pbr322  (New England Biolabs)


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    Name:
    pBR322 Vector
    Description:
    pBR322 Vector 250 ug
    Catalog Number:
    N3033L
    Price:
    302
    Category:
    Vectors Plasmids
    Size:
    250 ug
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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 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pbr322 - by Bioz Stars, 2021-05
    98/100 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 "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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    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 "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

    Related Articles

    Polymerase Chain Reaction:

    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 ▿
    Article Snippet: The SOE PCR was performed in a reaction mixture of 1× Pfu DNA polymerase buffer (with Mg2+ ), 200 nM dNTP, 20 μl of each purified PCR product, 1 U Pfu polymerase, and 0.5 U Taq DNA polymerase (Applied Biosystem, Foster City, CA). .. Final PCR products (inserts) and vector pBR322 were digested with NheI and EagI restriction enzymes (New England Biolabs, Ipswich, MA) and ligated with T4 DNA ligase (Invitrogen) under standard conditions ( ). .. Ligated products were introduced into 1836-2 and TOP10 competent cells with standard electroporation ( ).

    Plasmid Preparation:

    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 ▿
    Article Snippet: The SOE PCR was performed in a reaction mixture of 1× Pfu DNA polymerase buffer (with Mg2+ ), 200 nM dNTP, 20 μl of each purified PCR product, 1 U Pfu polymerase, and 0.5 U Taq DNA polymerase (Applied Biosystem, Foster City, CA). .. Final PCR products (inserts) and vector pBR322 were digested with NheI and EagI restriction enzymes (New England Biolabs, Ipswich, MA) and ligated with T4 DNA ligase (Invitrogen) under standard conditions ( ). .. Ligated products were introduced into 1836-2 and TOP10 competent cells with standard electroporation ( ).

    Article Title: Crystal structure of the modification-dependent SRA-HNH endonuclease TagI
    Article Snippet: TagI was purified by chromatography through chitin (NEB) and Hi-Trap heparin (GE Healthcare) columns. .. TagI restriction activity was first assessed by digestion of 0.5–1 μg of pBR322 (Dcm+ or Dcm− ), pBRFM+ (Dcm+ ) or pACYC-HpyCH4IVM+ plasmid in NEB buffer 2.1 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9) at 37°C for 1 h. After the reaction Protease K (1.6 U) was added at 37°C for 15 min. ..

    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).

    Activity Assay:

    Article Title: Crystal structure of the modification-dependent SRA-HNH endonuclease TagI
    Article Snippet: TagI was purified by chromatography through chitin (NEB) and Hi-Trap heparin (GE Healthcare) columns. .. TagI restriction activity was first assessed by digestion of 0.5–1 μg of pBR322 (Dcm+ or Dcm− ), pBRFM+ (Dcm+ ) or pACYC-HpyCH4IVM+ plasmid in NEB buffer 2.1 (50 mM NaCl, 10 mM Tris–HCl, 10 mM MgCl2 , 100 μg/ml BSA, pH 7.9) at 37°C for 1 h. After the reaction Protease K (1.6 U) was added at 37°C for 15 min. ..

    Irradiation:

    Article Title: Evaluating very high energy electron RBE from nanodosimetric pBR322 plasmid DNA damage
    Article Snippet: .. DSB yields were then used as the biological endpoint to calculate VHEE RBE. pBR322 plasmids irradiated in dry and aqueous environments showed little variation in DSB induction over 100–200 MeV, likely due to there being correspondingly little variation in LET (0.220–0.226 keV/μm). ..

    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).

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity
    Article Snippet: .. EcoRV binding reactions contained 2.5 nM EcoRV and 5 nM pBR322 in 100 mM NaCl, 50 mM Tris-HCl pH 7.5 and 10 mM calcium acetate. ..

    Clone Assay:

    Article Title: Construction of recB-recD genetic fusion and functional analysis of RecBDC fusion enzyme in Escherichia coli
    Article Snippet: The square root of ratio of the turbid plaques to clear plaques in Cross 1 to Cross 2 gives the Chi activity. .. Cloning of fusion mutants A 18.3 kb BamH1 fragment containing either wild type rec BCD or rec B-rec D fusion mutants was ligated into the BamHI site of pBR322 by T4 DNA ligase (New England BioLabs), and transformed into V2831 thy - -arg - cells to select thy + -arg + cells. .. Transformants were confirmed by sequencing.

    Transformation Assay:

    Article Title: Construction of recB-recD genetic fusion and functional analysis of RecBDC fusion enzyme in Escherichia coli
    Article Snippet: The square root of ratio of the turbid plaques to clear plaques in Cross 1 to Cross 2 gives the Chi activity. .. Cloning of fusion mutants A 18.3 kb BamH1 fragment containing either wild type rec BCD or rec B-rec D fusion mutants was ligated into the BamHI site of pBR322 by T4 DNA ligase (New England BioLabs), and transformed into V2831 thy - -arg - cells to select thy + -arg + cells. .. Transformants were confirmed by sequencing.

    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).

    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).

    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).

    Labeling:

    Article Title: Diabetes and aging alter bone marrow contributions to tissue maintenance
    Article Snippet: However, due to the close proximity of laminin to the satellite cell and myofiber nuclei, we were unable to unambiguously identify pBR322+ nuclei. .. Sections were instead labeled with anti-desmin to identify skeletal muscle followed by pBR322 in situ hybridization, which allowed us to identify pBR322+ nuclei. ..

    In Situ Hybridization:

    Article Title: Diabetes and aging alter bone marrow contributions to tissue maintenance
    Article Snippet: However, due to the close proximity of laminin to the satellite cell and myofiber nuclei, we were unable to unambiguously identify pBR322+ nuclei. .. Sections were instead labeled with anti-desmin to identify skeletal muscle followed by pBR322 in situ hybridization, which allowed us to identify pBR322+ nuclei. ..

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    New England Biolabs double stranded pbr322 plasmid dna
    Sequence-specific labeling of DNA with an oligodeoxynucleotide (ODN). (a) Modification of the DNA at the sequence 5′-TCGA-3′ using M. Taq I and AdoYnODN11 cofactor. The site was covalently labeled with an ODN containing 11 nucleotides (5′-TTATACATCTA-3′). (b) Distribution of the target sequence (5′-TCGA-3′) sites on <t>pBR322</t> plasmid DNA. (c) Confirmation of the modification using restriction enzymes. The left panel shows the distribution of the sites of the restriction enzymes. The right panel shows the analysis by agarose gel electrophoresis. (d) Linearization of the pBR322 DNA for nanopore measurement. The labeled DNA was linearized with the restriction endonuclease R. Ahd I which cleaves the pBR322 plasmid at a single site.
    Double Stranded Pbr322 Plasmid Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs pbr322 backbone
    Genomic configurations at the Ate1 locus of Cre-lox -based mouse strains constructed in the present work. (A) The 5′ end of the previously produced unconditional Ate1 − allele [10] , in which the Ate1 exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P Ate1 promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P PGK promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse Ate1 , indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of Ate1 homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of Ate1 exon 2, a “floxed”-hygromycin-resistance ( hph ) cassette, expressed from the P PGK promoter (blue arrow between two red triangles) downstream of Ate1 exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P HSV promoter (yellow arrow). Wavy line indicates an abutting sequence of the <t>pBR322</t> plasmid DNA. (D) The tri-lox Ate1 allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active Ate1 flox allele), whereas “flox-off” indicates a configuration depicted in panel F (the null Ate1 − allele). The functionally active, “flox-on” ( Ate1 flox ) allele, obtained by the removal of the hph cassette, using the in vivo expression of Cre-recombinase driven by the P EIIA promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” ( Ate1 − ) allele obtained by the inducible expression of CreER recombinase from the P Cagg promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and Materials and Methods ). H, approximate locations of HindIII sites used in Southern analyses with DNA probe A (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe D (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes A and D , respectively. (G) Southern hybridization analysis using DNA probe A and HindIII-digested genomic DNA. The wt Ate1 allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed [10] unconditionally null Ate1 − allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on ( Ate1 flox ) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, Ate1 +/+ ; lane 2, Ate1 +/− ; lane 3, Ate1 +/− ; lane 4, Ate1 flox/− . (H) Southern hybridization analysis using DNA probe D (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed [10] unconditionally null Ate1 − allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type Ate1 allele and the flox-on ( Ate1 flox ) allele yield the 9.7 kB fragment, whereas the null flox-off ( Ate1 − ) allele yields the characteristic 3.8 kb fragment. The use of DNA probe D and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated Ate1 flox/− ; CaggCreER mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off ( Ate1 − ) allele. For example, whereas no flox-on ( Ate1 flox ) allele could be detected in the kidney and brain of Ate1 flox/− ; CaggCreER mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on ( Ate1 flox ) and flox-off ( Ate1 − ) alleles were present in the heart of TM-treated Ate1 flox/− ; CaggCreER mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed [10] Ate1 +/− mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated Ate1 flox/− ; CaggCreER mice. Lane 8, same as lane 7, but from a TM-treated Ate1 flox/− mouse (lacking the CaggCreER transgene).
    Pbr322 Backbone, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sequence-specific labeling of DNA with an oligodeoxynucleotide (ODN). (a) Modification of the DNA at the sequence 5′-TCGA-3′ using M. Taq I and AdoYnODN11 cofactor. The site was covalently labeled with an ODN containing 11 nucleotides (5′-TTATACATCTA-3′). (b) Distribution of the target sequence (5′-TCGA-3′) sites on pBR322 plasmid DNA. (c) Confirmation of the modification using restriction enzymes. The left panel shows the distribution of the sites of the restriction enzymes. The right panel shows the analysis by agarose gel electrophoresis. (d) Linearization of the pBR322 DNA for nanopore measurement. The labeled DNA was linearized with the restriction endonuclease R. Ahd I which cleaves the pBR322 plasmid at a single site.

    Journal: ACS Nano

    Article Title: Electrical DNA Sequence Mapping Using Oligodeoxynucleotide Labels and Nanopores

    doi: 10.1021/acsnano.0c07947

    Figure Lengend Snippet: Sequence-specific labeling of DNA with an oligodeoxynucleotide (ODN). (a) Modification of the DNA at the sequence 5′-TCGA-3′ using M. Taq I and AdoYnODN11 cofactor. The site was covalently labeled with an ODN containing 11 nucleotides (5′-TTATACATCTA-3′). (b) Distribution of the target sequence (5′-TCGA-3′) sites on pBR322 plasmid DNA. (c) Confirmation of the modification using restriction enzymes. The left panel shows the distribution of the sites of the restriction enzymes. The right panel shows the analysis by agarose gel electrophoresis. (d) Linearization of the pBR322 DNA for nanopore measurement. The labeled DNA was linearized with the restriction endonuclease R. Ahd I which cleaves the pBR322 plasmid at a single site.

    Article Snippet: Sequence-Specific Labeling with ODN ODN-labeled DNA was prepared by incubating double-stranded pBR322 plasmid DNA (100 ng/μL, New England BioLabs (NEB), Ipswich, MA), ODN-modified AdoMet analogue AdoYnODN11 (10 μM) and M.Taq I (2.43 μM, 10 equiv of M.Taq I with respect to 5′-TCGA-3′ recognition sequences on the plasmid) in NEB buffer 4 (110 μL, 20 mM Tris–HCl, 50 mM KOAc, 10 mM Mg(OAc)2 , 1 mM DTT, pH 7.9) at 65 °C for 1 h. Plasmids were purified using the QIAquick PCR purification kit (QIAGEN, Hilden, Germany) according to the instructions of the manufacturer.

    Techniques: Sequencing, Labeling, Modification, Plasmid Preparation, Agarose Gel Electrophoresis

    Development of a universal bla TEM primer/probe set.

    Journal:

    Article Title: Quantifying Nonspecific TEM ?-Lactamase (blaTEM) Genes in a Wastewater Stream ▿

    doi: 10.1128/AEM.01254-08

    Figure Lengend Snippet: Development of a universal bla TEM primer/probe set.

    Article Snippet: The bla TEM standard pBR322 (New England Biolabs, Ipswich, MA), which contains TEM-1 , was diluted such that bla TEM copy numbers ranged from 1.67 × 103 to 1.71 × 106 per reaction mixture.

    Techniques: Transmission Electron Microscopy

    Primers and 5′-nuclease assay probe resulting from the alignment of the 135 bla TEM genes. Base pairs that are not conserved are in blue, primers are highlighted in red, and the probe is highlighted in green.

    Journal:

    Article Title: Quantifying Nonspecific TEM ?-Lactamase (blaTEM) Genes in a Wastewater Stream ▿

    doi: 10.1128/AEM.01254-08

    Figure Lengend Snippet: Primers and 5′-nuclease assay probe resulting from the alignment of the 135 bla TEM genes. Base pairs that are not conserved are in blue, primers are highlighted in red, and the probe is highlighted in green.

    Article Snippet: The bla TEM standard pBR322 (New England Biolabs, Ipswich, MA), which contains TEM-1 , was diluted such that bla TEM copy numbers ranged from 1.67 × 103 to 1.71 × 106 per reaction mixture.

    Techniques: Nuclease Assay, Transmission Electron Microscopy

    bla TEM copy number per ml of sample (A), relative to bacterial count (as estimated by 16S rRNA copy number) (B), and relative to biomass (as estimated by total DNA concentration) (C). Gene copy numbers were determined by quantitative PCR. Bars represent

    Journal:

    Article Title: Quantifying Nonspecific TEM ?-Lactamase (blaTEM) Genes in a Wastewater Stream ▿

    doi: 10.1128/AEM.01254-08

    Figure Lengend Snippet: bla TEM copy number per ml of sample (A), relative to bacterial count (as estimated by 16S rRNA copy number) (B), and relative to biomass (as estimated by total DNA concentration) (C). Gene copy numbers were determined by quantitative PCR. Bars represent

    Article Snippet: The bla TEM standard pBR322 (New England Biolabs, Ipswich, MA), which contains TEM-1 , was diluted such that bla TEM copy numbers ranged from 1.67 × 103 to 1.71 × 106 per reaction mixture.

    Techniques: Transmission Electron Microscopy, Concentration Assay, Real-time Polymerase Chain Reaction

    Mutant E-sites bind and inhibit topo IV. Supercoiled pBR322 DNA (400 ng) was incubated with topo IV and 0.17 µM gemifloxacin in the absence (lane B) or presence of 34-mer DNA duplexes comprising the E-site sequence (wt) or mutant sites M2C, M4C or M1A bearing −2C/+6G, −4C/+8G and −1A/+5T alterations, respectively (lanes 1–20). For each set of lanes 1–5, 6–10, 11–15 and 16–20, the concentrations of 34-mer were 1.5, 3, 6, 12 and 30 µM, respectively. Cleavage conditions were as described in Figure 3 . After SDS and proteinase K treatment, plasmid DNA products were separated and displayed by gel electrophoresis in 1% agarose. Lane SC, supercoiled pBR322 substrate; lane A, as lane B but omitting gemifloxacin. N, L and SC denote nicked, linear and supercoiled plasmid bands.

    Journal: Nucleic Acids Research

    Article Title: Functional determinants of gate-DNA selection and cleavage by bacterial type II topoisomerases

    doi: 10.1093/nar/gkt696

    Figure Lengend Snippet: Mutant E-sites bind and inhibit topo IV. Supercoiled pBR322 DNA (400 ng) was incubated with topo IV and 0.17 µM gemifloxacin in the absence (lane B) or presence of 34-mer DNA duplexes comprising the E-site sequence (wt) or mutant sites M2C, M4C or M1A bearing −2C/+6G, −4C/+8G and −1A/+5T alterations, respectively (lanes 1–20). For each set of lanes 1–5, 6–10, 11–15 and 16–20, the concentrations of 34-mer were 1.5, 3, 6, 12 and 30 µM, respectively. Cleavage conditions were as described in Figure 3 . After SDS and proteinase K treatment, plasmid DNA products were separated and displayed by gel electrophoresis in 1% agarose. Lane SC, supercoiled pBR322 substrate; lane A, as lane B but omitting gemifloxacin. N, L and SC denote nicked, linear and supercoiled plasmid bands.

    Article Snippet: Supercoiled pBR322 was from New England BioLabs.

    Techniques: Mutagenesis, Incubation, Sequencing, Plasmid Preparation, Nucleic Acid Electrophoresis

    Genomic configurations at the Ate1 locus of Cre-lox -based mouse strains constructed in the present work. (A) The 5′ end of the previously produced unconditional Ate1 − allele [10] , in which the Ate1 exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P Ate1 promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P PGK promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse Ate1 , indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of Ate1 homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of Ate1 exon 2, a “floxed”-hygromycin-resistance ( hph ) cassette, expressed from the P PGK promoter (blue arrow between two red triangles) downstream of Ate1 exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P HSV promoter (yellow arrow). Wavy line indicates an abutting sequence of the pBR322 plasmid DNA. (D) The tri-lox Ate1 allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active Ate1 flox allele), whereas “flox-off” indicates a configuration depicted in panel F (the null Ate1 − allele). The functionally active, “flox-on” ( Ate1 flox ) allele, obtained by the removal of the hph cassette, using the in vivo expression of Cre-recombinase driven by the P EIIA promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” ( Ate1 − ) allele obtained by the inducible expression of CreER recombinase from the P Cagg promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and Materials and Methods ). H, approximate locations of HindIII sites used in Southern analyses with DNA probe A (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe D (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes A and D , respectively. (G) Southern hybridization analysis using DNA probe A and HindIII-digested genomic DNA. The wt Ate1 allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed [10] unconditionally null Ate1 − allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on ( Ate1 flox ) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, Ate1 +/+ ; lane 2, Ate1 +/− ; lane 3, Ate1 +/− ; lane 4, Ate1 flox/− . (H) Southern hybridization analysis using DNA probe D (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed [10] unconditionally null Ate1 − allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type Ate1 allele and the flox-on ( Ate1 flox ) allele yield the 9.7 kB fragment, whereas the null flox-off ( Ate1 − ) allele yields the characteristic 3.8 kb fragment. The use of DNA probe D and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated Ate1 flox/− ; CaggCreER mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off ( Ate1 − ) allele. For example, whereas no flox-on ( Ate1 flox ) allele could be detected in the kidney and brain of Ate1 flox/− ; CaggCreER mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on ( Ate1 flox ) and flox-off ( Ate1 − ) alleles were present in the heart of TM-treated Ate1 flox/− ; CaggCreER mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed [10] Ate1 +/− mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated Ate1 flox/− ; CaggCreER mice. Lane 8, same as lane 7, but from a TM-treated Ate1 flox/− mouse (lacking the CaggCreER transgene).

    Journal: PLoS ONE

    Article Title: Ablation of Arginylation in the Mouse N-End Rule Pathway: Loss of Fat, Higher Metabolic Rate, Damaged Spermatogenesis, and Neurological Perturbations

    doi: 10.1371/journal.pone.0007757

    Figure Lengend Snippet: Genomic configurations at the Ate1 locus of Cre-lox -based mouse strains constructed in the present work. (A) The 5′ end of the previously produced unconditional Ate1 − allele [10] , in which the Ate1 exons 1b through 3 were replaced by a cassette encoding a promoter-lacking, NLS-containing LacZ (NLS-βgal) (it was expressed from the endogenous P Ate1 promoter) and the Neo selection marker expressed from the phosphoglycerate kinase P PGK promoter (green rectangles). (B) A diagram of the 5′ end of wild-type (wt) mouse Ate1 , indicating approximate locations of exons 1a through 5. (C) The ∼22.5 kb targeting construct containing a ∼6 kb long-arm region of Ate1 homology (shown as a shaded rectangle on the left); a single loxP site (red triangle) upstream of Ate1 exon 2, a “floxed”-hygromycin-resistance ( hph ) cassette, expressed from the P PGK promoter (blue arrow between two red triangles) downstream of Ate1 exon 4; a ∼2 kb short-arm region of homology (an inclined shaded rectangle), and the HSV thymidine kinase (tk) negative-selection cassette expressed from the P HSV promoter (yellow arrow). Wavy line indicates an abutting sequence of the pBR322 plasmid DNA. (D) The tri-lox Ate1 allele obtained after a correctly targeted double crossover event. (E) In the notations here and elsewhere in the paper, “flox-on” indicates a configuration depicted in this panel (the functionally active Ate1 flox allele), whereas “flox-off” indicates a configuration depicted in panel F (the null Ate1 − allele). The functionally active, “flox-on” ( Ate1 flox ) allele, obtained by the removal of the hph cassette, using the in vivo expression of Cre-recombinase driven by the P EIIA promoter, which is active only in pre-implantation blastocysts. (F) The null “flox-off” ( Ate1 − ) allele obtained by the inducible expression of CreER recombinase from the P Cagg promoter and posttranslationally induced by tamoxifen (TM) treatment (see the main text and Materials and Methods ). H, approximate locations of HindIII sites used in Southern analyses with DNA probe A (see panel G); E, approximate locations of EcoRI sites used in Southern analyses with DNA probe D (see panel H); black boxes marked “A” and “D” indicate the regions specific for DNA probes A and D , respectively. (G) Southern hybridization analysis using DNA probe A and HindIII-digested genomic DNA. The wt Ate1 allele (panel B) yields the 11.8 kb HindIII fragment. The previously constructed [10] unconditionally null Ate1 − allele (panel A), denoted as “null” on this panel, yields the 9.8 kb HindIII fragment. The functionally active flox-on ( Ate1 flox ) allele (panel E) yields the 6.3 kb HindIII fragment. Lane 1, Ate1 +/+ ; lane 2, Ate1 +/− ; lane 3, Ate1 +/− ; lane 4, Ate1 flox/− . (H) Southern hybridization analysis using DNA probe D (external to targeting vector) and EcoRI-digested genomic DNA. The previously constructed [10] unconditionally null Ate1 − allele (denoted as “null”) yields the 5.8 kb fragment. Both the wild-type Ate1 allele and the flox-on ( Ate1 flox ) allele yield the 9.7 kB fragment, whereas the null flox-off ( Ate1 − ) allele yields the characteristic 3.8 kb fragment. The use of DNA probe D and EcoRI-digested DNA from specific tissues of tamoxifen (TM)-treated Ate1 flox/− ; CaggCreER mice allowed approximate estimates of the levels of Cre-mediated recombination that produced the flox-off ( Ate1 − ) allele. For example, whereas no flox-on ( Ate1 flox ) allele could be detected in the kidney and brain of Ate1 flox/− ; CaggCreER mice after TM treatment (lanes 5, 6), approximately equal amounts of flox-on ( Ate1 flox ) and flox-off ( Ate1 − ) alleles were present in the heart of TM-treated Ate1 flox/− ; CaggCreER mice. Lanes 1–3, 1,000, 250, and 25 ng of EcoRI-digested wt mouse genomic DNA (from a tail biopsy), respectively. Lane 4, EcoRI-digested genomic DNA from the tail of a previously constructed [10] Ate1 +/− mouse. Lanes 5–7, EcoRI-digested genomic DNA from the indicated tissues of TM-treated Ate1 flox/− ; CaggCreER mice. Lane 8, same as lane 7, but from a TM-treated Ate1 flox/− mouse (lacking the CaggCreER transgene).

    Article Snippet: The entire ∼12 kb HindIII fragment and a part of the ∼2.9 kb fragment were modified as described below and assembled into a final ∼22.5 kb targeting vector consisting of the following parts ( ): ( i ) pBR322 backbone (New England Biolabs, Ipswich, MA); ( ii ) a ∼6.3 kb “long arm” of Ate1 homology containing the Ate1 exon 1a, the bidirectional PAte1 promoter , and exon 1b; ( iii ) A single loxP site ∼300 bp upstream of Ate1 exon 2; ( iv ) a ∼2 kb fragment that contains, 50 bp downstream of Ate1 exon 4, a “floxed” Hph (hygromycin) antibiotic-resistance marker, expressed from the PPGK promoter ; ( v ) a ∼1.2 kb “short arm” of Ate1 homology that spans most of the intron between exons 4 and 5; ( vi ) a gene encoding HSV-TK (herpes simplex virus thymidine kinase), expressed from the PPGK promoter.

    Techniques: Construct, Produced, Selection, Marker, Sequencing, Plasmid Preparation, In Vivo, Expressing, Hybridization, Mouse Assay