supercoiled dna  (New England Biolabs)


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    Name:
    Supercoiled DNA Ladder
    Description:
    Supercoiled DNA Ladder 100 gel lanes
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    n0472s
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    100 gel lanes
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    DNA Ladders
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    New England Biolabs supercoiled dna
    Supercoiled DNA Ladder
    Supercoiled DNA Ladder 100 gel lanes
    https://www.bioz.com/result/supercoiled dna/product/New England Biolabs
    Average 95 stars, based on 75 article reviews
    Price from $9.99 to $1999.99
    supercoiled dna - by Bioz Stars, 2020-02
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    Images

    1) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    2) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    Trypanosoma brucei cells contain ECTRs. Undigested genomic DNA (5 μg) from WT (uninduced) cells was subjected to neutral-neutral 2D-gel electrophoresis in duplicates and dried. One gel ( A ) was hybridized with a radioactively labeled C-probe (AACCCT) 3 , and the other ( B ) was hybridized with a G-probe (AGGGTT) 3 , first under native conditions to detect single-stranded G-rich and C-rich telomeric repeats, respectively. Then, the DNA in the gels was denatured and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Indicated are single-stranded G- and C-rich telomeric sequences associated with linear dsDNA (G- and C-overhangs) and t-circles (G- and C-circles), and ssDNA (SS-G and SS-C). Note that after denaturation the hybridization signal was stronger, thus much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) Ethidium bromide staining of the gel in (A) shows circular and linear dsDNA markers. Nicked circular DNA (circles) was generated by UV irradiation of supercoiled ladder, and single-stranded DNA (ss linear) was generated by UV irradiation followed by heat denaturation and snap cooling.
    Figure Legend Snippet: Trypanosoma brucei cells contain ECTRs. Undigested genomic DNA (5 μg) from WT (uninduced) cells was subjected to neutral-neutral 2D-gel electrophoresis in duplicates and dried. One gel ( A ) was hybridized with a radioactively labeled C-probe (AACCCT) 3 , and the other ( B ) was hybridized with a G-probe (AGGGTT) 3 , first under native conditions to detect single-stranded G-rich and C-rich telomeric repeats, respectively. Then, the DNA in the gels was denatured and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Indicated are single-stranded G- and C-rich telomeric sequences associated with linear dsDNA (G- and C-overhangs) and t-circles (G- and C-circles), and ssDNA (SS-G and SS-C). Note that after denaturation the hybridization signal was stronger, thus much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) Ethidium bromide staining of the gel in (A) shows circular and linear dsDNA markers. Nicked circular DNA (circles) was generated by UV irradiation of supercoiled ladder, and single-stranded DNA (ss linear) was generated by UV irradiation followed by heat denaturation and snap cooling.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, Hybridization, Staining, Generated, Irradiation

    3) Product Images from "Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR"

    Article Title: Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2016.19

    Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.
    Figure Legend Snippet: Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.

    Techniques Used: Plasmid Preparation, CTG Assay, Agarose Gel Electrophoresis, Electrophoresis, Concentration Assay, Spectrophotometry

    4) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    5) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    6) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    7) Product Images from "Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection"

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky597

    Trypanosoma brucei cells contain ECTRs. Undigested genomic DNA (5 μg) from WT (uninduced) cells was subjected to neutral-neutral 2D-gel electrophoresis in duplicates and dried. One gel ( A ) was hybridized with a radioactively labeled C-probe (AACCCT) 3 , and the other ( B ) was hybridized with a G-probe (AGGGTT) 3 , first under native conditions to detect single-stranded G-rich and C-rich telomeric repeats, respectively. Then, the DNA in the gels was denatured and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Indicated are single-stranded G- and C-rich telomeric sequences associated with linear dsDNA (G- and C-overhangs) and t-circles (G- and C-circles), and ssDNA (SS-G and SS-C). Note that after denaturation the hybridization signal was stronger, thus much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) Ethidium bromide staining of the gel in (A) shows circular and linear dsDNA markers. Nicked circular DNA (circles) was generated by UV irradiation of supercoiled ladder, and single-stranded DNA (ss linear) was generated by UV irradiation followed by heat denaturation and snap cooling.
    Figure Legend Snippet: Trypanosoma brucei cells contain ECTRs. Undigested genomic DNA (5 μg) from WT (uninduced) cells was subjected to neutral-neutral 2D-gel electrophoresis in duplicates and dried. One gel ( A ) was hybridized with a radioactively labeled C-probe (AACCCT) 3 , and the other ( B ) was hybridized with a G-probe (AGGGTT) 3 , first under native conditions to detect single-stranded G-rich and C-rich telomeric repeats, respectively. Then, the DNA in the gels was denatured and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Indicated are single-stranded G- and C-rich telomeric sequences associated with linear dsDNA (G- and C-overhangs) and t-circles (G- and C-circles), and ssDNA (SS-G and SS-C). Note that after denaturation the hybridization signal was stronger, thus much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) Ethidium bromide staining of the gel in (A) shows circular and linear dsDNA markers. Nicked circular DNA (circles) was generated by UV irradiation of supercoiled ladder, and single-stranded DNA (ss linear) was generated by UV irradiation followed by heat denaturation and snap cooling.

    Techniques Used: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, Hybridization, Staining, Generated, Irradiation

    8) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    9) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    10) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    11) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    12) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    13) Product Images from "Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR"

    Article Title: Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2016.19

    Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.
    Figure Legend Snippet: Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.

    Techniques Used: Plasmid Preparation, CTG Assay, Agarose Gel Electrophoresis, Electrophoresis, Concentration Assay, Spectrophotometry

    14) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    15) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    16) Product Images from "The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9"

    Article Title: The Impact of DNA Topology and Guide Length on Target Selection by a Cytosine-Specific Cas9

    Journal: ACS synthetic biology

    doi: 10.1021/acssynbio.7b00050

    Plasmid protospacer specificity of AceCas9. 3 nM of plasmid substrates were incubated with 500 nM of AceCas9:sgRNA for 1 h and the cleavage products were separated and visualized on a 1.0% agarose gel. Fraction of cleavage was calculated based on integrated band intensities. (A) Sequences and names of a series of protospacer mutants in the pUC19 substrate for AceCas9:sgRNA. Mutated base pairs are shown in bold letters. (B) Comparison of DNA cleavage by AceCas9:sgRNA between the wild-type and mutants for the Bam HI-prelinearized and supercoiled plasmids and for reaction temperatures of 50 and 37 °C. (C) Quantified cleavage activities from reactions shown in (B). For quantification, the intensity of the 3kb linearized DNA plasmid and that of the 2.5 kb large cleavage product bands were obtained by integration and the fraction of cleavage was calculated by taking the ratio of the two. Similarly, the intensity of the supercoiled DNA plasmid and that of the linearized cleavage product were used to determine fraction of cleavage for the supercoiled substrates. The fraction of cleavage for the wild-type plasmid was normalized to 100%.
    Figure Legend Snippet: Plasmid protospacer specificity of AceCas9. 3 nM of plasmid substrates were incubated with 500 nM of AceCas9:sgRNA for 1 h and the cleavage products were separated and visualized on a 1.0% agarose gel. Fraction of cleavage was calculated based on integrated band intensities. (A) Sequences and names of a series of protospacer mutants in the pUC19 substrate for AceCas9:sgRNA. Mutated base pairs are shown in bold letters. (B) Comparison of DNA cleavage by AceCas9:sgRNA between the wild-type and mutants for the Bam HI-prelinearized and supercoiled plasmids and for reaction temperatures of 50 and 37 °C. (C) Quantified cleavage activities from reactions shown in (B). For quantification, the intensity of the 3kb linearized DNA plasmid and that of the 2.5 kb large cleavage product bands were obtained by integration and the fraction of cleavage was calculated by taking the ratio of the two. Similarly, the intensity of the supercoiled DNA plasmid and that of the linearized cleavage product were used to determine fraction of cleavage for the supercoiled substrates. The fraction of cleavage for the wild-type plasmid was normalized to 100%.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis

    17) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    18) Product Images from "A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site"

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl636

    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.
    Figure Legend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Techniques Used: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.
    Figure Legend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Techniques Used: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Figure Legend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Techniques Used: Incubation

    19) Product Images from "Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles"

    Article Title: Bending modes of DNA directly addressed by cryo-electron microscopy of DNA minicircles

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp137

    Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).
    Figure Legend Snippet: Bal31 nuclease detects a destabilized duplex DNA structure in covalently closed supercoiled DNA molecules but does not reveal the presence of kinks in 94-bp covalently closed DNA minicircles. A 2.5% agarose gel run in the presence of ethidium bromide (0.5 µg/ml) reveals that while negatively supercoiled DNA (lane 6) and nonligated nicked rings are completely digested by Bal31 nuclease (see lanes 2–4), the covalently closed monomeric, dimerc, trimeric and tetrameric circles (indicated with arrows) remain resistant to action of Bal31 nuclease (compare lanes 4 and 3).

    Techniques Used: Agarose Gel Electrophoresis

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    Article Title: Spit and venom from Scytodes spiders: a diverse and distinct cocktail
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    Article Snippet: SeqA protein binding to DNA fragments was determined by electrophoretic mobility shift assays (EMSAs) or by 1,10-phenanthroline– copper footprinting as previously described ( ; ). .. Linear DNA was prepared by digesting the supercoiled DNA with Nde I (New England Biolabs) as suggested by the manufacturer, and purifying the cut DNA using the QIAquick PCR purification kit (Qiagen).

    Generated:

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: .. Linear pBR322 was generated by digesting negatively supercoiled DNA with EcoRI (New England BioLabs), followed by phenol extraction and ethanol precipitation. .. Positively supercoiled pBR322 was generated by incubating relaxed pBR322 with a large excess of DNA gyrase under relaxation conditions (no ATP): 35 mM Tris–HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2 , 5 mM DTT, 1.8 mM spermidine, 6.5% (v/v) glycerol, 0.36 mg/ml acetylated BSA for 3 h at 37°C, followed by phenol extraction and ethanol precipitation.

    other:

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: With linear DNA as substrate, CcdB stabilization of cleavage complexes is ATP-dependent; with negatively supercoiled DNA, the presence of at least the non-hydrolysable analogue of ATP, ADPNP (5′-adenylyl β,γ-imidodiphosphate), is required ( ).

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: Gyrase with a truncated GyrA (A592 B2 ) can still perform relaxation of negatively supercoiled DNA in both an ATP-independent ( ) and ATP-dependent manner ( ) manner.

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: We suggest that the earlier observations are a consequence of the ‘bottom-up’ strand passage that occurs during relaxation of negatively supercoiled DNA by gyrase, generating intermediate 5 from 7 in .

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: CcdB stabilized the A592 B2 cleavage complex and inhibited the relaxation of negatively supercoiled DNA by the enzyme ( ).

    Polymerase Chain Reaction:

    Article Title: Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae
    Article Snippet: All the transconjugants were screened using PCR for the plasmid replicons and antibiotic resistance genes in their donors. .. Supercoiled DNA ladder (New England Biolabs, UK) was used as a molecular weight standard for the plasmid size estimation.

    Article Title: Spit and venom from Scytodes spiders: a diverse and distinct cocktail
    Article Snippet: Clones with PCR products > 500 bp were converted from the λ vector to plasmids (pTriplEx2) using E. coli BM25.8 cells provided with the library kit (Clontech). .. In total, 1,728 colonies were screened for cDNAs ≥ 700 bp by extracting DNA with phenol:chloroform, and visualizing recombinant plasmids on an agarose gel, along with supercoiled DNA ladder (NEB).

    Article Title: Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA
    Article Snippet: Digoxigenin (DIG)-labeled probes were amplified from mid-rearrangement genomic DNA using the PCR DIG Probe Synthesis Kit (Roche) according to manufacturer's instructions, with the exception of using ¼ of the standard amount of DIG-labeled nucleotides. .. Supercoiled DNA ladder (New England BioLabs) and 1 Kb Plus DNA ladder (Invitrogen) were used as spike-in standards.

    Article Title: A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation
    Article Snippet: .. Linear DNA was prepared by digesting the supercoiled DNA with Nde I (New England Biolabs) as suggested by the manufacturer, and purifying the cut DNA using the QIAquick PCR purification kit (Qiagen). .. Completed reaction mixtures were incubated for 30 min at 30°C, filtered through 25 mm diameter nitrocellulose filters and washed once with 500 µl of binding buffer at room temperature.

    Recombinant:

    Article Title: Spit and venom from Scytodes spiders: a diverse and distinct cocktail
    Article Snippet: .. In total, 1,728 colonies were screened for cDNAs ≥ 700 bp by extracting DNA with phenol:chloroform, and visualizing recombinant plasmids on an agarose gel, along with supercoiled DNA ladder (NEB). .. Size-selected plasmids were then purified and cDNA inserts were sequenced with either T7 or Sp6 universal primers.

    Molecular Weight:

    Article Title: Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae
    Article Snippet: .. Supercoiled DNA ladder (New England Biolabs, UK) was used as a molecular weight standard for the plasmid size estimation. .. Electrophoresis was performed in 0.5X TBE buffer at 80 V for 3 hr.

    Nucleic Acid Electrophoresis:

    Article Title: Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae
    Article Snippet: Supercoiled DNA ladder (New England Biolabs, UK) was used as a molecular weight standard for the plasmid size estimation. .. Restriction fragments were separated on a 0.8% agarose gel prestained with 0.5 μg/ml ethidium bromide (Thermo Scientific, US) at 80 V for 3 hrs and visualized following gel electrophoresis.

    Magnetic Beads:

    Article Title: Spit and venom from Scytodes spiders: a diverse and distinct cocktail
    Article Snippet: At UML, two total RNA extractions were made from the combined venom glands of eight females and eleven males by homogenization in TRIzol (Invitrogen) followed by purification using the RNeasy Mini-kit (Qiagen). mRNA was isolated from the combined RNA extractions using oligo-(dT)25 -tagged magnetic beads (Invitrogen) and SuperScript III (Invitrogen) used to make cDNA with an oligo-(dT)18 V primer as an anchor. .. In total, 1,728 colonies were screened for cDNAs ≥ 700 bp by extracting DNA with phenol:chloroform, and visualizing recombinant plasmids on an agarose gel, along with supercoiled DNA ladder (NEB).

    Isolation:

    Article Title: Spit and venom from Scytodes spiders: a diverse and distinct cocktail
    Article Snippet: Paragraph title: Venom gland and RNA isolation, cDNA library construction and screening ... In total, 1,728 colonies were screened for cDNAs ≥ 700 bp by extracting DNA with phenol:chloroform, and visualizing recombinant plasmids on an agarose gel, along with supercoiled DNA ladder (NEB).

    Labeling:

    Article Title: Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution
    Article Snippet: Lambda DNA, HindIII digested Lambda DNA, 1 kb DNA Ladder, and Supercoiled DNA Ladder (all from New England Biolabs, Inc.) were used as double stranded DNA samples. .. Labeled oligos were ordered with either Alexa 647 or Cy5 covalently attached.

    Article Title: A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation
    Article Snippet: For the assays using tritiated supercoiled DNA, the 25 µl reactions also contained 2 µg of poly(dI–dC) and 25 or 250 ng of labeled pGB2 plasmid DNA (1000 c.p.m.). .. Linear DNA was prepared by digesting the supercoiled DNA with Nde I (New England Biolabs) as suggested by the manufacturer, and purifying the cut DNA using the QIAquick PCR purification kit (Qiagen).

    Purification:

    Article Title: Spit and venom from Scytodes spiders: a diverse and distinct cocktail
    Article Snippet: At UML, two total RNA extractions were made from the combined venom glands of eight females and eleven males by homogenization in TRIzol (Invitrogen) followed by purification using the RNeasy Mini-kit (Qiagen). mRNA was isolated from the combined RNA extractions using oligo-(dT)25 -tagged magnetic beads (Invitrogen) and SuperScript III (Invitrogen) used to make cDNA with an oligo-(dT)18 V primer as an anchor. .. In total, 1,728 colonies were screened for cDNAs ≥ 700 bp by extracting DNA with phenol:chloroform, and visualizing recombinant plasmids on an agarose gel, along with supercoiled DNA ladder (NEB).

    Article Title: A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation
    Article Snippet: .. Linear DNA was prepared by digesting the supercoiled DNA with Nde I (New England Biolabs) as suggested by the manufacturer, and purifying the cut DNA using the QIAquick PCR purification kit (Qiagen). .. Completed reaction mixtures were incubated for 30 min at 30°C, filtered through 25 mm diameter nitrocellulose filters and washed once with 500 µl of binding buffer at room temperature.

    Sequencing:

    Article Title: Spit and venom from Scytodes spiders: a diverse and distinct cocktail
    Article Snippet: We trimmed vector sequence and assembled all sequences using Sequencher 4.7 (Gene Codes Corp.), resulting in 765 sequences for transcriptomic analysis (“SCY” sequences). .. In total, 1,728 colonies were screened for cDNAs ≥ 700 bp by extracting DNA with phenol:chloroform, and visualizing recombinant plasmids on an agarose gel, along with supercoiled DNA ladder (NEB).

    Article Title: Construction of a shuttle expression vector for lactic acid bacteria
    Article Snippet: Agarose gel electrophoresis of various plasmids used in the study, (a) Lane 1: NEB Supercoiled DNA ladder; Lane 2: Shuttle vector pPBT-GFP; Lane 3: Plasmid pLES003; Lane 4: Plasmid pCP289; Lane 5: Vector pMK-RQ. .. Sequence of Shuttle Vector pPBT-GFP, 9.6 kb.

    Electrophoretic Mobility Shift Assay:

    Article Title: A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation
    Article Snippet: SeqA protein binding to DNA fragments was determined by electrophoretic mobility shift assays (EMSAs) or by 1,10-phenanthroline– copper footprinting as previously described ( ; ). .. Linear DNA was prepared by digesting the supercoiled DNA with Nde I (New England Biolabs) as suggested by the manufacturer, and purifying the cut DNA using the QIAquick PCR purification kit (Qiagen).

    cDNA Library Assay:

    Article Title: Spit and venom from Scytodes spiders: a diverse and distinct cocktail
    Article Snippet: Paragraph title: Venom gland and RNA isolation, cDNA library construction and screening ... In total, 1,728 colonies were screened for cDNAs ≥ 700 bp by extracting DNA with phenol:chloroform, and visualizing recombinant plasmids on an agarose gel, along with supercoiled DNA ladder (NEB).

    Agarose Gel Electrophoresis:

    Article Title: Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae
    Article Snippet: To determine the plasmid size and number, extracted plasmids from donors and transconjugants were separated on a 0.8% agarose gel prestained with 0.5 μg/ml ethidium bromide (Thermo Scientific, US). .. Supercoiled DNA ladder (New England Biolabs, UK) was used as a molecular weight standard for the plasmid size estimation.

    Article Title: Spit and venom from Scytodes spiders: a diverse and distinct cocktail
    Article Snippet: .. In total, 1,728 colonies were screened for cDNAs ≥ 700 bp by extracting DNA with phenol:chloroform, and visualizing recombinant plasmids on an agarose gel, along with supercoiled DNA ladder (NEB). .. Size-selected plasmids were then purified and cDNA inserts were sequenced with either T7 or Sp6 universal primers.

    Article Title: Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA
    Article Snippet: Paragraph title: Two-dimensional agarose gel electrophoresis and Southern blotting ... Supercoiled DNA ladder (New England BioLabs) and 1 Kb Plus DNA ladder (Invitrogen) were used as spike-in standards.

    Article Title: Construction of a shuttle expression vector for lactic acid bacteria
    Article Snippet: .. Agarose gel electrophoresis of various plasmids used in the study, (a) Lane 1: NEB Supercoiled DNA ladder; Lane 2: Shuttle vector pPBT-GFP; Lane 3: Plasmid pLES003; Lane 4: Plasmid pCP289; Lane 5: Vector pMK-RQ. ..

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection
    Article Snippet: A total of 5 μg genomic DNA samples, either undigested or digested with HinfI, were electrophoresed on a 0.4% agarose gel (first dimension) at room temperature and 1V/cm for 16 h. The lanes were cut (removing the first 1 cm from the wells), embedded in a second dimension gel (1.2% agarose with 0.3 μg/ml ethidium bromide) and electrophoresed at 4.5V/cm for 6 h. The gel was dried, cut to separate the duplicated samples and hybridized side by side with C- and G-probes to detect single-stranded telomeric DNA, as described for in-gel analysis. .. Supercoiled DNA ladder (NEB Inc), containing nine supercoiled plasmids (2–10 kb), was used as reference.

    Plasmid Preparation:

    Article Title: Molecular Analysis of Antibiotic Resistance Determinants and Plasmids in Malaysian Isolates of Multidrug Resistant Klebsiella pneumoniae
    Article Snippet: .. Supercoiled DNA ladder (New England Biolabs, UK) was used as a molecular weight standard for the plasmid size estimation. .. Electrophoresis was performed in 0.5X TBE buffer at 80 V for 3 hr.

    Article Title: Spit and venom from Scytodes spiders: a diverse and distinct cocktail
    Article Snippet: The cDNA was size fractionated using ChromaSpin 1000 columns (Clontech) to enrich for transcripts ≥1000 bp. cDNAs were then cloned into the pZErO-2 plasmid using blunt-end ligation at EcoRV-digested sites and recombinant plasmids transformed into Escherichia coli TOP10 competent cells (Invitrogen) by electroporation. .. In total, 1,728 colonies were screened for cDNAs ≥ 700 bp by extracting DNA with phenol:chloroform, and visualizing recombinant plasmids on an agarose gel, along with supercoiled DNA ladder (NEB).

    Article Title: Construction of a shuttle expression vector for lactic acid bacteria
    Article Snippet: .. Agarose gel electrophoresis of various plasmids used in the study, (a) Lane 1: NEB Supercoiled DNA ladder; Lane 2: Shuttle vector pPBT-GFP; Lane 3: Plasmid pLES003; Lane 4: Plasmid pCP289; Lane 5: Vector pMK-RQ. ..

    Article Title: A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation
    Article Snippet: For the assays using tritiated supercoiled DNA, the 25 µl reactions also contained 2 µg of poly(dI–dC) and 25 or 250 ng of labeled pGB2 plasmid DNA (1000 c.p.m.). .. Linear DNA was prepared by digesting the supercoiled DNA with Nde I (New England Biolabs) as suggested by the manufacturer, and purifying the cut DNA using the QIAquick PCR purification kit (Qiagen).

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: DNA and drugs Negatively supercoiled and relaxed pBR322 plasmid DNA were kind gifts from Mrs A. J. Howells (John Innes Enterprises). .. Linear pBR322 was generated by digesting negatively supercoiled DNA with EcoRI (New England BioLabs), followed by phenol extraction and ethanol precipitation.

    Binding Assay:

    Article Title: A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation
    Article Snippet: The binding buffer was 90 mM potassium glutamate, 1 mM magnesium acetate, 20 mM Tris acetate pH 7.5, 1 mM dithiothreitol, 0.1 mM EDTA, 5% (v/v) glycerol. .. Linear DNA was prepared by digesting the supercoiled DNA with Nde I (New England Biolabs) as suggested by the manufacturer, and purifying the cut DNA using the QIAquick PCR purification kit (Qiagen).

    Sample Prep:

    Article Title: Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution
    Article Snippet: Paragraph title: Sample Preparation ... Lambda DNA, HindIII digested Lambda DNA, 1 kb DNA Ladder, and Supercoiled DNA Ladder (all from New England Biolabs, Inc.) were used as double stranded DNA samples.

    Homogenization:

    Article Title: Spit and venom from Scytodes spiders: a diverse and distinct cocktail
    Article Snippet: At UML, two total RNA extractions were made from the combined venom glands of eight females and eleven males by homogenization in TRIzol (Invitrogen) followed by purification using the RNeasy Mini-kit (Qiagen). mRNA was isolated from the combined RNA extractions using oligo-(dT)25 -tagged magnetic beads (Invitrogen) and SuperScript III (Invitrogen) used to make cDNA with an oligo-(dT)18 V primer as an anchor. .. In total, 1,728 colonies were screened for cDNAs ≥ 700 bp by extracting DNA with phenol:chloroform, and visualizing recombinant plasmids on an agarose gel, along with supercoiled DNA ladder (NEB).

    Protein Binding:

    Article Title: A case for sliding SeqA tracts at anchored replication forks during Escherichia coli chromosome replication and segregation
    Article Snippet: Paragraph title: Assays of SeqA protein binding ... Linear DNA was prepared by digesting the supercoiled DNA with Nde I (New England Biolabs) as suggested by the manufacturer, and purifying the cut DNA using the QIAquick PCR purification kit (Qiagen).

    Concentration Assay:

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: .. CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA. .. CcdB stabilized the A592 B2 cleavage complex and inhibited the relaxation of negatively supercoiled DNA by the enzyme ( ).

    Article Title: Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution
    Article Snippet: Lambda DNA, HindIII digested Lambda DNA, 1 kb DNA Ladder, and Supercoiled DNA Ladder (all from New England Biolabs, Inc.) were used as double stranded DNA samples. .. Staining was performed at 5 or 10 ng/μL total dsDNA concentration and 1 μM TOTO-3 Iodide (Life Technologies) for at least 1 hour in the dark.

    Two-Dimensional Gel Electrophoresis:

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection
    Article Snippet: Paragraph title: Two-dimensional gel electrophoresis ... Supercoiled DNA ladder (NEB Inc), containing nine supercoiled plasmids (2–10 kb), was used as reference.

    Ethanol Precipitation:

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site
    Article Snippet: .. Linear pBR322 was generated by digesting negatively supercoiled DNA with EcoRI (New England BioLabs), followed by phenol extraction and ethanol precipitation. .. Positively supercoiled pBR322 was generated by incubating relaxed pBR322 with a large excess of DNA gyrase under relaxation conditions (no ATP): 35 mM Tris–HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2 , 5 mM DTT, 1.8 mM spermidine, 6.5% (v/v) glycerol, 0.36 mg/ml acetylated BSA for 3 h at 37°C, followed by phenol extraction and ethanol precipitation.

    Staining:

    Article Title: Single Molecule Hydrodynamic Separation Allows Sensitive and Quantitative Analysis of DNA Conformation and Binding Interactions in Free Solution
    Article Snippet: Lambda DNA, HindIII digested Lambda DNA, 1 kb DNA Ladder, and Supercoiled DNA Ladder (all from New England Biolabs, Inc.) were used as double stranded DNA samples. .. Staining was performed at 5 or 10 ng/μL total dsDNA concentration and 1 μM TOTO-3 Iodide (Life Technologies) for at least 1 hour in the dark.

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    New England Biolabs supercoiled dna
    CcdB can inhibit catalytic relaxation of <t>DNA</t> by gyrase. Negatively <t>supercoiled</t> pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).
    Supercoiled Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 73 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Journal: Nucleic Acids Research

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    doi: 10.1093/nar/gkl636

    Figure Lengend Snippet: CcdB can inhibit catalytic relaxation of DNA by gyrase. Negatively supercoiled pBR322 (3.5 nM) was incubated with gyrase (20 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 4 h at 25°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Article Snippet: CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA.

    Techniques: Incubation

    Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Journal: Nucleic Acids Research

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    doi: 10.1093/nar/gkl636

    Figure Lengend Snippet: Estimation of IC 50 s for CcdB inhibition of the catalytic reactions of DNA gyrase. Relaxed ( A ) or negatively supercoiled ( B ) pN01 (3.5 nM) was incubated with gyrase [1.5 nM (A); 20 nM (B)], ATP [1.4 mM (A); 0 mM (B)] and various concentrations of CFX (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 5 and 10 μM) or CcdB (0, 0.1, 0.2, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and 12.5 μM) as indicated, for 4 h at 25°C. DNA was subjected to phenol extraction and analysed on 1% agarose gels, or triplex formation was quantitatively analysed by SYBR fluorescence and data plotted ( 36 ). Data were fitted with (A) single exponential decay curves or (B) sigmoidal curves.

    Article Snippet: CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA.

    Techniques: Inhibition, Incubation, Fluorescence

    CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Journal: Nucleic Acids Research

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    doi: 10.1093/nar/gkl636

    Figure Lengend Snippet: CcdB can inhibit the ATP-independent relaxation of DNA by an A 2 B47 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A 2 B47 2 complex (200 nM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 2 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Article Snippet: CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA.

    Techniques: Incubation

    Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Journal: Nucleic Acids Research

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    doi: 10.1093/nar/gkl636

    Figure Lengend Snippet: Nucleotide-dependence of CcdB-stabilized, DNA gyrase-mediated cleavage of DNA. Relaxed, negatively supercoiled (−ve s/c), linear or positively supercoiled (+ve s/c) pBR322 (3.5 nM) was incubated with gyrase (30 nM), either with no nucleotide, ATP (1.4 mM) or ADPNP (1.4 mM) and either CFX (13.5 μM) or CcdB (3.6 μM), as indicated, for 1 h at 37°C. Cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels. L, linear; N, nicked; R, relaxed; SC, supercoiled.

    Article Snippet: CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA.

    Techniques: Incubation

    CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Journal: Nucleic Acids Research

    Article Title: A strand-passage conformation of DNA gyrase is required to allow the bacterial toxin, CcdB, to access its binding site

    doi: 10.1093/nar/gkl636

    Figure Lengend Snippet: CcdB can inhibit the ATP-dependent relaxation of DNA by an A59 2 B 2 gyrase complex. Negatively supercoiled pBR322 (3.5 nM) was incubated with A59 2 B 2 (100 nM), ATP (1.4 mM) and various concentrations of CFX and CcdB (0, 0.1, 0.2, 0.5, 1, 2, 5 and 10 μM) as indicated, for 1 h at 37°C. Assays were either ( A ) stopped or ( B ) cleavage complexes were revealed by the addition of SDS and proteinase K and incubation at 37°C for 30 min. DNA was subjected to phenol extraction and analysed on 1% agarose gels run in the (A) absence or (B) presence of ethidium bromide (1 μg/ml).

    Article Snippet: CcdB's ability to stabilize the A592 B2 cleavage complex and inhibit the relaxation of negatively supercoiled DNA by the A592 B2 enzyme was investigated by titrating CcdB into a fixed concentration of A592 B2 and DNA.

    Techniques: Incubation

    Trypanosoma brucei cells contain ECTRs. Undigested genomic DNA (5 μg) from WT (uninduced) cells was subjected to neutral-neutral 2D-gel electrophoresis in duplicates and dried. One gel ( A ) was hybridized with a radioactively labeled C-probe (AACCCT) 3 , and the other ( B ) was hybridized with a G-probe (AGGGTT) 3 , first under native conditions to detect single-stranded G-rich and C-rich telomeric repeats, respectively. Then, the DNA in the gels was denatured and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Indicated are single-stranded G- and C-rich telomeric sequences associated with linear dsDNA (G- and C-overhangs) and t-circles (G- and C-circles), and ssDNA (SS-G and SS-C). Note that after denaturation the hybridization signal was stronger, thus much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) Ethidium bromide staining of the gel in (A) shows circular and linear dsDNA markers. Nicked circular DNA (circles) was generated by UV irradiation of supercoiled ladder, and single-stranded DNA (ss linear) was generated by UV irradiation followed by heat denaturation and snap cooling.

    Journal: Nucleic Acids Research

    Article Title: Trypanosoma brucei UMSBP2 is a single-stranded telomeric DNA binding protein essential for chromosome end protection

    doi: 10.1093/nar/gky597

    Figure Lengend Snippet: Trypanosoma brucei cells contain ECTRs. Undigested genomic DNA (5 μg) from WT (uninduced) cells was subjected to neutral-neutral 2D-gel electrophoresis in duplicates and dried. One gel ( A ) was hybridized with a radioactively labeled C-probe (AACCCT) 3 , and the other ( B ) was hybridized with a G-probe (AGGGTT) 3 , first under native conditions to detect single-stranded G-rich and C-rich telomeric repeats, respectively. Then, the DNA in the gels was denatured and re-hybridized to the same probes to detect both single- and double-stranded telomeric repeats. Indicated are single-stranded G- and C-rich telomeric sequences associated with linear dsDNA (G- and C-overhangs) and t-circles (G- and C-circles), and ssDNA (SS-G and SS-C). Note that after denaturation the hybridization signal was stronger, thus much shorter exposure was sufficient to visualize the dsDNA and thus ssDNA appears weaker or disappeared. ( C ) Ethidium bromide staining of the gel in (A) shows circular and linear dsDNA markers. Nicked circular DNA (circles) was generated by UV irradiation of supercoiled ladder, and single-stranded DNA (ss linear) was generated by UV irradiation followed by heat denaturation and snap cooling.

    Article Snippet: Supercoiled DNA ladder (NEB Inc), containing nine supercoiled plasmids (2–10 kb), was used as reference.

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, Hybridization, Staining, Generated, Irradiation

    Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    doi: 10.1038/mtm.2016.19

    Figure Lengend Snippet: Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.

    Article Snippet: One hundred ng of each supercoiled and linearized plasmids were analyzed on a 1% native agarose gel alongside a 2-log DNA ladder and supercoiled ladder (New England BioLabs Cat N3200S and N0472S, Ipswich, MA) to confirm complete digestion and purity, and to confirm concentration.

    Techniques: Plasmid Preparation, CTG Assay, Agarose Gel Electrophoresis, Electrophoresis, Concentration Assay, Spectrophotometry