. (c) Densitometry tracing of gel electrophoretic results in Lane 3. (d) Plot of Gauss fit on the data shown in Fig. 1 c, which gave rise to − 6.34 as its mean value of ΔLk (e) Densitometry tracing of gel electrophoretic results in Lane 4. (f) Plot of Gauss fit on the data shown in Fig. 1 e, which gave rise to − 6.07 as its mean value of ΔLk .
    Figure Legend Snippet: (a) Illustration of our preparations of Structure 10 and Structure 12 in the presence of 0.75 mM spermidine. (b) Chloroquine-based agarose gel electrophoretic analysis on Structure 10 and Structure 12. Lane 1: molecular weight markers; Lane 2: relaxed forms of pBR322; Lane 3: Structure 10 and Lane 4: Structure 12. (c) Densitometry tracing of gel electrophoretic results in Lane 3. (d) Plot of Gauss fit on the data shown in Fig. 1 c, which gave rise to − 6.34 as its mean value of ΔLk (e) Densitometry tracing of gel electrophoretic results in Lane 4. (f) Plot of Gauss fit on the data shown in Fig. 1 e, which gave rise to − 6.07 as its mean value of ΔLk .

    Techniques Used: Agarose Gel Electrophoresis, Molecular Weight

    (a) Illustration of our preparations of Structure 18 and Structure 20 in the presence of 2 mM spermidine. (b) Chloroquine-based agarose gel electrophoretic analysis on Structure 18 and Structure 20. Lane 1: molecular weight markers; Lane 2: relaxed forms of pBR322; Lane 3: Structure 18 and Lane 4: Structure 20. (c) Densitometry tracing of gel electrophoretic results in Lane 3. (d) Plot of Gauss fit on the data shown in Fig. 3 c, which gave rise to − 6.41 as its mean value of ΔLk (e) Densitometry tracing of gel electrophoretic results in Lane 4. (f) Plot of Gauss fit on the data shown in Fig. 3 e, which gave rise to − 5.98 as its mean value of ΔLk .
    Figure Legend Snippet: (a) Illustration of our preparations of Structure 18 and Structure 20 in the presence of 2 mM spermidine. (b) Chloroquine-based agarose gel electrophoretic analysis on Structure 18 and Structure 20. Lane 1: molecular weight markers; Lane 2: relaxed forms of pBR322; Lane 3: Structure 18 and Lane 4: Structure 20. (c) Densitometry tracing of gel electrophoretic results in Lane 3. (d) Plot of Gauss fit on the data shown in Fig. 3 c, which gave rise to − 6.41 as its mean value of ΔLk (e) Densitometry tracing of gel electrophoretic results in Lane 4. (f) Plot of Gauss fit on the data shown in Fig. 3 e, which gave rise to − 5.98 as its mean value of ΔLk .

    Techniques Used: Agarose Gel Electrophoresis, Molecular Weight

    (a) Illustration of our preparations of Structure 14 and Structure 16 in the presence of 0 mM spermidine. (b) Chloroquine-based agarose gel electrophoretic analysis on Structure 14 and Structure 16. Lane 1: molecular weight markers; Lane 2: relaxed forms of pBR322; Lane 3: Structure 14 and Lane 4: Structure 16. (c) Densitometry tracing of gel electrophoretic results in Lane 3. (d) Plot of Gauss fit on the data shown in Fig. 2 c, which gave rise to − 6.01 as its mean value of ΔLk (e) Densitometry tracing of gel electrophoretic results in Lane 4. (f) Plot of Gauss fit on the data shown in Fig. 2 e, which gave rise to − 5.98 as its mean value of ΔLk .
    Figure Legend Snippet: (a) Illustration of our preparations of Structure 14 and Structure 16 in the presence of 0 mM spermidine. (b) Chloroquine-based agarose gel electrophoretic analysis on Structure 14 and Structure 16. Lane 1: molecular weight markers; Lane 2: relaxed forms of pBR322; Lane 3: Structure 14 and Lane 4: Structure 16. (c) Densitometry tracing of gel electrophoretic results in Lane 3. (d) Plot of Gauss fit on the data shown in Fig. 2 c, which gave rise to − 6.01 as its mean value of ΔLk (e) Densitometry tracing of gel electrophoretic results in Lane 4. (f) Plot of Gauss fit on the data shown in Fig. 2 e, which gave rise to − 5.98 as its mean value of ΔLk .

    Techniques Used: Agarose Gel Electrophoresis, Molecular Weight

    (a) Illustration of our preparations of Structure 22 and Structure 24 in the presence of 3 mM spermidine. (b) Chloroquine-based agarose gel electrophoretic analysis on Structure 22 and Structure 24. Lane 1: molecular weight markers; Lane 2: relaxed forms of pBR322; Lane 3: Structure 22 and Lane 4: Structure 24. (c) Densitometry tracing of gel electrophoretic results in Lane 3. (d) Plot of Gauss fit on the data shown in Fig. 4 c, which gave rise to − 5.48 as its mean value of ΔLk (e) Densitometry tracing of gel electrophoretic results in Lane 4. (f) Plot of Gauss fit on the data shown in Fig. 4 e, which gave rise to − 5.19 as its mean value of ΔLk .
    Figure Legend Snippet: (a) Illustration of our preparations of Structure 22 and Structure 24 in the presence of 3 mM spermidine. (b) Chloroquine-based agarose gel electrophoretic analysis on Structure 22 and Structure 24. Lane 1: molecular weight markers; Lane 2: relaxed forms of pBR322; Lane 3: Structure 22 and Lane 4: Structure 24. (c) Densitometry tracing of gel electrophoretic results in Lane 3. (d) Plot of Gauss fit on the data shown in Fig. 4 c, which gave rise to − 5.48 as its mean value of ΔLk (e) Densitometry tracing of gel electrophoretic results in Lane 4. (f) Plot of Gauss fit on the data shown in Fig. 4 e, which gave rise to − 5.19 as its mean value of ΔLk .

    Techniques Used: Agarose Gel Electrophoresis, Molecular Weight

    24) Product Images from "Extrachromosomal circular DNA is common in yeast"

    Article Title: Extrachromosomal circular DNA is common in yeast

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1508825112

    Detection of known circular DNA elements. ( A ) Scatter plot shows fraction of uniquely mapped reads for plasmids spiked into samples after cell lysis and before column purification. Plasmids added in ratios per cell: pBR322 (crosses) 1:1, pUC19 (circles)
    Figure Legend Snippet: Detection of known circular DNA elements. ( A ) Scatter plot shows fraction of uniquely mapped reads for plasmids spiked into samples after cell lysis and before column purification. Plasmids added in ratios per cell: pBR322 (crosses) 1:1, pUC19 (circles)

    Techniques Used: Lysis, Purification

    25) Product Images from "Alleviating transcript insufficiency caused by Friedreich's ataxia triplet repeats"

    Article Title: Alleviating transcript insufficiency caused by Friedreich's ataxia triplet repeats

    Journal: Nucleic Acids Research

    doi:

    Relative triplex strength determines polymerase pause sites. The gel image shows the products of transcription at the indicated pH from supercoiled templates producing an RNA with the (TRS) 88 indicated above each lane. The large black arrow to the left points to the full-length product. The gray bracket immediately below full-length indicates short products that come from deletions present within the TRS in the templates. The black bracket indicates the location of the triplets in the transcript. The TRS in the GAA-RNA is slightly offset from the other two; it starts at base 55, the others at base 67 in the transcripts. Small black arrowheads point to specific truncated products in the distal end of the TRS; hollow arrowheads denote their absence. The numbers to the right indicate the size in bases of selected bands of an Msp I digest of pBR322. To the right of the gel image and aligned with it are scans of the TRS portion of lanes 2, 3 and 8. The arrows at the top indicate the lane scanned and highlight the relative peaks at the end of the TRS.
    Figure Legend Snippet: Relative triplex strength determines polymerase pause sites. The gel image shows the products of transcription at the indicated pH from supercoiled templates producing an RNA with the (TRS) 88 indicated above each lane. The large black arrow to the left points to the full-length product. The gray bracket immediately below full-length indicates short products that come from deletions present within the TRS in the templates. The black bracket indicates the location of the triplets in the transcript. The TRS in the GAA-RNA is slightly offset from the other two; it starts at base 55, the others at base 67 in the transcripts. Small black arrowheads point to specific truncated products in the distal end of the TRS; hollow arrowheads denote their absence. The numbers to the right indicate the size in bases of selected bands of an Msp I digest of pBR322. To the right of the gel image and aligned with it are scans of the TRS portion of lanes 2, 3 and 8. The arrows at the top indicate the lane scanned and highlight the relative peaks at the end of the TRS.

    Techniques Used:

    ODNs effective in enhancing transcription produce specific changes in truncations. The gel image shows the products of transcription, in the presence of the ODNs indicated at the top, from supercoiled templates producing an RNA with the (TRS) 88 indicated above each lane. The large black arrow to the left points to the full-length product. The gray bracket immediately below the arrow indicates short products from deletions present within the TRS in the templates. The black bracket indicates the location of the triplets in the transcript. The GAA tract starts at base 55, the others at base 67 in the transcripts. Small black arrowheads point to bands in the distal part of the GAA tract in lanes 3 and 6. A hollow arrowhead highlights the absence of the distal bands in lane 9. The numbers to the right indicate the size in bases of selected bands of an Msp I digest of pBR322. The ODN concentration was 2.5 µM, when present. The (TTC) 7 ODN produced a ladder of products that can be seen in lane 7 and is superimposed on the truncations present in lanes 8 and 9.
    Figure Legend Snippet: ODNs effective in enhancing transcription produce specific changes in truncations. The gel image shows the products of transcription, in the presence of the ODNs indicated at the top, from supercoiled templates producing an RNA with the (TRS) 88 indicated above each lane. The large black arrow to the left points to the full-length product. The gray bracket immediately below the arrow indicates short products from deletions present within the TRS in the templates. The black bracket indicates the location of the triplets in the transcript. The GAA tract starts at base 55, the others at base 67 in the transcripts. Small black arrowheads point to bands in the distal part of the GAA tract in lanes 3 and 6. A hollow arrowhead highlights the absence of the distal bands in lane 9. The numbers to the right indicate the size in bases of selected bands of an Msp I digest of pBR322. The ODN concentration was 2.5 µM, when present. The (TTC) 7 ODN produced a ladder of products that can be seen in lane 7 and is superimposed on the truncations present in lanes 8 and 9.

    Techniques Used: Concentration Assay, Produced

    26) Product Images from "High Performance DNA Purification using a Novel Ion Exchange Matrix"

    Article Title: High Performance DNA Purification using a Novel Ion Exchange Matrix

    Journal: Journal of Biomolecular Techniques : JBT

    doi:

    Isolation of plasmids with anion exchange membranes. A: Purification of high-copy plasmid pUC19 (lanes 1–4) and low-copy plasmid pBR322 (lanes 5–8) using IEXM. Fractions were precipitated by isopropanol to remove excess salts and redissolved
    Figure Legend Snippet: Isolation of plasmids with anion exchange membranes. A: Purification of high-copy plasmid pUC19 (lanes 1–4) and low-copy plasmid pBR322 (lanes 5–8) using IEXM. Fractions were precipitated by isopropanol to remove excess salts and redissolved

    Techniques Used: Isolation, Purification, Plasmid Preparation

    27) Product Images from "A System for the Analysis of BKV Non-coding Control Regions: Application to Clinical Isolates from an HIV/AIDS Patient"

    Article Title: A System for the Analysis of BKV Non-coding Control Regions: Application to Clinical Isolates from an HIV/AIDS Patient

    Journal: Virology

    doi: 10.1016/j.virol.2010.08.032

    The NCCR determines replication efficiency of BKV. (A) Schematic of the swap genome with the archetype virus (Dik) NCCR. SpeI and SacII sites were inserted into the pBR322-Dunlop or –Dik vectors flanking the majority of the NCCR and a PmlI site was inserted between the early and late regions. (B) RPTE cells were transfected with recircularized viral genome and low molecular weight DNA was harvested 5 dpt. Samples were linearized, digested with DpnI, and analyzed by Southern blotting. The left panel shows Dik and Dunlop wt replication compared to the swap vectors containing the three inserted restriction enzyme sites, Dik3 and Dun3 respectively. The right panel shows all possible swap combinations. Each construct is designated by a three letter abbreviation. A=archetype and R=rearranged. The first letter denotes the NCCR; second letter, early region; third letter, late region. Marker, HindIII digest of pGEM-TU; Mock, mock transfection.
    Figure Legend Snippet: The NCCR determines replication efficiency of BKV. (A) Schematic of the swap genome with the archetype virus (Dik) NCCR. SpeI and SacII sites were inserted into the pBR322-Dunlop or –Dik vectors flanking the majority of the NCCR and a PmlI site was inserted between the early and late regions. (B) RPTE cells were transfected with recircularized viral genome and low molecular weight DNA was harvested 5 dpt. Samples were linearized, digested with DpnI, and analyzed by Southern blotting. The left panel shows Dik and Dunlop wt replication compared to the swap vectors containing the three inserted restriction enzyme sites, Dik3 and Dun3 respectively. The right panel shows all possible swap combinations. Each construct is designated by a three letter abbreviation. A=archetype and R=rearranged. The first letter denotes the NCCR; second letter, early region; third letter, late region. Marker, HindIII digest of pGEM-TU; Mock, mock transfection.

    Techniques Used: Transfection, Molecular Weight, Southern Blot, Construct, Marker

    28) Product Images from "mRNA and DNA selection via protein multimerization: YB-1 as a case study"

    Article Title: mRNA and DNA selection via protein multimerization: YB-1 as a case study

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv822

    YB-1 binds preferentially supercoiled DNA at crosses ( A ) Left panel: The interaction of YB-1 (500 nM) with nicked or supercoiled pBR322 DNA (2 nM) was probed by AFM on mica. YB-1 forms short multimers on supercoiled pBR322 DNA, especially at DNA crosses, while the binding of YB-1 to relaxed plasmids was not detected. Scale bar: 200 nm. Right panel: Analysis of the heights at DNA crosses of supercoiled DNA in the presence or absence of YB-1. The significant increase in height of DNA crosses in the presence of YB-1 reveals its multimerization at crosses. ( B ) Upper panel: Gel mobility shift assay probing the binding of YB-1 to an equimolar mixture of supercoiled and nicked pBR322 DNA (70 fmoles each). A significant decrease in the mobility of supercoiled DNA occurs at lower concentration than for relaxed plasmid. Two non-exclusive explanations can be advanced for this: (i) YB-1 binds to both supercoiled and relaxed DNA but only reduces the mobility of supercoiled DNA by modifying its conformation, (ii) YB-1 preferentially binds to supercoiled DNA. AFM data (see lower panel) indicate that YB-1 indeed preferentially binds to DNA crosses. Lower panel: AFM image of linearized and supercoiled pBR322 DNA (1 nM each) after their incubation in the presence of 500 nM YB-1. We observed that YB-1 preferentially binds to supercoiled DNA at DNA crosses in the presence of linear DNA. Scale bar: 150 nm. ( C ) Cellular location of the indicated GFP-labeled YB-1 constructs after their expression in normal rat kidney cells. YB-1-tr, CTD, CTD1, CTD2 were rather located in the nucleus in contrast with the cytoplasmic location of YB-1 and the homogenous distribution of CSD. ( D ) The presence of the indicated YB-1 constructs on supercoiled pBR322 DNA (2 nM) was probed by AFM. YB-1-tr (500 nM) form short multimers at DNA crosses in contrast with CTD1 (100 nM) and CTD2 (500 nM). CTD (500 nM) and CTD1 (500 nM, not shown) lead to the formation of mRNA-containing granules. In the presence of CSD, no interaction with supercoiled DNA was observed. Scale bar: 100 nm. ( E ) Gel mobility shift assay representing the binding of the indicated YB-1 constructs with supercoiled pBR322 DNA concentration. In agreement with the AFM results ( D ), a significant shift was detected with YB-1-tr. The shift is less marked for CTD2 and for CTD1 than YB-1 and YB-1-tr, before aggregation takes place. No shift was detected with CSD in the conditions explored here.
    Figure Legend Snippet: YB-1 binds preferentially supercoiled DNA at crosses ( A ) Left panel: The interaction of YB-1 (500 nM) with nicked or supercoiled pBR322 DNA (2 nM) was probed by AFM on mica. YB-1 forms short multimers on supercoiled pBR322 DNA, especially at DNA crosses, while the binding of YB-1 to relaxed plasmids was not detected. Scale bar: 200 nm. Right panel: Analysis of the heights at DNA crosses of supercoiled DNA in the presence or absence of YB-1. The significant increase in height of DNA crosses in the presence of YB-1 reveals its multimerization at crosses. ( B ) Upper panel: Gel mobility shift assay probing the binding of YB-1 to an equimolar mixture of supercoiled and nicked pBR322 DNA (70 fmoles each). A significant decrease in the mobility of supercoiled DNA occurs at lower concentration than for relaxed plasmid. Two non-exclusive explanations can be advanced for this: (i) YB-1 binds to both supercoiled and relaxed DNA but only reduces the mobility of supercoiled DNA by modifying its conformation, (ii) YB-1 preferentially binds to supercoiled DNA. AFM data (see lower panel) indicate that YB-1 indeed preferentially binds to DNA crosses. Lower panel: AFM image of linearized and supercoiled pBR322 DNA (1 nM each) after their incubation in the presence of 500 nM YB-1. We observed that YB-1 preferentially binds to supercoiled DNA at DNA crosses in the presence of linear DNA. Scale bar: 150 nm. ( C ) Cellular location of the indicated GFP-labeled YB-1 constructs after their expression in normal rat kidney cells. YB-1-tr, CTD, CTD1, CTD2 were rather located in the nucleus in contrast with the cytoplasmic location of YB-1 and the homogenous distribution of CSD. ( D ) The presence of the indicated YB-1 constructs on supercoiled pBR322 DNA (2 nM) was probed by AFM. YB-1-tr (500 nM) form short multimers at DNA crosses in contrast with CTD1 (100 nM) and CTD2 (500 nM). CTD (500 nM) and CTD1 (500 nM, not shown) lead to the formation of mRNA-containing granules. In the presence of CSD, no interaction with supercoiled DNA was observed. Scale bar: 100 nm. ( E ) Gel mobility shift assay representing the binding of the indicated YB-1 constructs with supercoiled pBR322 DNA concentration. In agreement with the AFM results ( D ), a significant shift was detected with YB-1-tr. The shift is less marked for CTD2 and for CTD1 than YB-1 and YB-1-tr, before aggregation takes place. No shift was detected with CSD in the conditions explored here.

    Techniques Used: Binding Assay, Mobility Shift, Concentration Assay, Plasmid Preparation, Incubation, Labeling, Construct, Expressing

    29) Product Images from "A robust assay to measure DNA topology-dependent protein binding affinity"

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1381

    Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).
    Figure Legend Snippet: Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).

    Techniques Used: Binding Assay, SYBR Green Assay, Staining, Electrophoresis, Agarose Gel Electrophoresis, Plasmid Preparation, Mutagenesis

    (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).
    Figure Legend Snippet: (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).

    Techniques Used: Binding Assay

    30) Product Images from "Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV "

    Article Title: Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00113-09

    Assay for DNA unwinding of closed circular DNA by PD 0305970 and ciprofloxacin. Relaxed pBR322 DNA (0.8 μg) was preincubated with calf thymus DNA topo I (10 U) at room temperature for 10 min in the presence of MgCl 2 at the concentrations (mM)
    Figure Legend Snippet: Assay for DNA unwinding of closed circular DNA by PD 0305970 and ciprofloxacin. Relaxed pBR322 DNA (0.8 μg) was preincubated with calf thymus DNA topo I (10 U) at room temperature for 10 min in the presence of MgCl 2 at the concentrations (mM)

    Techniques Used:

    Reversal of drug-stabilized DNA cleavage in the absence (top) or presence (bottom) of ATP. (Top) Supercoiled plasmid pBR322 (0.45 μg) was incubated with S. pneumoniae GyrA (0.45 μg) and GyrB (1.7 μg) in a DNA cleavage assay in
    Figure Legend Snippet: Reversal of drug-stabilized DNA cleavage in the absence (top) or presence (bottom) of ATP. (Top) Supercoiled plasmid pBR322 (0.45 μg) was incubated with S. pneumoniae GyrA (0.45 μg) and GyrB (1.7 μg) in a DNA cleavage assay in

    Techniques Used: Plasmid Preparation, Incubation, DNA Cleavage Assay

    Inhibitory activity of PD 0305970 against wild-type and mutant S. pneumoniae type II topoisomerases. Inhibition of DNA supercoiling by DNA gyrase. Relaxed pBR322 plasmid DNA (0.4 μg) was incubated with wild-type (wt) gyrase activity (1 U) or mutant
    Figure Legend Snippet: Inhibitory activity of PD 0305970 against wild-type and mutant S. pneumoniae type II topoisomerases. Inhibition of DNA supercoiling by DNA gyrase. Relaxed pBR322 plasmid DNA (0.4 μg) was incubated with wild-type (wt) gyrase activity (1 U) or mutant

    Techniques Used: Activity Assay, Mutagenesis, Inhibition, Plasmid Preparation, Incubation

    PD 0305970 induces site-specific DNA cleavage by gyrase. (A) PD 0305970 promotes gyrase-mediated double-stranded DNA cleavage. pBR322 DNA linearized with EcoRI was employed as a substrate in a cleavage assay with wild-type S. pneumoniae gyrase (as described
    Figure Legend Snippet: PD 0305970 induces site-specific DNA cleavage by gyrase. (A) PD 0305970 promotes gyrase-mediated double-stranded DNA cleavage. pBR322 DNA linearized with EcoRI was employed as a substrate in a cleavage assay with wild-type S. pneumoniae gyrase (as described

    Techniques Used: Cleavage Assay

    31) Product Images from "Crystal structure of the modification-dependent SRA-HNH endonuclease TagI"

    Article Title: Crystal structure of the modification-dependent SRA-HNH endonuclease TagI

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky781

    Sequence logo for TagI ds cleavage (left) or nicking (right) activity. 16 double strand cleavage sites ( A ) and 31 nicking sites ( B ) were combined from pBR322, pBRFM + , and 5hm C PCR DNA cleavage performed at 37°C. The arrow denotes the site of DNA cleavage or nicking. Note a systematic bias in the determination of the sequence logos. An A base immediately downstream of the cleavage site is detected with lower efficiency (because the polymerase incorporates the correct base, albeit in a template-independent manner).
    Figure Legend Snippet: Sequence logo for TagI ds cleavage (left) or nicking (right) activity. 16 double strand cleavage sites ( A ) and 31 nicking sites ( B ) were combined from pBR322, pBRFM + , and 5hm C PCR DNA cleavage performed at 37°C. The arrow denotes the site of DNA cleavage or nicking. Note a systematic bias in the determination of the sequence logos. An A base immediately downstream of the cleavage site is detected with lower efficiency (because the polymerase incorporates the correct base, albeit in a template-independent manner).

    Techniques Used: Sequencing, Activity Assay, Polymerase Chain Reaction

    32) Product Images from "Diabetes and aging alter bone marrow contributions to tissue maintenance"

    Article Title: Diabetes and aging alter bone marrow contributions to tissue maintenance

    Journal: International Journal of Physiology, Pathophysiology and Pharmacology

    doi:

    Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and
    Figure Legend Snippet: Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and

    Techniques Used: Derivative Assay, In Situ Hybridization, Labeling, Mouse Assay

    Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young
    Figure Legend Snippet: Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young

    Techniques Used: Quantitation Assay, Derivative Assay, Mouse Assay, Generated

    33) Product Images from "ClpXP protease targets long-lived DNA translocation states of a helicase-like motor to cause restriction alleviation"

    Article Title: ClpXP protease targets long-lived DNA translocation states of a helicase-like motor to cause restriction alleviation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku851

    Proteolysis of HsdR is dependent on translocation and not DNA cleavage. ( A ) Quantified data from 60-min reactions on pLKS5 in the presence or absence of ClpXP, and with EcoKI MTase without HsdR, with wild-type HsdR or with HsdRs mutated in either a nuclease (Nuc-) or ATPase (ATP-) motif. Standard deviation error bars from three repeat experiments. ( B ) Transformation screen to measure RA by ClpXP. Colony forming units (c.f.u.) were measured in separate transformation reactions using pBR322 or plasmids carrying the complete EcoKI operon (WT), the genes for the MTase alone (ΔHsdR) or the EcoKI operon with an HsdR mutated within the nuclease domain (D298E). The first two rows show the quotient of the c.f.u. for the EcoKI plasmid and pBR322 in strains with active ClpXP (NK311) or without active ClpXP (NK312). The third row is the quotient of the transformation efficiency in the presence and absence of ClpXP, with elevated values indicating ClpXP-dependent RA. The grey box indicates that plasmid preparations from the successful transformants showed loss of DNA in 100% of cases examined. The smaller DNA could retransform NK312 more efficiently than the original DNA, indicating loss of EcoKI activity.
    Figure Legend Snippet: Proteolysis of HsdR is dependent on translocation and not DNA cleavage. ( A ) Quantified data from 60-min reactions on pLKS5 in the presence or absence of ClpXP, and with EcoKI MTase without HsdR, with wild-type HsdR or with HsdRs mutated in either a nuclease (Nuc-) or ATPase (ATP-) motif. Standard deviation error bars from three repeat experiments. ( B ) Transformation screen to measure RA by ClpXP. Colony forming units (c.f.u.) were measured in separate transformation reactions using pBR322 or plasmids carrying the complete EcoKI operon (WT), the genes for the MTase alone (ΔHsdR) or the EcoKI operon with an HsdR mutated within the nuclease domain (D298E). The first two rows show the quotient of the c.f.u. for the EcoKI plasmid and pBR322 in strains with active ClpXP (NK311) or without active ClpXP (NK312). The third row is the quotient of the transformation efficiency in the presence and absence of ClpXP, with elevated values indicating ClpXP-dependent RA. The grey box indicates that plasmid preparations from the successful transformants showed loss of DNA in 100% of cases examined. The smaller DNA could retransform NK312 more efficiently than the original DNA, indicating loss of EcoKI activity.

    Techniques Used: Translocation Assay, Standard Deviation, Transformation Assay, Plasmid Preparation, Activity Assay

    34) Product Images from "Visible Light-Induced Radical Mediated DNA Damage"

    Article Title: Visible Light-Induced Radical Mediated DNA Damage

    Journal: Photochemistry and photobiology

    doi: 10.1111/php.12890

    Effect of photolysis of methylcobalamin before the addition of plasmid DNA on the cleavage of DNA. Methylcobalamin (1 mM) mediated pBR322 DNA (10 μM/bp in pH 7.4 phosphate buffer) cleavage was measured after DNA was added to photolyzed methylcobalamin. Lane 1 pBR322 DNA; lane 2 pBR322 DNA and methylcobalamin; and lane methylcobalamin illuminated with filtered green light ( > 500 nm, 450 W medium pressure Hg arc lamp) for 15 min before DNA was added in the dark.
    Figure Legend Snippet: Effect of photolysis of methylcobalamin before the addition of plasmid DNA on the cleavage of DNA. Methylcobalamin (1 mM) mediated pBR322 DNA (10 μM/bp in pH 7.4 phosphate buffer) cleavage was measured after DNA was added to photolyzed methylcobalamin. Lane 1 pBR322 DNA; lane 2 pBR322 DNA and methylcobalamin; and lane methylcobalamin illuminated with filtered green light ( > 500 nm, 450 W medium pressure Hg arc lamp) for 15 min before DNA was added in the dark.

    Techniques Used: Plasmid Preparation

    Anaerobic cleavage of pBR322 DNA (10 μM/bp in pH 7.4 PBS) by vitamin Co(II)bl. Anaerobic DNA cleavage by vitamin Co(II)bl in the presence and absence of TEMPO was assessed. Lane 1, DNA alone; lane 2, DNA and vitamin Co(II)bl (1 mM); lane 3, DNA, vitamin Co(II)bl (1 mM); and TEMPO (50 mM). Co(II)bl was produced by the reduction of hydroxocobalamin by formate. Samples were incubated in dark for 5 min.
    Figure Legend Snippet: Anaerobic cleavage of pBR322 DNA (10 μM/bp in pH 7.4 PBS) by vitamin Co(II)bl. Anaerobic DNA cleavage by vitamin Co(II)bl in the presence and absence of TEMPO was assessed. Lane 1, DNA alone; lane 2, DNA and vitamin Co(II)bl (1 mM); lane 3, DNA, vitamin Co(II)bl (1 mM); and TEMPO (50 mM). Co(II)bl was produced by the reduction of hydroxocobalamin by formate. Samples were incubated in dark for 5 min.

    Techniques Used: Produced, Incubation

    Visible light-mediated cleavage of pBR322 DNA (10 μM/bp in pH 7.4 phosphate buffer) by spermine-cobalamin conjugate ( 2 ). Lanes 1 and 2, DNA alone; lanes 3 – 12, DNA and spermine-cobalamin conjugate (250, 250, 175, 122, 85, 60, 42, 29, 20, and 14 μM, respectively). Samples in lanes 1 and 3 were incubated in the dark, and those in lane 2 and lanes 4 – 12 were illuminated with filtered green light ( > 500 nm, 450 W medium pressure Hg arc lamp) for 10 min.
    Figure Legend Snippet: Visible light-mediated cleavage of pBR322 DNA (10 μM/bp in pH 7.4 phosphate buffer) by spermine-cobalamin conjugate ( 2 ). Lanes 1 and 2, DNA alone; lanes 3 – 12, DNA and spermine-cobalamin conjugate (250, 250, 175, 122, 85, 60, 42, 29, 20, and 14 μM, respectively). Samples in lanes 1 and 3 were incubated in the dark, and those in lane 2 and lanes 4 – 12 were illuminated with filtered green light ( > 500 nm, 450 W medium pressure Hg arc lamp) for 10 min.

    Techniques Used: Incubation

    Anaerobic visible light-mediated DNA cleavage by methylcobalamin in the presence of TEMPO. Methylcobalamin (1 mM) mediated pBR322 DNA (10 μM/bp in pH 7.4 PBS) cleavage was measured in the absence of oxygen. Lanes 1 and 2, DNA alone; lanes 3, and 4 DNA and methylcobalamin; lane 5 DNA, methylcobalamin, and TEMPO (50 mM). Nitrogen on was bubbled through the buffer of the deoxygenated samples (lanes 4 and 5) for 30 min. Samples in lanes 1 and 3 were stored in the dark and samples in lanes 2, 4, and 5 were irradiated with filtered green light (450 W medium pressure Hg arc lamp) for 10 min.
    Figure Legend Snippet: Anaerobic visible light-mediated DNA cleavage by methylcobalamin in the presence of TEMPO. Methylcobalamin (1 mM) mediated pBR322 DNA (10 μM/bp in pH 7.4 PBS) cleavage was measured in the absence of oxygen. Lanes 1 and 2, DNA alone; lanes 3, and 4 DNA and methylcobalamin; lane 5 DNA, methylcobalamin, and TEMPO (50 mM). Nitrogen on was bubbled through the buffer of the deoxygenated samples (lanes 4 and 5) for 30 min. Samples in lanes 1 and 3 were stored in the dark and samples in lanes 2, 4, and 5 were irradiated with filtered green light (450 W medium pressure Hg arc lamp) for 10 min.

    Techniques Used: Irradiation

    Visible light-mediated cleavage of pBR322 DNA (10 μM/bp in pH 7.4 phosphate buffer) by methylcobalamin. Lanes 1 and 2, DNA alone; lanes 3 – 8, DNA and methylcobalamin (1000, 1000, 750, 563, 422, and 316 μM, respectively). Samples in lanes 1 and 3 were incubated in the dark, and those in lane 2 and lanes 4 – 8 were illuminated with filtered green light (450 W medium pressure Hg arc lamp) for 45 min.
    Figure Legend Snippet: Visible light-mediated cleavage of pBR322 DNA (10 μM/bp in pH 7.4 phosphate buffer) by methylcobalamin. Lanes 1 and 2, DNA alone; lanes 3 – 8, DNA and methylcobalamin (1000, 1000, 750, 563, 422, and 316 μM, respectively). Samples in lanes 1 and 3 were incubated in the dark, and those in lane 2 and lanes 4 – 8 were illuminated with filtered green light (450 W medium pressure Hg arc lamp) for 45 min.

    Techniques Used: Incubation

    Radical scavenging of methylcobalamin-mediated DNA cleavage. The effect of TEMPO on the cleavage of pBR322 DNA (10 μM/bp in pH 7.4 PBS) by methylcobalamin was assessed. Lanes 1 and 2, DNA alone; lanes 3 and 4, DNA and methylcobalamin (0.500 mM); lane 5 DNA, methylcobalamin (0.500 mM), and TEMPO (50.0 mM). The samples in lanes 1 and 3 were incubated in the dark, and those in lanes 2 and 4 - 6 were illuminated with filtered green light ( > 500 nm, 450 W medium pressure Hg arc lamp) for 10 min.
    Figure Legend Snippet: Radical scavenging of methylcobalamin-mediated DNA cleavage. The effect of TEMPO on the cleavage of pBR322 DNA (10 μM/bp in pH 7.4 PBS) by methylcobalamin was assessed. Lanes 1 and 2, DNA alone; lanes 3 and 4, DNA and methylcobalamin (0.500 mM); lane 5 DNA, methylcobalamin (0.500 mM), and TEMPO (50.0 mM). The samples in lanes 1 and 3 were incubated in the dark, and those in lanes 2 and 4 - 6 were illuminated with filtered green light ( > 500 nm, 450 W medium pressure Hg arc lamp) for 10 min.

    Techniques Used: Incubation

    Visible light-mediated DNA cleavage by methylcobalamin in the absence of oxygen. Methylcobalamin (1 mM) mediated pBR322 DNA (10 μM/bp in pH 7.4 phosphate buffer) cleavage was measured in the presence and absence of oxygen. Lanes 1 and 2, DNA alone; lanes 3, 4 and 5, DNA and methylcobalamin (1.00 mM). a) Nitrogen was bubbled through the buffer of the deoxygenated sample (lane 5) for 30 min. b) The degassed sample (Lane 5) underwent 5 freeze-pump-thaw cycles before being illuminated with light while under vacuum. Samples in lanes 1 and 3 were stored in the dark and samples in lanes 2, 4, and 5 were irradiated with filtered green light ( > 500 nm, 450 W medium pressure Hg arc lamp) for 10 min.
    Figure Legend Snippet: Visible light-mediated DNA cleavage by methylcobalamin in the absence of oxygen. Methylcobalamin (1 mM) mediated pBR322 DNA (10 μM/bp in pH 7.4 phosphate buffer) cleavage was measured in the presence and absence of oxygen. Lanes 1 and 2, DNA alone; lanes 3, 4 and 5, DNA and methylcobalamin (1.00 mM). a) Nitrogen was bubbled through the buffer of the deoxygenated sample (lane 5) for 30 min. b) The degassed sample (Lane 5) underwent 5 freeze-pump-thaw cycles before being illuminated with light while under vacuum. Samples in lanes 1 and 3 were stored in the dark and samples in lanes 2, 4, and 5 were irradiated with filtered green light ( > 500 nm, 450 W medium pressure Hg arc lamp) for 10 min.

    Techniques Used: Irradiation

    Hydroxocobalamin is ineffective at causing cleavage of pBR322 DNA (10 μM/bp in pH 7.4 PBS) when illuminated with visible light. Lanes 1 and 2, DNA alone; lanes 3 – 8, DNA and hydroxocobalamin (1.00, 0.750, 0.500, and 0.250 mM, respectively). Samples in lanes 1 and 3 were incubated in the dark, and those in lane 2 and lanes 4 – 8 were illuminated with filtered green light ( > 500 nm, 450 W medium pressure Hg arc lamp) for 45 min.
    Figure Legend Snippet: Hydroxocobalamin is ineffective at causing cleavage of pBR322 DNA (10 μM/bp in pH 7.4 PBS) when illuminated with visible light. Lanes 1 and 2, DNA alone; lanes 3 – 8, DNA and hydroxocobalamin (1.00, 0.750, 0.500, and 0.250 mM, respectively). Samples in lanes 1 and 3 were incubated in the dark, and those in lane 2 and lanes 4 – 8 were illuminated with filtered green light ( > 500 nm, 450 W medium pressure Hg arc lamp) for 45 min.

    Techniques Used: Incubation

    35) Product Images from "Crystal structure of the modification-dependent SRA-HNH endonuclease TagI"

    Article Title: Crystal structure of the modification-dependent SRA-HNH endonuclease TagI

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky781

    Sequence logo for TagI ds cleavage (left) or nicking (right) activity. 16 double strand cleavage sites ( A ) and 31 nicking sites ( B ) were combined from pBR322, pBRFM + , and 5hm C PCR DNA cleavage performed at 37°C. The arrow denotes the site of DNA cleavage or nicking. Note a systematic bias in the determination of the sequence logos. An A base immediately downstream of the cleavage site is detected with lower efficiency (because the polymerase incorporates the correct base, albeit in a template-independent manner).
    Figure Legend Snippet: Sequence logo for TagI ds cleavage (left) or nicking (right) activity. 16 double strand cleavage sites ( A ) and 31 nicking sites ( B ) were combined from pBR322, pBRFM + , and 5hm C PCR DNA cleavage performed at 37°C. The arrow denotes the site of DNA cleavage or nicking. Note a systematic bias in the determination of the sequence logos. An A base immediately downstream of the cleavage site is detected with lower efficiency (because the polymerase incorporates the correct base, albeit in a template-independent manner).

    Techniques Used: Sequencing, Activity Assay, Polymerase Chain Reaction

    36) Product Images from "Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB"

    Article Title: Perturbed structural dynamics underlie inhibition and altered specificity of the multidrug efflux pump AcrB

    Journal: bioRxiv

    doi: 10.1101/2020.04.27.063511

    AcrB G288D is inhibited by the EPI PAβN. ( a ) Molecular docking and multi-copy μs-long MD simulations reveal stable interactions of CIP (orange) and PAβN (cyan) to AcrB G288D T-state monomer and show their likely binding locations. The pose and its orientation are the same as shown for AcrB WT in Fig. 3c . EG = exit channel gate (blue spheres), SL = switch-loop (yellow), and HT = hydrophobic trap (purple). All computational data, including binding free energies be found in Supplementary Table 3 and Supplementary Fig. 6, 12-13. ( b ) MIC assays of Escherichia coli containing AcrB WT or AcrB G288D in the presence of inhibitors and antibiotics. AcrB was overexpressed in MG1655 ΔacrB from a pBR322 plasmid containing its corresponding acrAB genes, natural promoter and ‘marbox’ sequence. Minocycline = MIN, Ciprofloxacin = CIP, and phenylalanine-arginine-β-naphthylamide = PAβN. † PAβN was added at a concentration of 50 μg/ml.
    Figure Legend Snippet: AcrB G288D is inhibited by the EPI PAβN. ( a ) Molecular docking and multi-copy μs-long MD simulations reveal stable interactions of CIP (orange) and PAβN (cyan) to AcrB G288D T-state monomer and show their likely binding locations. The pose and its orientation are the same as shown for AcrB WT in Fig. 3c . EG = exit channel gate (blue spheres), SL = switch-loop (yellow), and HT = hydrophobic trap (purple). All computational data, including binding free energies be found in Supplementary Table 3 and Supplementary Fig. 6, 12-13. ( b ) MIC assays of Escherichia coli containing AcrB WT or AcrB G288D in the presence of inhibitors and antibiotics. AcrB was overexpressed in MG1655 ΔacrB from a pBR322 plasmid containing its corresponding acrAB genes, natural promoter and ‘marbox’ sequence. Minocycline = MIN, Ciprofloxacin = CIP, and phenylalanine-arginine-β-naphthylamide = PAβN. † PAβN was added at a concentration of 50 μg/ml.

    Techniques Used: Binding Assay, Plasmid Preparation, Sequencing, Concentration Assay

    37) Product Images from "Diabetes and aging alter bone marrow contributions to tissue maintenance"

    Article Title: Diabetes and aging alter bone marrow contributions to tissue maintenance

    Journal: International Journal of Physiology, Pathophysiology and Pharmacology

    doi:

    Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and
    Figure Legend Snippet: Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and

    Techniques Used: Derivative Assay, In Situ Hybridization, Labeling, Mouse Assay

    Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young
    Figure Legend Snippet: Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young

    Techniques Used: Quantitation Assay, Derivative Assay, Mouse Assay, Generated

    38) Product Images from "Diabetes and aging alter bone marrow contributions to tissue maintenance"

    Article Title: Diabetes and aging alter bone marrow contributions to tissue maintenance

    Journal: International Journal of Physiology, Pathophysiology and Pharmacology

    doi:

    Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and
    Figure Legend Snippet: Bone marrow-derived cells in quiescent tissue. Brightfield images of tissue sections after in situ hybridization with digoxygenin labeled pBR322 probe to detect bone marrow-derived cells in chimeric mice. A B) Liver section of young (A) and

    Techniques Used: Derivative Assay, In Situ Hybridization, Labeling, Mouse Assay

    Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young
    Figure Legend Snippet: Quantitation of BM-derived nuclei in quiescent hepatocytes, skeletal muscle, and cardiomyocytes in chimeric mice generated by lethally irradiating mice and replacing their BM with cells carrying multiple copies of pBR322 sequences in their genome. Young

    Techniques Used: Quantitation Assay, Derivative Assay, Mouse Assay, Generated

    39) Product Images from "Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair"

    Article Title: Analysis of the Xenopus Werner syndrome protein in DNA double-strand break repair

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.200502077

    Establishment of SSA in NPE. (A) Preparation of the SSA substrate pRW4′. Plasmid pRW4 was digested with Xho I and then partially filled in by TTP and dCTP with Klenow (exo - ; NEB, NE). (B) pRW4′ (12 ng/μl) was incubated in NPE at room temperature. Samples were taken at the indicated times, treated with SDS/proteinase K, and separated on a 1% agarose gel. Lanes 1–4: time points of the reaction in NPE; lane 5: Xho I-digested pRW4 ligated with T4 DNA ligase; lane 6: uncut pRW4; lane 7: pRW4′; lane 8: pRW4′ ligated with T4 DNA ligase. Bands indicated by (*) are NHEJ products. (C) Restriction digestion of the 10-kb repair product (indicated by the line in B). Left: predicted digestion pattern by Sal I and EcoR I; middle and right: gel electrophoresis of the digested DNA. The faint bands above the 4.36 band are due to partial digestion. (D) Restriction digestion of the cloned EcoR I fragment. Left: gel electrophoresis of the digested plasmid; right: predicted digestion patterns of the pBR322 plasmid and pRW4 plasmid. X: Xho I site. (E) Gel electrophoresis of the junction DNA directly amplified from the 10-kb repair product.
    Figure Legend Snippet: Establishment of SSA in NPE. (A) Preparation of the SSA substrate pRW4′. Plasmid pRW4 was digested with Xho I and then partially filled in by TTP and dCTP with Klenow (exo - ; NEB, NE). (B) pRW4′ (12 ng/μl) was incubated in NPE at room temperature. Samples were taken at the indicated times, treated with SDS/proteinase K, and separated on a 1% agarose gel. Lanes 1–4: time points of the reaction in NPE; lane 5: Xho I-digested pRW4 ligated with T4 DNA ligase; lane 6: uncut pRW4; lane 7: pRW4′; lane 8: pRW4′ ligated with T4 DNA ligase. Bands indicated by (*) are NHEJ products. (C) Restriction digestion of the 10-kb repair product (indicated by the line in B). Left: predicted digestion pattern by Sal I and EcoR I; middle and right: gel electrophoresis of the digested DNA. The faint bands above the 4.36 band are due to partial digestion. (D) Restriction digestion of the cloned EcoR I fragment. Left: gel electrophoresis of the digested plasmid; right: predicted digestion patterns of the pBR322 plasmid and pRW4 plasmid. X: Xho I site. (E) Gel electrophoresis of the junction DNA directly amplified from the 10-kb repair product.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Non-Homologous End Joining, Nucleic Acid Electrophoresis, Clone Assay, Amplification

    Related Articles

    Amplification:

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    Article Snippet: .. Primers used for amplification included MM578, which is complementary to the bottom strand of coding sequence, and one of four primers complementary to different parts of pBR322, 1238, 1239, 1240 and 1241 (New England Biolabs). .. PCR and cloning (TA Topo cloning kit, Invitrogen) were carried out as described previously ( ).

    Sequencing:

    Article Title: Inverse transposition by the RAG1 and RAG2 proteins: role reversal of donor and target DNA
    Article Snippet: .. Primers used for amplification included MM578, which is complementary to the bottom strand of coding sequence, and one of four primers complementary to different parts of pBR322, 1238, 1239, 1240 and 1241 (New England Biolabs). .. PCR and cloning (TA Topo cloning kit, Invitrogen) were carried out as described previously ( ).

    Electrophoretic Mobility Shift Assay:

    Article Title: Investigation into the antimicrobial action and mechanism of a novel endogenous peptide β-casein 197 from human milk
    Article Snippet: .. DNA binding assay Gel-retardation experiments were performed using 5 µL of a 25 µg mL−1 pBR322 vector from E. coli (BioLabs, New England). .. Plasmid DNA (pDNA) was exposed to 5 µL of different concentrations of β-casein 197 at 37 °C for 1 h prior to gel electrophoresis of the reaction mixtures through a 0.7% agarose gel in Tris–acetate EDTA buffer.

    Purification:

    Article Title: The Fitness Landscapes of cis-Acting Binding Sites in Different Promoter and Environmental Contexts
    Article Snippet: .. The DNA was made double stranded by second strand synthesis with Klenow (NEB), and the fragments were purified with a QIAquick PCR purification kit (Qiagen). pBR322 , pBR322 , Ins , and Ins were cut with Eco RI and Cla I (New England Biolabs) for two hours at C and gel purified using a QIAquick gel extraction kit (Qiagen). .. All four combinations of plasmids and inserts were mixed and ligated overnight at C with T4 DNA ligase (NEB) generating 4 libraries (Mar:TAT, Anti:TAT, Mar:TTT and Anti:TTT).

    DNA Binding Assay:

    Article Title: Investigation into the antimicrobial action and mechanism of a novel endogenous peptide β-casein 197 from human milk
    Article Snippet: .. DNA binding assay Gel-retardation experiments were performed using 5 µL of a 25 µg mL−1 pBR322 vector from E. coli (BioLabs, New England). .. Plasmid DNA (pDNA) was exposed to 5 µL of different concentrations of β-casein 197 at 37 °C for 1 h prior to gel electrophoresis of the reaction mixtures through a 0.7% agarose gel in Tris–acetate EDTA buffer.

    Concentration Assay:

    Article Title: Pyridine and p-Nitrophenyl Oxime Esters with Possible Photochemotherapeutic Activity: Synthesis, DNA Photocleavage and DNA Binding Studies
    Article Snippet: .. The CT DNA concentration was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M−1 cm−1 [ ]. pBluescipt KS II plasmid DNA purification was performed using the Nucleospin plasmid kit, according to the protocol provided by the manufacturer (Macherey-Nagel, Duren, Germany). pBR322 was purchased from New England BioLabs (Ipswich, MA, USA).UV-visible (UV-vis) spectra were recorded on a U-2001 dual beam spectrophotometer (Hitachi, Tokyo, Japan). .. Fluorescence emission spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan).

    other:

    Article Title: Diabetes and aging alter bone marrow contributions to tissue maintenance
    Article Snippet: However, no bi-nucleate cardiomyocytes with pBR322+ nuclei were observed in any conditions.

    Article Title: Diabetes and aging alter bone marrow contributions to tissue maintenance
    Article Snippet: We also saw no pBR322+ nuclei in bi-nucleate hepatocytes.

    Spectrophotometry:

    Article Title: Pyridine and p-Nitrophenyl Oxime Esters with Possible Photochemotherapeutic Activity: Synthesis, DNA Photocleavage and DNA Binding Studies
    Article Snippet: .. The CT DNA concentration was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M−1 cm−1 [ ]. pBluescipt KS II plasmid DNA purification was performed using the Nucleospin plasmid kit, according to the protocol provided by the manufacturer (Macherey-Nagel, Duren, Germany). pBR322 was purchased from New England BioLabs (Ipswich, MA, USA).UV-visible (UV-vis) spectra were recorded on a U-2001 dual beam spectrophotometer (Hitachi, Tokyo, Japan). .. Fluorescence emission spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan).

    DNA Purification:

    Article Title: Pyridine and p-Nitrophenyl Oxime Esters with Possible Photochemotherapeutic Activity: Synthesis, DNA Photocleavage and DNA Binding Studies
    Article Snippet: .. The CT DNA concentration was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M−1 cm−1 [ ]. pBluescipt KS II plasmid DNA purification was performed using the Nucleospin plasmid kit, according to the protocol provided by the manufacturer (Macherey-Nagel, Duren, Germany). pBR322 was purchased from New England BioLabs (Ipswich, MA, USA).UV-visible (UV-vis) spectra were recorded on a U-2001 dual beam spectrophotometer (Hitachi, Tokyo, Japan). .. Fluorescence emission spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan).

    Polymerase Chain Reaction:

    Article Title: The Fitness Landscapes of cis-Acting Binding Sites in Different Promoter and Environmental Contexts
    Article Snippet: .. The DNA was made double stranded by second strand synthesis with Klenow (NEB), and the fragments were purified with a QIAquick PCR purification kit (Qiagen). pBR322 , pBR322 , Ins , and Ins were cut with Eco RI and Cla I (New England Biolabs) for two hours at C and gel purified using a QIAquick gel extraction kit (Qiagen). .. All four combinations of plasmids and inserts were mixed and ligated overnight at C with T4 DNA ligase (NEB) generating 4 libraries (Mar:TAT, Anti:TAT, Mar:TTT and Anti:TTT).

    Gel Extraction:

    Article Title: The Fitness Landscapes of cis-Acting Binding Sites in Different Promoter and Environmental Contexts
    Article Snippet: .. The DNA was made double stranded by second strand synthesis with Klenow (NEB), and the fragments were purified with a QIAquick PCR purification kit (Qiagen). pBR322 , pBR322 , Ins , and Ins were cut with Eco RI and Cla I (New England Biolabs) for two hours at C and gel purified using a QIAquick gel extraction kit (Qiagen). .. All four combinations of plasmids and inserts were mixed and ligated overnight at C with T4 DNA ligase (NEB) generating 4 libraries (Mar:TAT, Anti:TAT, Mar:TTT and Anti:TTT).

    Binding Assay:

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity
    Article Snippet: .. Tfam binding reactions contained 10 nM Tfam and 10 nM pBR322 in 150 mM NaCl, 10 mM Tris pH 7.5, 10 nM MgCl2 and 1 mM DTT based on the buffer conditions used in ( ). .. Topo IV binding reactions for both the wild type and Y120F constructs contained 2 nM Topo IV and 10 nM pBR322 in 40 mM Tris-HCl pH 7.5, 6 mM MgCl2 , 100 mM potassium acetate, 1 mM DTT and 0.1 mM ethylenediaminetetraacetic acid (EDTA).

    Plasmid Preparation:

    Article Title: Investigation into the antimicrobial action and mechanism of a novel endogenous peptide β-casein 197 from human milk
    Article Snippet: .. DNA binding assay Gel-retardation experiments were performed using 5 µL of a 25 µg mL−1 pBR322 vector from E. coli (BioLabs, New England). .. Plasmid DNA (pDNA) was exposed to 5 µL of different concentrations of β-casein 197 at 37 °C for 1 h prior to gel electrophoresis of the reaction mixtures through a 0.7% agarose gel in Tris–acetate EDTA buffer.

    Article Title: Pyridine and p-Nitrophenyl Oxime Esters with Possible Photochemotherapeutic Activity: Synthesis, DNA Photocleavage and DNA Binding Studies
    Article Snippet: .. The CT DNA concentration was determined by the UV absorbance at 260 nm after 1:20 dilution using ε = 6600 M−1 cm−1 [ ]. pBluescipt KS II plasmid DNA purification was performed using the Nucleospin plasmid kit, according to the protocol provided by the manufacturer (Macherey-Nagel, Duren, Germany). pBR322 was purchased from New England BioLabs (Ipswich, MA, USA).UV-visible (UV-vis) spectra were recorded on a U-2001 dual beam spectrophotometer (Hitachi, Tokyo, Japan). .. Fluorescence emission spectra were recorded in solution on a Hitachi F-7000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan).

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    New England Biolabs pbr322
    Topology-dependent binding of Topoisomerase IV to <t>pBR322.</t> (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).
    Pbr322, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).

    Journal: Nucleic Acids Research

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity

    doi: 10.1093/nar/gku1381

    Figure Lengend Snippet: Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).

    Article Snippet: Tfam binding reactions contained 10 nM Tfam and 10 nM pBR322 in 150 mM NaCl, 10 mM Tris pH 7.5, 10 nM MgCl2 and 1 mM DTT based on the buffer conditions used in ( ).

    Techniques: Binding Assay, SYBR Green Assay, Staining, Electrophoresis, Agarose Gel Electrophoresis, Plasmid Preparation, Mutagenesis

    (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).

    Journal: Nucleic Acids Research

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity

    doi: 10.1093/nar/gku1381

    Figure Lengend Snippet: (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).

    Article Snippet: Tfam binding reactions contained 10 nM Tfam and 10 nM pBR322 in 150 mM NaCl, 10 mM Tris pH 7.5, 10 nM MgCl2 and 1 mM DTT based on the buffer conditions used in ( ).

    Techniques: Binding Assay

    Fig. 2. Transposition of non-RSS sequence mediated by RAG1 and RAG2 proteins. ( A ) Schematic drawings of oligonucleotides used in the plasmid assay for inverse transposition. The position of radioactive label on the bottom strand of an RSS-containing oligonucleotide is indicated by an asterisk. The top and bottom strands are defined by the orientation of the RSS sequence. Either intact (substrates 2 and 4) or pre-nicked dideoxy (substrates 1 and 3) oligonucleotides were used. The labeled oligonucleotide was incubated with supercoiled pBR322 DNA in the presence of RAG1/2, HMG1 and Mg 2+ at 37°C for 2 h. Reaction samples were deproteinized and analyzed by agarose gel electrophoresis. ( B ) Strand transfer of the coding sequence to a non-RSS plasmid requires both RAG1 and RAG2 proteins. In addition to 3′- 32 P-labeled intact substrate (substrate 4), pBR322 plasmid and reaction buffer, lanes 1, 2 and 3 (all from the same experiment) contain purified RAG1, purified RAG2 and co-expressed RAG1/2 proteins, respectively. The reactions were analyzed on native agarose gels. N, nicked circular plasmid; L, linear plasmid; M, molecular marker of λ Hin dIII ladder. ( C ) Covalent strand connection of the inverse- transposition products was analyzed by comparing the deproteinized inverse-transposition products on a native gel (1.2%, left panel) and an alkaline denaturing gel (0.8%, right panel). The substrate for each reaction is indicated. 1× and 2× indicate the products with a single and double length of the linear single-stranded plasmid, respectively.

    Journal: The EMBO Journal

    Article Title: Inverse transposition by the RAG1 and RAG2 proteins: role reversal of donor and target DNA

    doi: 10.1093/emboj/cdf630

    Figure Lengend Snippet: Fig. 2. Transposition of non-RSS sequence mediated by RAG1 and RAG2 proteins. ( A ) Schematic drawings of oligonucleotides used in the plasmid assay for inverse transposition. The position of radioactive label on the bottom strand of an RSS-containing oligonucleotide is indicated by an asterisk. The top and bottom strands are defined by the orientation of the RSS sequence. Either intact (substrates 2 and 4) or pre-nicked dideoxy (substrates 1 and 3) oligonucleotides were used. The labeled oligonucleotide was incubated with supercoiled pBR322 DNA in the presence of RAG1/2, HMG1 and Mg 2+ at 37°C for 2 h. Reaction samples were deproteinized and analyzed by agarose gel electrophoresis. ( B ) Strand transfer of the coding sequence to a non-RSS plasmid requires both RAG1 and RAG2 proteins. In addition to 3′- 32 P-labeled intact substrate (substrate 4), pBR322 plasmid and reaction buffer, lanes 1, 2 and 3 (all from the same experiment) contain purified RAG1, purified RAG2 and co-expressed RAG1/2 proteins, respectively. The reactions were analyzed on native agarose gels. N, nicked circular plasmid; L, linear plasmid; M, molecular marker of λ Hin dIII ladder. ( C ) Covalent strand connection of the inverse- transposition products was analyzed by comparing the deproteinized inverse-transposition products on a native gel (1.2%, left panel) and an alkaline denaturing gel (0.8%, right panel). The substrate for each reaction is indicated. 1× and 2× indicate the products with a single and double length of the linear single-stranded plasmid, respectively.

    Article Snippet: Primers used for amplification included MM578, which is complementary to the bottom strand of coding sequence, and one of four primers complementary to different parts of pBR322, 1238, 1239, 1240 and 1241 (New England Biolabs).

    Techniques: Sequencing, Plasmid Preparation, Labeling, Incubation, Agarose Gel Electrophoresis, Purification, Marker

    Fig. 3. Hairpin structure on the plasmid DNA resulting from inverse transposition. ( A ) Inverse-transposition reactions with an intact 3′- 32 P-12 RSS oligonucleotide (*) and supercoiled pBR322 were carried out in the presence of RAG1/2, HMG1 and 5 mM Mg 2+ /0.5 mM Mn 2+ (Mn 2+ conditions) at 37°C for 2 h, as described in Materials and methods. Predicted configurations of the inverse-transposition products are shown. ( B ) Two-dimensional gel analysis of the inverse-transposition product. The reaction product was treated with mung bean nuclease or with mock digestion before deproteinization, and analyzed by two-dimensional gel electrophoresis. The first dimension was fractionated on a 1.2% native agarose gel, and the second dimension on a 1% alkaline denaturing gel. Three species, 1L, 2L and 1N, were detected. The numbers on the left indicate the positions of molecular markers.

    Journal: The EMBO Journal

    Article Title: Inverse transposition by the RAG1 and RAG2 proteins: role reversal of donor and target DNA

    doi: 10.1093/emboj/cdf630

    Figure Lengend Snippet: Fig. 3. Hairpin structure on the plasmid DNA resulting from inverse transposition. ( A ) Inverse-transposition reactions with an intact 3′- 32 P-12 RSS oligonucleotide (*) and supercoiled pBR322 were carried out in the presence of RAG1/2, HMG1 and 5 mM Mg 2+ /0.5 mM Mn 2+ (Mn 2+ conditions) at 37°C for 2 h, as described in Materials and methods. Predicted configurations of the inverse-transposition products are shown. ( B ) Two-dimensional gel analysis of the inverse-transposition product. The reaction product was treated with mung bean nuclease or with mock digestion before deproteinization, and analyzed by two-dimensional gel electrophoresis. The first dimension was fractionated on a 1.2% native agarose gel, and the second dimension on a 1% alkaline denaturing gel. Three species, 1L, 2L and 1N, were detected. The numbers on the left indicate the positions of molecular markers.

    Article Snippet: Primers used for amplification included MM578, which is complementary to the bottom strand of coding sequence, and one of four primers complementary to different parts of pBR322, 1238, 1239, 1240 and 1241 (New England Biolabs).

    Techniques: Plasmid Preparation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Agarose Gel Electrophoresis

    Schematic Diagram of selection promoter. Sequences of the four randomized promoter libraries (top), and a diagram mapping the promoter components (bottom). The MarA or sites were varied (blue boxes). Spacing between the binding sites may affect transcriptional output [5] . We used the same sequence between the and found in the tet promoter of pBR322 in our selection system because it has the optimal spacing [11] . We used a slight variation of the spacer between the MarA binding site and the from the mar gene [22] . Spacer sequences are shown in gray. Restriction sites used to clone synthesized libraries into the selection plasmid are marked in orange. All libraries have 6 randomized bases at the hexamer (green box).

    Journal: PLoS Genetics

    Article Title: The Fitness Landscapes of cis-Acting Binding Sites in Different Promoter and Environmental Contexts

    doi: 10.1371/journal.pgen.1001042

    Figure Lengend Snippet: Schematic Diagram of selection promoter. Sequences of the four randomized promoter libraries (top), and a diagram mapping the promoter components (bottom). The MarA or sites were varied (blue boxes). Spacing between the binding sites may affect transcriptional output [5] . We used the same sequence between the and found in the tet promoter of pBR322 in our selection system because it has the optimal spacing [11] . We used a slight variation of the spacer between the MarA binding site and the from the mar gene [22] . Spacer sequences are shown in gray. Restriction sites used to clone synthesized libraries into the selection plasmid are marked in orange. All libraries have 6 randomized bases at the hexamer (green box).

    Article Snippet: The DNA was made double stranded by second strand synthesis with Klenow (NEB), and the fragments were purified with a QIAquick PCR purification kit (Qiagen). pBR322 , pBR322 , Ins , and Ins were cut with Eco RI and Cla I (New England Biolabs) for two hours at C and gel purified using a QIAquick gel extraction kit (Qiagen).

    Techniques: Selection, Binding Assay, Sequencing, Synthesized, Plasmid Preparation

    Assay for DNA unwinding of closed circular DNA by PD 0305970 and ciprofloxacin. Relaxed pBR322 DNA (0.8 μg) was preincubated with calf thymus DNA topo I (10 U) at room temperature for 10 min in the presence of MgCl 2 at the concentrations (mM)

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV

    doi: 10.1128/AAC.00113-09

    Figure Lengend Snippet: Assay for DNA unwinding of closed circular DNA by PD 0305970 and ciprofloxacin. Relaxed pBR322 DNA (0.8 μg) was preincubated with calf thymus DNA topo I (10 U) at room temperature for 10 min in the presence of MgCl 2 at the concentrations (mM)

    Article Snippet: Supercoiled pBR322 and relaxed pBR322 were from New England BioLabs and John Innes Enterprises, Ltd. Kinetoplast DNA was purchased from TopoGEN.

    Techniques:

    Reversal of drug-stabilized DNA cleavage in the absence (top) or presence (bottom) of ATP. (Top) Supercoiled plasmid pBR322 (0.45 μg) was incubated with S. pneumoniae GyrA (0.45 μg) and GyrB (1.7 μg) in a DNA cleavage assay in

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV

    doi: 10.1128/AAC.00113-09

    Figure Lengend Snippet: Reversal of drug-stabilized DNA cleavage in the absence (top) or presence (bottom) of ATP. (Top) Supercoiled plasmid pBR322 (0.45 μg) was incubated with S. pneumoniae GyrA (0.45 μg) and GyrB (1.7 μg) in a DNA cleavage assay in

    Article Snippet: Supercoiled pBR322 and relaxed pBR322 were from New England BioLabs and John Innes Enterprises, Ltd. Kinetoplast DNA was purchased from TopoGEN.

    Techniques: Plasmid Preparation, Incubation, DNA Cleavage Assay

    Inhibitory activity of PD 0305970 against wild-type and mutant S. pneumoniae type II topoisomerases. Inhibition of DNA supercoiling by DNA gyrase. Relaxed pBR322 plasmid DNA (0.4 μg) was incubated with wild-type (wt) gyrase activity (1 U) or mutant

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV

    doi: 10.1128/AAC.00113-09

    Figure Lengend Snippet: Inhibitory activity of PD 0305970 against wild-type and mutant S. pneumoniae type II topoisomerases. Inhibition of DNA supercoiling by DNA gyrase. Relaxed pBR322 plasmid DNA (0.4 μg) was incubated with wild-type (wt) gyrase activity (1 U) or mutant

    Article Snippet: Supercoiled pBR322 and relaxed pBR322 were from New England BioLabs and John Innes Enterprises, Ltd. Kinetoplast DNA was purchased from TopoGEN.

    Techniques: Activity Assay, Mutagenesis, Inhibition, Plasmid Preparation, Incubation

    PD 0305970 induces site-specific DNA cleavage by gyrase. (A) PD 0305970 promotes gyrase-mediated double-stranded DNA cleavage. pBR322 DNA linearized with EcoRI was employed as a substrate in a cleavage assay with wild-type S. pneumoniae gyrase (as described

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Probing the Differential Interactions of Quinazolinedione PD 0305970 and Quinolones with Gyrase and Topoisomerase IV

    doi: 10.1128/AAC.00113-09

    Figure Lengend Snippet: PD 0305970 induces site-specific DNA cleavage by gyrase. (A) PD 0305970 promotes gyrase-mediated double-stranded DNA cleavage. pBR322 DNA linearized with EcoRI was employed as a substrate in a cleavage assay with wild-type S. pneumoniae gyrase (as described

    Article Snippet: Supercoiled pBR322 and relaxed pBR322 were from New England BioLabs and John Innes Enterprises, Ltd. Kinetoplast DNA was purchased from TopoGEN.

    Techniques: Cleavage Assay