pbr322 plasmid  (New England Biolabs)


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    Structured Review

    New England Biolabs pbr322 plasmid
    DdrC binds to ssDNA and dsDNA with a preference for ssDNA. A Binding of recombinant DdrC to plasmid or viral DNA analyzed by EMSA. 200 ng of supercoiled or linear <t>pBR322</t> DNA as well as 200 ng of RFI or single-stranded DNA of phiX174 virion (31 μM nucleotides of each DNA) were incubated with increasing concentrations of DdrC as indicated in the figure. DNA-protein complexes were separated in 1.2% agarose gels. Products loaded in the right lane of the left panel were treated with SDS and proteinase K. sc: supercoiled dsDNA, oc: open circle dsDNA, Li: linear dsDNA. B Binding of DdrC to oligonucleotides. Increasing concentrations of DdrC were incubated with 3.3 nM of a single-stranded (ss) 67-mer fluorescent oligonucleotide (left panel) or 3.3 nM of the corresponding ds oligonucleotide (right panel). The products of the reactions were separated in 6% native polyacrylamide gels. Lanes C: DNA control without DdrC.
    Pbr322 Plasmid, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 89/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pbr322 plasmid/product/New England Biolabs
    Average 89 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    pbr322 plasmid - by Bioz Stars, 2020-07
    89/100 stars

    Images

    1) Product Images from "In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium"

    Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177751

    DdrC binds to ssDNA and dsDNA with a preference for ssDNA. A Binding of recombinant DdrC to plasmid or viral DNA analyzed by EMSA. 200 ng of supercoiled or linear pBR322 DNA as well as 200 ng of RFI or single-stranded DNA of phiX174 virion (31 μM nucleotides of each DNA) were incubated with increasing concentrations of DdrC as indicated in the figure. DNA-protein complexes were separated in 1.2% agarose gels. Products loaded in the right lane of the left panel were treated with SDS and proteinase K. sc: supercoiled dsDNA, oc: open circle dsDNA, Li: linear dsDNA. B Binding of DdrC to oligonucleotides. Increasing concentrations of DdrC were incubated with 3.3 nM of a single-stranded (ss) 67-mer fluorescent oligonucleotide (left panel) or 3.3 nM of the corresponding ds oligonucleotide (right panel). The products of the reactions were separated in 6% native polyacrylamide gels. Lanes C: DNA control without DdrC.
    Figure Legend Snippet: DdrC binds to ssDNA and dsDNA with a preference for ssDNA. A Binding of recombinant DdrC to plasmid or viral DNA analyzed by EMSA. 200 ng of supercoiled or linear pBR322 DNA as well as 200 ng of RFI or single-stranded DNA of phiX174 virion (31 μM nucleotides of each DNA) were incubated with increasing concentrations of DdrC as indicated in the figure. DNA-protein complexes were separated in 1.2% agarose gels. Products loaded in the right lane of the left panel were treated with SDS and proteinase K. sc: supercoiled dsDNA, oc: open circle dsDNA, Li: linear dsDNA. B Binding of DdrC to oligonucleotides. Increasing concentrations of DdrC were incubated with 3.3 nM of a single-stranded (ss) 67-mer fluorescent oligonucleotide (left panel) or 3.3 nM of the corresponding ds oligonucleotide (right panel). The products of the reactions were separated in 6% native polyacrylamide gels. Lanes C: DNA control without DdrC.

    Techniques Used: Binding Assay, Recombinant, Plasmid Preparation, Incubation

    Circularization of pBR322 (cohesive ends) and pUC19 (blunt ends) plasmids mediated by DdrC visualized by electron microscopy. Panel a: Control pBR322 DNA linearized by PstI. Panels b-e: pBR322 circularization mediated by DdrC. Panel f. Control pUC19 DNA linearized by Ssp11. Panels g-j: pUC19 circularization mediated by DdrC. 1 μM DdrC was mixed with 2 nM molecules of linear pBR322 or pUC19 plasmid, containing cohesive or blunt ends, respectively. The shapes are similar at 0.5 μM, 1 μM or 2 μM of DdrC. Magnification = 85,000. Some loci of plasmid circularization are indicated by arrows.
    Figure Legend Snippet: Circularization of pBR322 (cohesive ends) and pUC19 (blunt ends) plasmids mediated by DdrC visualized by electron microscopy. Panel a: Control pBR322 DNA linearized by PstI. Panels b-e: pBR322 circularization mediated by DdrC. Panel f. Control pUC19 DNA linearized by Ssp11. Panels g-j: pUC19 circularization mediated by DdrC. 1 μM DdrC was mixed with 2 nM molecules of linear pBR322 or pUC19 plasmid, containing cohesive or blunt ends, respectively. The shapes are similar at 0.5 μM, 1 μM or 2 μM of DdrC. Magnification = 85,000. Some loci of plasmid circularization are indicated by arrows.

    Techniques Used: Electron Microscopy, Plasmid Preparation

    Visualization of DdrC-DNA complexes by transmission electron microscopy. A PhiX174 ssDNA (1.4 nM, 7.5 μM nucleotides) was incubated with 1 μM (panels b-d) or 2 μM (panels f-h) of DdrC. Panel a: phiX174 ssDNA control without DdrC. Panel e: Interaction of E . coli SSB protein (1 μM) with ssDNA. Magnification = 85,000. B Supercoiled pBR322 DNA (1.7 nM, 7.5 μM base pairs) incubated with 1 μM (panel b and c) or 2 μM (panel d) of DdrC. Panel a: pBR322 DNA control without protein. Magnification = 85,000. Some“bridge” structures, forming loops or kinks, are indicated by arrows.
    Figure Legend Snippet: Visualization of DdrC-DNA complexes by transmission electron microscopy. A PhiX174 ssDNA (1.4 nM, 7.5 μM nucleotides) was incubated with 1 μM (panels b-d) or 2 μM (panels f-h) of DdrC. Panel a: phiX174 ssDNA control without DdrC. Panel e: Interaction of E . coli SSB protein (1 μM) with ssDNA. Magnification = 85,000. B Supercoiled pBR322 DNA (1.7 nM, 7.5 μM base pairs) incubated with 1 μM (panel b and c) or 2 μM (panel d) of DdrC. Panel a: pBR322 DNA control without protein. Magnification = 85,000. Some“bridge” structures, forming loops or kinks, are indicated by arrows.

    Techniques Used: Transmission Assay, Electron Microscopy, Incubation

    DdrC protects DNA against degradation by nucleases. Protection of supercoiled pBR322 plasmid (3.5 nM) from DNase I activity (0.1 U) (panel a), linear pBR322 (3.5 nM) from Exonuclease III activity (200 U) (panel b) and phiX174 ssDNA (5.9 nM) from Mung Bean Nuclease activity (1 U) (panel c) by 7 μM, 7 μM, and 2 μM DdrC, respectively. Lanes C: DNA controls without protein. Lanes 1: DNA incubation with nuclease alone. Lanes 2: DNA incubation with DdrC alone. Lanes 3: DNA pre-incubated with DdrC 15 min at 4°C before addition of nuclease. Lanes 4: Reaction products corresponding to lane 3 were further treated with Proteinase K/SDS. Panel a, lane 5: DdrC and DNase I were simultaneously incubated with supercoiled DNA before treatment with Proteinase K/SDS.
    Figure Legend Snippet: DdrC protects DNA against degradation by nucleases. Protection of supercoiled pBR322 plasmid (3.5 nM) from DNase I activity (0.1 U) (panel a), linear pBR322 (3.5 nM) from Exonuclease III activity (200 U) (panel b) and phiX174 ssDNA (5.9 nM) from Mung Bean Nuclease activity (1 U) (panel c) by 7 μM, 7 μM, and 2 μM DdrC, respectively. Lanes C: DNA controls without protein. Lanes 1: DNA incubation with nuclease alone. Lanes 2: DNA incubation with DdrC alone. Lanes 3: DNA pre-incubated with DdrC 15 min at 4°C before addition of nuclease. Lanes 4: Reaction products corresponding to lane 3 were further treated with Proteinase K/SDS. Panel a, lane 5: DdrC and DNase I were simultaneously incubated with supercoiled DNA before treatment with Proteinase K/SDS.

    Techniques Used: Plasmid Preparation, Activity Assay, Incubation

    2) Product Images from "Reconstituting ParA/ParB-mediated transport of DNA cargo"

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo

    Journal: Methods in cell biology

    doi: 10.1016/bs.mcb.2015.01.021

    Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.
    Figure Legend Snippet: Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.

    Techniques Used: Labeling, Plasmid Preparation, Flow Cytometry, Ligation

    Cell-free setup for visualizing SopAB-mediated dynamics of plasmids or beads on a DNA carpet. (A) SopA and SopB proteins are preincubated with plasmid or bead cargo bearing the sopC -centromere site in Sop buffer containing ATP. The sample is then flowed into the DNA-carpeted flow cell. Flow is stopped to visualize how SopA and SopB mediate cargo dynamics on the DNA carpet. (B) Excitation lasers are directed onto the flow cell at an angle that allows total internal reflection (TIRF). The evanescent wave (blue haze) produced by the incident and reflected light propagates into the flow cell ~100 nm. SopA–GFP (green) is covisualized with either SopBeAlexa647, or one of two possible cargos labeled with Alexa647, the pBR322:: sopC plasmid or sopC -coated beads. (i) ATP-bound SopA binds the DNA carpet and tethers the plasmid by SopA–SopB interaction, (ii) until SopB locally stimulates the removal of SopA. (iii) Once all SopA anchor points are removed, the plasmid is released and is no longer observable via TIRFM. A magnet above the flow cell surface confines sopC -coated magnetic beads on the DNA carpet, which supports persistent SopA removal and directed bead movement. (See color plate)
    Figure Legend Snippet: Cell-free setup for visualizing SopAB-mediated dynamics of plasmids or beads on a DNA carpet. (A) SopA and SopB proteins are preincubated with plasmid or bead cargo bearing the sopC -centromere site in Sop buffer containing ATP. The sample is then flowed into the DNA-carpeted flow cell. Flow is stopped to visualize how SopA and SopB mediate cargo dynamics on the DNA carpet. (B) Excitation lasers are directed onto the flow cell at an angle that allows total internal reflection (TIRF). The evanescent wave (blue haze) produced by the incident and reflected light propagates into the flow cell ~100 nm. SopA–GFP (green) is covisualized with either SopBeAlexa647, or one of two possible cargos labeled with Alexa647, the pBR322:: sopC plasmid or sopC -coated beads. (i) ATP-bound SopA binds the DNA carpet and tethers the plasmid by SopA–SopB interaction, (ii) until SopB locally stimulates the removal of SopA. (iii) Once all SopA anchor points are removed, the plasmid is released and is no longer observable via TIRFM. A magnet above the flow cell surface confines sopC -coated magnetic beads on the DNA carpet, which supports persistent SopA removal and directed bead movement. (See color plate)

    Techniques Used: Plasmid Preparation, Flow Cytometry, Produced, Labeling, Magnetic Beads

    Related Articles

    Amplification:

    Article Title: Developing E. coli-E. coli co-cultures to overcome barriers of heterologous tryptamine biosynthesis
    Article Snippet: .. To construct plasmid pTS, three DNA fragments, including tnaC sequence with the tnaCAB operon promoter PCR amplified from the E. coli K12 chromosome by primers Tnac-Gf and Tnac-Gr, the tetA fragment PCR amplified from pBR322 plasmid by primers Teta-Gf and Teta-Gr, and plasmid pET21c backbone digested with SphI/NotI, were assembled together using the Gibson assembly kit (New England Biolabs). .. To construct plasmid pTC, the tetA gene with the promoter was PCR amplified from pBR322 using primer Tet-f and Tet-r, digested with NdeI/XhoI, and then ligated with plasmid pET21c treated with the same enzymes.

    Article Title: Multiple Kinesins Induce Tension for Smooth Cargo Transport
    Article Snippet: .. Rupture force experiment with optical tweezerDouble-stranded DNA was synthesized through PCR amplification of a 1.565-kbp segment of the pBR322 plasmid (New England Biolabs), using forward and reverse primers conjugated with a 5’ biotin and a 5’ digoxigenin, respectively (Integrated DNA Technologies) and a high-fidelity master mix (New England Biolabs). .. The PCR product was purified with a PCR cleanup kit (QIAGEN).

    Synthesized:

    Article Title: Multiple Kinesins Induce Tension for Smooth Cargo Transport
    Article Snippet: .. Rupture force experiment with optical tweezerDouble-stranded DNA was synthesized through PCR amplification of a 1.565-kbp segment of the pBR322 plasmid (New England Biolabs), using forward and reverse primers conjugated with a 5’ biotin and a 5’ digoxigenin, respectively (Integrated DNA Technologies) and a high-fidelity master mix (New England Biolabs). .. The PCR product was purified with a PCR cleanup kit (QIAGEN).

    Construct:

    Article Title: Developing E. coli-E. coli co-cultures to overcome barriers of heterologous tryptamine biosynthesis
    Article Snippet: .. To construct plasmid pTS, three DNA fragments, including tnaC sequence with the tnaCAB operon promoter PCR amplified from the E. coli K12 chromosome by primers Tnac-Gf and Tnac-Gr, the tetA fragment PCR amplified from pBR322 plasmid by primers Teta-Gf and Teta-Gr, and plasmid pET21c backbone digested with SphI/NotI, were assembled together using the Gibson assembly kit (New England Biolabs). .. To construct plasmid pTC, the tetA gene with the promoter was PCR amplified from pBR322 using primer Tet-f and Tet-r, digested with NdeI/XhoI, and then ligated with plasmid pET21c treated with the same enzymes.

    Purification:

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322 plasmid (4.4 Kb) pBR322:: sopC plasmid (4.7 Kb) Nt BspQI Nicking Enzyme (New England Biolabs, Cat. # R0644S) Phenol:CHCl3 :Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol Deoxyribonucleotide (dNTP) mix (Life Technologies; Cat. # 18427–013) dCTP (Life Technologies; Cat. # 18253–013) Alexa Fluor 647–12-OBEA-dCTP (Life Technologies; Cat. # ) DNA Polymerase I (Life Technologies, Cat. # 18010–025) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) QIAquick polymerase chain reaction (PCR) Purification Kit (Qiagen, Cat. # 28104) Ethidium Bromide 400 U/μL T4 Ligase (New England Biolabs, Cat. # M0202S) .. Water Bath Vortex Table top centrifuge Nanodrop Spectrophotometer Typhoon Imager (GE Healthcare).

    Sequencing:

    Article Title: Developing E. coli-E. coli co-cultures to overcome barriers of heterologous tryptamine biosynthesis
    Article Snippet: .. To construct plasmid pTS, three DNA fragments, including tnaC sequence with the tnaCAB operon promoter PCR amplified from the E. coli K12 chromosome by primers Tnac-Gf and Tnac-Gr, the tetA fragment PCR amplified from pBR322 plasmid by primers Teta-Gf and Teta-Gr, and plasmid pET21c backbone digested with SphI/NotI, were assembled together using the Gibson assembly kit (New England Biolabs). .. To construct plasmid pTC, the tetA gene with the promoter was PCR amplified from pBR322 using primer Tet-f and Tet-r, digested with NdeI/XhoI, and then ligated with plasmid pET21c treated with the same enzymes.

    Polymerase Chain Reaction:

    Article Title: Developing E. coli-E. coli co-cultures to overcome barriers of heterologous tryptamine biosynthesis
    Article Snippet: .. To construct plasmid pTS, three DNA fragments, including tnaC sequence with the tnaCAB operon promoter PCR amplified from the E. coli K12 chromosome by primers Tnac-Gf and Tnac-Gr, the tetA fragment PCR amplified from pBR322 plasmid by primers Teta-Gf and Teta-Gr, and plasmid pET21c backbone digested with SphI/NotI, were assembled together using the Gibson assembly kit (New England Biolabs). .. To construct plasmid pTC, the tetA gene with the promoter was PCR amplified from pBR322 using primer Tet-f and Tet-r, digested with NdeI/XhoI, and then ligated with plasmid pET21c treated with the same enzymes.

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322 plasmid (4.4 Kb) pBR322:: sopC plasmid (4.7 Kb) Nt BspQI Nicking Enzyme (New England Biolabs, Cat. # R0644S) Phenol:CHCl3 :Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol Deoxyribonucleotide (dNTP) mix (Life Technologies; Cat. # 18427–013) dCTP (Life Technologies; Cat. # 18253–013) Alexa Fluor 647–12-OBEA-dCTP (Life Technologies; Cat. # ) DNA Polymerase I (Life Technologies, Cat. # 18010–025) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) QIAquick polymerase chain reaction (PCR) Purification Kit (Qiagen, Cat. # 28104) Ethidium Bromide 400 U/μL T4 Ligase (New England Biolabs, Cat. # M0202S) .. Water Bath Vortex Table top centrifuge Nanodrop Spectrophotometer Typhoon Imager (GE Healthcare).

    Article Title: Multiple Kinesins Induce Tension for Smooth Cargo Transport
    Article Snippet: .. Rupture force experiment with optical tweezerDouble-stranded DNA was synthesized through PCR amplification of a 1.565-kbp segment of the pBR322 plasmid (New England Biolabs), using forward and reverse primers conjugated with a 5’ biotin and a 5’ digoxigenin, respectively (Integrated DNA Technologies) and a high-fidelity master mix (New England Biolabs). .. The PCR product was purified with a PCR cleanup kit (QIAGEN).

    Staining:

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322 plasmid (4.4 Kb) pBR322:: sopC plasmid (4.7 Kb) Nt BspQI Nicking Enzyme (New England Biolabs, Cat. # R0644S) Phenol:CHCl3 :Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol Deoxyribonucleotide (dNTP) mix (Life Technologies; Cat. # 18427–013) dCTP (Life Technologies; Cat. # 18253–013) Alexa Fluor 647–12-OBEA-dCTP (Life Technologies; Cat. # ) DNA Polymerase I (Life Technologies, Cat. # 18010–025) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) QIAquick polymerase chain reaction (PCR) Purification Kit (Qiagen, Cat. # 28104) Ethidium Bromide 400 U/μL T4 Ligase (New England Biolabs, Cat. # M0202S) .. Water Bath Vortex Table top centrifuge Nanodrop Spectrophotometer Typhoon Imager (GE Healthcare).

    Plasmid Preparation:

    Article Title: Using Single-Turnover Kinetics with Osmotic Stress to Characterize the EcoRV Cleavage Reaction
    Article Snippet: .. Cleavage of the 310 bp fragment at the EcoRV recognition site yields DNA fragments of 107 bp and 203 bp long. pBR322 plasmid and restriction enzymes were ordered from New England Biolabs. .. The primer oligonucleotides were purchased from Invitrogen.

    Article Title: Developing E. coli-E. coli co-cultures to overcome barriers of heterologous tryptamine biosynthesis
    Article Snippet: .. To construct plasmid pTS, three DNA fragments, including tnaC sequence with the tnaCAB operon promoter PCR amplified from the E. coli K12 chromosome by primers Tnac-Gf and Tnac-Gr, the tetA fragment PCR amplified from pBR322 plasmid by primers Teta-Gf and Teta-Gr, and plasmid pET21c backbone digested with SphI/NotI, were assembled together using the Gibson assembly kit (New England Biolabs). .. To construct plasmid pTC, the tetA gene with the promoter was PCR amplified from pBR322 using primer Tet-f and Tet-r, digested with NdeI/XhoI, and then ligated with plasmid pET21c treated with the same enzymes.

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo
    Article Snippet: .. pBR322 plasmid (4.4 Kb) pBR322:: sopC plasmid (4.7 Kb) Nt BspQI Nicking Enzyme (New England Biolabs, Cat. # R0644S) Phenol:CHCl3 :Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol Deoxyribonucleotide (dNTP) mix (Life Technologies; Cat. # 18427–013) dCTP (Life Technologies; Cat. # 18253–013) Alexa Fluor 647–12-OBEA-dCTP (Life Technologies; Cat. # ) DNA Polymerase I (Life Technologies, Cat. # 18010–025) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) QIAquick polymerase chain reaction (PCR) Purification Kit (Qiagen, Cat. # 28104) Ethidium Bromide 400 U/μL T4 Ligase (New England Biolabs, Cat. # M0202S) .. Water Bath Vortex Table top centrifuge Nanodrop Spectrophotometer Typhoon Imager (GE Healthcare).

    Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium
    Article Snippet: .. To analyze interaction of DdrC with linear DNA fragment, pUC19 plasmid (New England Biolabs) was linearized with SspI (New England Biolabs) restriction enzyme producing blunt ends and pBR322 plasmid was linearized with PstI (New England Biolabs) producing 3’ overhang cohesive ends. .. For TEM experiments, all DNA molecules were purified on a MiniQ anion exchange column (GE Healthcare) with a chromatography SMART system.

    Article Title: Multiple Kinesins Induce Tension for Smooth Cargo Transport
    Article Snippet: .. Rupture force experiment with optical tweezerDouble-stranded DNA was synthesized through PCR amplification of a 1.565-kbp segment of the pBR322 plasmid (New England Biolabs), using forward and reverse primers conjugated with a 5’ biotin and a 5’ digoxigenin, respectively (Integrated DNA Technologies) and a high-fidelity master mix (New England Biolabs). .. The PCR product was purified with a PCR cleanup kit (QIAGEN).

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV
    Article Snippet: .. The pBR322 plasmid and restriction enzymes SphI and HindIII were purchased from New England Biolabs. .. The primer oligonucleotides were purchased from Invitrogen.

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    New England Biolabs plasmid pbr322
    RecA stimulates Topo I-catalyzed relaxation reaction. ( A ) The standard relaxation reaction mixtures containing 100 fmol (as molecule) <t>pBR322</t> form I DNA and the indicated amounts of Topo I were incubated in the absence (−) or presence (+) of 5.6 μg (3 nt per RecA monomer) of RecA and the DNA products were analyzed as described in Materials and Methods. The assay was repeated three times and virtually identical results were obtained. Representative results are shown. ( B ) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and either 0 fmol (−) or 33 fmol (+) of Topo I were incubated in the presence of the indicated amounts of RecA and the DNA products were analyzed as described in Materials and Methods. Two independent experiments showed essentially identical results and representative results are shown.
    Plasmid Pbr322, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid pbr322/product/New England Biolabs
    Average 90 stars, based on 36 article reviews
    Price from $9.99 to $1999.99
    plasmid pbr322 - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

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    RecA stimulates Topo I-catalyzed relaxation reaction. ( A ) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and the indicated amounts of Topo I were incubated in the absence (−) or presence (+) of 5.6 μg (3 nt per RecA monomer) of RecA and the DNA products were analyzed as described in Materials and Methods. The assay was repeated three times and virtually identical results were obtained. Representative results are shown. ( B ) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and either 0 fmol (−) or 33 fmol (+) of Topo I were incubated in the presence of the indicated amounts of RecA and the DNA products were analyzed as described in Materials and Methods. Two independent experiments showed essentially identical results and representative results are shown.

    Journal: Nucleic Acids Research

    Article Title: RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli

    doi: 10.1093/nar/gkl981

    Figure Lengend Snippet: RecA stimulates Topo I-catalyzed relaxation reaction. ( A ) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and the indicated amounts of Topo I were incubated in the absence (−) or presence (+) of 5.6 μg (3 nt per RecA monomer) of RecA and the DNA products were analyzed as described in Materials and Methods. The assay was repeated three times and virtually identical results were obtained. Representative results are shown. ( B ) The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA and either 0 fmol (−) or 33 fmol (+) of Topo I were incubated in the presence of the indicated amounts of RecA and the DNA products were analyzed as described in Materials and Methods. Two independent experiments showed essentially identical results and representative results are shown.

    Article Snippet: Supercoiling reaction Covalently closed, negatively supercoiled double-stranded circular (form I) DNA of plasmid pBR322 (4361 bp) was purchased from New England Biolabs (Beverly, MA).

    Techniques: Incubation

    Formation of active RecA filaments is required for the stimulation of Topo I-catalyzed relaxation activity by RecA. The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA, 5 mM ADP, ATP ( A ), dATP, or ATPγS ( B ), as indicated, and either 0 fmol (−) or 100 fmol (+) of Topo I were incubated in the presence of the indicated amounts of RecA and the DNA products were analyzed as described in Materials and Methods.

    Journal: Nucleic Acids Research

    Article Title: RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli

    doi: 10.1093/nar/gkl981

    Figure Lengend Snippet: Formation of active RecA filaments is required for the stimulation of Topo I-catalyzed relaxation activity by RecA. The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA, 5 mM ADP, ATP ( A ), dATP, or ATPγS ( B ), as indicated, and either 0 fmol (−) or 100 fmol (+) of Topo I were incubated in the presence of the indicated amounts of RecA and the DNA products were analyzed as described in Materials and Methods.

    Article Snippet: Supercoiling reaction Covalently closed, negatively supercoiled double-stranded circular (form I) DNA of plasmid pBR322 (4361 bp) was purchased from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay, Incubation

    RecA has no effect on the supercoiling activity of gyrase. The standard supercoiling reaction mixtures containing 100 fmol (as molecule) pBR322 form I′ DNA and the indicated amounts (as tetramer) of the wild-type gyrase were incubated in the absence (−) or presence (+) of 5.6 μg (3 nt per RecA monomer) of RecA and the DNA products were analyzed as described in Materials and Methods. Two independent experiments exhibited essentially identical results and representative results are shown.

    Journal: Nucleic Acids Research

    Article Title: RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli

    doi: 10.1093/nar/gkl981

    Figure Lengend Snippet: RecA has no effect on the supercoiling activity of gyrase. The standard supercoiling reaction mixtures containing 100 fmol (as molecule) pBR322 form I′ DNA and the indicated amounts (as tetramer) of the wild-type gyrase were incubated in the absence (−) or presence (+) of 5.6 μg (3 nt per RecA monomer) of RecA and the DNA products were analyzed as described in Materials and Methods. Two independent experiments exhibited essentially identical results and representative results are shown.

    Article Snippet: Supercoiling reaction Covalently closed, negatively supercoiled double-stranded circular (form I) DNA of plasmid pBR322 (4361 bp) was purchased from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay, Incubation

    The D82G mutation in the GyrA subunit reduces the supercoiling activity of DNA gyrase. ( A ) The standard supercoiling reaction mixtures containing 100 fmol (as molecule) pBR322 form I′ DNA and the indicated amounts (as tetramer) of either the wild-type or a mutant gyrase were incubated and the DNA products were analyzed as described in Materials and Methods. ( B ) The standard supercoiling reaction mixtures containing 100 fmol (as molecule) pBR322 form I′ DNA, 10 fmol (as tetramer) of either the wild-type or a mutant gyrase, and the indicated concentrations of norfloxacin (Norf) were incubated and the DNA products were analyzed as described in Materials and Methods. Both experiments were repeated three times and virtually identical results were obtained. Representative results are shown. Lane 1 contains the substrate alone. wt, the wild-type gyrase; D82G, GyrA D82G gyrase; S83W, GyrA S83W gyrase.

    Journal: Nucleic Acids Research

    Article Title: RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli

    doi: 10.1093/nar/gkl981

    Figure Lengend Snippet: The D82G mutation in the GyrA subunit reduces the supercoiling activity of DNA gyrase. ( A ) The standard supercoiling reaction mixtures containing 100 fmol (as molecule) pBR322 form I′ DNA and the indicated amounts (as tetramer) of either the wild-type or a mutant gyrase were incubated and the DNA products were analyzed as described in Materials and Methods. ( B ) The standard supercoiling reaction mixtures containing 100 fmol (as molecule) pBR322 form I′ DNA, 10 fmol (as tetramer) of either the wild-type or a mutant gyrase, and the indicated concentrations of norfloxacin (Norf) were incubated and the DNA products were analyzed as described in Materials and Methods. Both experiments were repeated three times and virtually identical results were obtained. Representative results are shown. Lane 1 contains the substrate alone. wt, the wild-type gyrase; D82G, GyrA D82G gyrase; S83W, GyrA S83W gyrase.

    Article Snippet: Supercoiling reaction Covalently closed, negatively supercoiled double-stranded circular (form I) DNA of plasmid pBR322 (4361 bp) was purchased from New England Biolabs (Beverly, MA).

    Techniques: Mutagenesis, Activity Assay, Incubation

    E.coli RecA fails to stimulate the relaxation activity of either E.coli Topo III or S.aureus Topo I. The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA, 5 mM ATP and the indicated amounts of E.coli Topo III ( A ), S.aureus (Sa) Topo I ( B ), or B.cereus (Bc) Topo I ( C ) were incubated in the absence (−) or presence (+) of 5.6 μg (3 nt per RecA monomer) of RecA and the DNA products were analyzed as described in Materials and Methods.

    Journal: Nucleic Acids Research

    Article Title: RecA can stimulate the relaxation activity of topoisomerase I: Molecular basis of topoisomerase-mediated genome-wide transcriptional responses in Escherichia coli

    doi: 10.1093/nar/gkl981

    Figure Lengend Snippet: E.coli RecA fails to stimulate the relaxation activity of either E.coli Topo III or S.aureus Topo I. The standard relaxation reaction mixtures containing 100 fmol (as molecule) pBR322 form I DNA, 5 mM ATP and the indicated amounts of E.coli Topo III ( A ), S.aureus (Sa) Topo I ( B ), or B.cereus (Bc) Topo I ( C ) were incubated in the absence (−) or presence (+) of 5.6 μg (3 nt per RecA monomer) of RecA and the DNA products were analyzed as described in Materials and Methods.

    Article Snippet: Supercoiling reaction Covalently closed, negatively supercoiled double-stranded circular (form I) DNA of plasmid pBR322 (4361 bp) was purchased from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay, Incubation

    Activity and expression of endonucleases in PTE cells. ( A ) In the total protein extracts isolated from DNase I WT mice, the strongest endonuclease activity could be obtained when Ca 2+ and Mg 2+ ions were added together, resulting in digested DNA. This is characteristic to DNase I, which therefore provides most of the endonuclease activity in the normal kidney. In the kidney tissue extracts obtained from DNase I KO mice, Mn-dependent endonuclease was the most prominent, suggesting that EndoG is the second major endonuclease in the absence of DNase I (Widlak et al ). Vertical row: O, open circular DNA; L, linear DNA; C, covalently closed circular DNA; D, digested DNA. Horizontal row: control nondigested pBR322 DNA; Ca 2+ , 2 mM CaCl 2 , pH 7.5; Mg 2+ , 2 mM MgCl 2 , pH 7.5; CM [Ca 2+ +Mg 2+ ], 2 mM CaCl 2 + 2 mM MgCl 2 ; Mn 2+ , 2 mM MnCl 2 , pH 7.5; E5, 2 mM EDTA, no cations, pH 5 (to measure DNase II activity). ( B ) Expression of endonucleases in WT, EndoG KO, and DNase I KO cells measured using real-time reverse transcriptase polymerase chain reaction. DNase I expression is wiped out almost completely, while EndoG KO is partially inhibited because these cells were isolated from heterozygous animals ( n = 4, * p

    Journal: DNA and Cell Biology

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases

    doi: 10.1089/dna.2008.0850

    Figure Lengend Snippet: Activity and expression of endonucleases in PTE cells. ( A ) In the total protein extracts isolated from DNase I WT mice, the strongest endonuclease activity could be obtained when Ca 2+ and Mg 2+ ions were added together, resulting in digested DNA. This is characteristic to DNase I, which therefore provides most of the endonuclease activity in the normal kidney. In the kidney tissue extracts obtained from DNase I KO mice, Mn-dependent endonuclease was the most prominent, suggesting that EndoG is the second major endonuclease in the absence of DNase I (Widlak et al ). Vertical row: O, open circular DNA; L, linear DNA; C, covalently closed circular DNA; D, digested DNA. Horizontal row: control nondigested pBR322 DNA; Ca 2+ , 2 mM CaCl 2 , pH 7.5; Mg 2+ , 2 mM MgCl 2 , pH 7.5; CM [Ca 2+ +Mg 2+ ], 2 mM CaCl 2 + 2 mM MgCl 2 ; Mn 2+ , 2 mM MnCl 2 , pH 7.5; E5, 2 mM EDTA, no cations, pH 5 (to measure DNase II activity). ( B ) Expression of endonucleases in WT, EndoG KO, and DNase I KO cells measured using real-time reverse transcriptase polymerase chain reaction. DNase I expression is wiped out almost completely, while EndoG KO is partially inhibited because these cells were isolated from heterozygous animals ( n = 4, * p

    Article Snippet: To determine whether Lipofectamine is protecting plasmid DNA from degradation by endonucleases, pBR322 plasmid was pretreated with Lipofectamine before it was exposed to the culture medium.

    Techniques: Activity Assay, Expressing, Isolation, Mouse Assay, Polymerase Chain Reaction

    Endonuclease activity in tubular epithelial cell extract and culture medium. The activity was measured using pBR322 PIA as described in the Materials and Methods section. ( A ) Endonuclease activity is present both in the cellular protein extracts and in the culture medium (left and middle panels). Pretreatment with Lipofectamine does not protect plasmid DNA against in vitro digestion by endonucleases (right panel). Dilutions (1–6) of cell extract or medium 1:1, 1:5, 1:25, 1:125, 1:625, and 1:3125, respectively. O, open circular DNA (with one or more single-strand DNA breaks but no double-strand breaks); L, linear DNA (with one double-strand DNA break); C, covalently closed circular DNA (without DNA breaks), which is the primary substrate for endonucleases. Endonuclease activity is seen only in the first two dilutions in cell extract, and in nondiluted culture medium. ( B ) Endonuclease activity in immortalized TKPTS cells and PTE cells. Primary cells have a higher total endonuclease activity (25 ± 2 units/μg protein in primary cells vs. 7 ± 3 units/μg protein in TKPTS cells, n = 3–6, p

    Journal: DNA and Cell Biology

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases

    doi: 10.1089/dna.2008.0850

    Figure Lengend Snippet: Endonuclease activity in tubular epithelial cell extract and culture medium. The activity was measured using pBR322 PIA as described in the Materials and Methods section. ( A ) Endonuclease activity is present both in the cellular protein extracts and in the culture medium (left and middle panels). Pretreatment with Lipofectamine does not protect plasmid DNA against in vitro digestion by endonucleases (right panel). Dilutions (1–6) of cell extract or medium 1:1, 1:5, 1:25, 1:125, 1:625, and 1:3125, respectively. O, open circular DNA (with one or more single-strand DNA breaks but no double-strand breaks); L, linear DNA (with one double-strand DNA break); C, covalently closed circular DNA (without DNA breaks), which is the primary substrate for endonucleases. Endonuclease activity is seen only in the first two dilutions in cell extract, and in nondiluted culture medium. ( B ) Endonuclease activity in immortalized TKPTS cells and PTE cells. Primary cells have a higher total endonuclease activity (25 ± 2 units/μg protein in primary cells vs. 7 ± 3 units/μg protein in TKPTS cells, n = 3–6, p

    Article Snippet: To determine whether Lipofectamine is protecting plasmid DNA from degradation by endonucleases, pBR322 plasmid was pretreated with Lipofectamine before it was exposed to the culture medium.

    Techniques: Activity Assay, Plasmid Preparation, In Vitro

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

    Journal: AMB Express

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

    doi: 10.1186/s13568-017-0409-y

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

    Article Snippet: DNA binding assay Gel-retardation experiments were performed using 5 µL of a 25 µg mL−1 pBR322 vector from E. coli (BioLabs, New England).

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

    Gel electrophoresis analysis for single- and double-stranded DNA breaks of plasmid DNA after exposure to M. albus volatile compounds. Plasmid pBR322 (lanes 1 and 6) was exposed to M. albus VOCs in Tris-EDTA, pH 8.0, for 24 h (lane 2) and 48 h (lane 3).

    Journal: Applied and Environmental Microbiology

    Article Title: Mycofumigation by the Volatile Organic Compound-Producing Fungus Muscodor albus Induces Bacterial Cell Death through DNA Damage

    doi: 10.1128/AEM.03294-14

    Figure Lengend Snippet: Gel electrophoresis analysis for single- and double-stranded DNA breaks of plasmid DNA after exposure to M. albus volatile compounds. Plasmid pBR322 (lanes 1 and 6) was exposed to M. albus VOCs in Tris-EDTA, pH 8.0, for 24 h (lane 2) and 48 h (lane 3).

    Article Snippet: Gel electrophoresis was performed on plasmid pBR322 (New England BioLabs Inc.) as previously described ( ).

    Techniques: Nucleic Acid Electrophoresis, Plasmid Preparation