calf thymus topoisomerase i  (Thermo Fisher)


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    Name:
    Topoisomerase I
    Description:
    Topoisomerase I DNA relaxing enzyme catalyzes the removal of superhelical turns from covalently closed DNA by a transient breakage and rejoining of phosphodiester bonds Topoisomerase I is active in the presence of EDTA Applications Relaxing positively and negatively supercoiled DNA 1 Producing DNA topoisomers 2 Source Purified from calf thymus Performance and Quality Testing Endodeoxyribonuclease 3 and 5 exodeoxyribonuclease and phosphatase assays conversion of super coiled DNA to relaxed DNA Unit Definition One unit catalyzes the conversion of 0 5 µg of superhelical Φ X174 RF DNA to a relaxed state in 30 min at 37°C Unit Reaction Conditions 50 mM Tris HCl pH 7 5 50 mM KCl 10 mM MgCl2 0 1 mM EDTA 0 5 mM DTT 30 µg ml BSA 0 5 µgΦ X174 RF DNA and enzyme in 50 µl for 30 min at 37°C
    Catalog Number:
    38042024
    Price:
    None
    Applications:
    Cloning|Restriction Enzyme Cloning
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher calf thymus topoisomerase i
    Topoisomerase I-linked assay. Reaction scheme for detecting Rad51 presynaptic filament disruption. “Hel.” stands for the helicase of interest. Topo I stands for calf-thymus topoisomerase I. PK stands for proteinase K.
    Topoisomerase I DNA relaxing enzyme catalyzes the removal of superhelical turns from covalently closed DNA by a transient breakage and rejoining of phosphodiester bonds Topoisomerase I is active in the presence of EDTA Applications Relaxing positively and negatively supercoiled DNA 1 Producing DNA topoisomers 2 Source Purified from calf thymus Performance and Quality Testing Endodeoxyribonuclease 3 and 5 exodeoxyribonuclease and phosphatase assays conversion of super coiled DNA to relaxed DNA Unit Definition One unit catalyzes the conversion of 0 5 µg of superhelical Φ X174 RF DNA to a relaxed state in 30 min at 37°C Unit Reaction Conditions 50 mM Tris HCl pH 7 5 50 mM KCl 10 mM MgCl2 0 1 mM EDTA 0 5 mM DTT 30 µg ml BSA 0 5 µgΦ X174 RF DNA and enzyme in 50 µl for 30 min at 37°C
    https://www.bioz.com/result/calf thymus topoisomerase i/product/Thermo Fisher
    Average 99 stars, based on 3 article reviews
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    calf thymus topoisomerase i - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Promotion and Regulation of Homologous Recombination by DNA Helicases"

    Article Title: Promotion and Regulation of Homologous Recombination by DNA Helicases

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2010.02.009

    Topoisomerase I-linked assay. Reaction scheme for detecting Rad51 presynaptic filament disruption. “Hel.” stands for the helicase of interest. Topo I stands for calf-thymus topoisomerase I. PK stands for proteinase K.
    Figure Legend Snippet: Topoisomerase I-linked assay. Reaction scheme for detecting Rad51 presynaptic filament disruption. “Hel.” stands for the helicase of interest. Topo I stands for calf-thymus topoisomerase I. PK stands for proteinase K.

    Techniques Used:

    2) Product Images from "Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *"

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.032953

    Resistance of the rad51 Y388H and rad51 G393D presynaptic filaments to Srs2. A , the schematic of the topoisomerase I-linked assay is shown in  panel I. Panel II  shows the results obtained when presynaptic filaments of Rad51, rad51 A320V, rad51 Y388H, or
    Figure Legend Snippet: Resistance of the rad51 Y388H and rad51 G393D presynaptic filaments to Srs2. A , the schematic of the topoisomerase I-linked assay is shown in panel I. Panel II shows the results obtained when presynaptic filaments of Rad51, rad51 A320V, rad51 Y388H, or

    Techniques Used:

    3) Product Images from "Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *"

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.032953

    Resistance of the rad51 Y388H and rad51 G393D presynaptic filaments to Srs2. A , the schematic of the topoisomerase I-linked assay is shown in  panel I. Panel II  shows the results obtained when presynaptic filaments of Rad51, rad51 A320V, rad51 Y388H, or
    Figure Legend Snippet: Resistance of the rad51 Y388H and rad51 G393D presynaptic filaments to Srs2. A , the schematic of the topoisomerase I-linked assay is shown in panel I. Panel II shows the results obtained when presynaptic filaments of Rad51, rad51 A320V, rad51 Y388H, or

    Techniques Used:

    4) Product Images from "Ciprofloxacin Dimers Target Gyrase in Streptococcus pneumoniae"

    Article Title: Ciprofloxacin Dimers Target Gyrase in Streptococcus pneumoniae

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.48.6.2108-2115.2004

    Ciprofloxacin and its symmetric dimer (dimer 2) induce unwinding of closed circular DNA. Relaxed plasmid pBR322 (0.8 μg) was incubated with calf thymus DNA topoisomerase I (12 U) in the presence of 10 mM MgCl 2 at room temperature for 10 min before addition of ciprofloxacin (CIP) and dimer 2 at the indicated concentrations. After incubation for 1 h at 37°C, the topoisomerase I was inactivated and the DNA was extracted with phenol to remove any bound ligand prior to agarose gel electrophoresis in Tris-phosphate-EDTA buffer. Half of each DNA sample was run in the absence of chloroquine (−CQ); the other half was electrophoresed in the presence of chloroquine (+CQ). Under the conditions of the gel without chloroquine, all the DNA samples are positively supercoiled and are made more positively coiled by the inclusion of chloroquine. Lanes a, pBR322 DNA (previously relaxed in the absence of Mg 2+ ions), used as the substrate.
    Figure Legend Snippet: Ciprofloxacin and its symmetric dimer (dimer 2) induce unwinding of closed circular DNA. Relaxed plasmid pBR322 (0.8 μg) was incubated with calf thymus DNA topoisomerase I (12 U) in the presence of 10 mM MgCl 2 at room temperature for 10 min before addition of ciprofloxacin (CIP) and dimer 2 at the indicated concentrations. After incubation for 1 h at 37°C, the topoisomerase I was inactivated and the DNA was extracted with phenol to remove any bound ligand prior to agarose gel electrophoresis in Tris-phosphate-EDTA buffer. Half of each DNA sample was run in the absence of chloroquine (−CQ); the other half was electrophoresed in the presence of chloroquine (+CQ). Under the conditions of the gel without chloroquine, all the DNA samples are positively supercoiled and are made more positively coiled by the inclusion of chloroquine. Lanes a, pBR322 DNA (previously relaxed in the absence of Mg 2+ ions), used as the substrate.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis

    5) Product Images from "Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption"

    Article Title: Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp748

    Rad51 presynaptic filament disruption as measured by a topoisomerase-linked assay. ( A ) Reaction scheme for detecting Rad51 presynaptic filament disruption. ( B ) Rad51 presynaptic filaments were incubated without or with Srs2, srs2 1–910, srs2 Δ875–902, srs2 1–875 or srs2 1–860 (60 and 80 nM) before the addition of relaxed DNA and topoisomerase I (lanes 3–13). Lane 2 contains Form U DNA made by incubating topologically relaxed DNA with Rad51 and topoisomerase I. Lane 1 contains topologically relaxed DNA.
    Figure Legend Snippet: Rad51 presynaptic filament disruption as measured by a topoisomerase-linked assay. ( A ) Reaction scheme for detecting Rad51 presynaptic filament disruption. ( B ) Rad51 presynaptic filaments were incubated without or with Srs2, srs2 1–910, srs2 Δ875–902, srs2 1–875 or srs2 1–860 (60 and 80 nM) before the addition of relaxed DNA and topoisomerase I (lanes 3–13). Lane 2 contains Form U DNA made by incubating topologically relaxed DNA with Rad51 and topoisomerase I. Lane 1 contains topologically relaxed DNA.

    Techniques Used: Incubation

    6) Product Images from "Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules"

    Article Title: Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119368

    Annealing of complementary single stranded circles (
    Figure Legend Snippet: Annealing of complementary single stranded circles ("Form V" DNA) by wheat germ topoisomerase I. The two complementary single stranded DNA circles prepared from minicircle HC03 were mixed and allowed to hybridize into Form V DNA, then annealed into a regular B-form covalently-closed DNA circle by incubation with various amounts of wheat germ topoisomerase I, either in the absence or in the presence of Mg ++ in the incubation buffer. Samples were then split in two parts and analyzed on a polyacrylamide gel, either native (left), or denatured by alkali (right). Note that a strong stimulation of annealing by Topoisomerase I is observed in the presence of magnesium (compare lanes 1–4 with lanes 6–9).

    Techniques Used: Incubation

    Assay of topoisomerases I with double strand hemicatenanes and single strand catenanes. Material from bands D and S ( Fig. 6C D ), expected to correspond respectively to hemicatenanes and single strand catenanes, were incubated with topoisomerase I from E. coli, wheat germ, and calf thymus. E. coli topoisomerase I, a type IA topoisomerase, is able to decatenate the single strand circles as expected, but has no effect on double strand hemicatenanes. The other two enzymes, type IB topoisomerases, show no activity on either substrate.
    Figure Legend Snippet: Assay of topoisomerases I with double strand hemicatenanes and single strand catenanes. Material from bands D and S ( Fig. 6C D ), expected to correspond respectively to hemicatenanes and single strand catenanes, were incubated with topoisomerase I from E. coli, wheat germ, and calf thymus. E. coli topoisomerase I, a type IA topoisomerase, is able to decatenate the single strand circles as expected, but has no effect on double strand hemicatenanes. The other two enzymes, type IB topoisomerases, show no activity on either substrate.

    Techniques Used: Incubation, IA, Activity Assay

    7) Product Images from "Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract"

    Article Title: Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh107

    Topological and enzymatic characterization of the minichromosomes. ( A ) Time course of chromatin assembly. Supercoiled pMMTV plasmid (1.5 μg) was assembled under standard conditions. At the indicated times, aliquots containing 100 ng of DNA were removed and processed. Samples were co-electrophoresed with topoisomerase-I-relaxed plasmid (lane 1) in a 1% agarose 1× TBE gel at 60 V for 14 h and then stained with ethidium bromide. ( B ) Two-dimensional electrophoresis. Assembled minichromosome (100 ng) was deproteinized and co-electrophoresed with the standard DNA topoisomer ladder (reference pattern) and the relaxed DNA. To obtain a good matching, the deproteinized minichromosome was first loaded, electrophoresed for 90 min and then, the standard ladder was loaded in the same well. The relaxed DNA was loaded at the same time as the standard but several wells to the right. The gel was electrophoresed and processed as described in Materials and methods. The linking-number change in the minichromosomes was determined by counting in the topoisomer ladder the number of spots from the relaxed DNA (0). N p , N m and N r are the nicked form of the reference pattern, minichromosomes and relaxed DNA, respectively. ( C ) Micrococcal nuclease digestion of assembled minichromosomes. Relaxed pUC18, 1.5 μg (as in lane 1), was incubated with yScS116 and added core histones under standard conditions. After 6 h, 75 ng was removed and deproteinized for topological analysis (lane 3). To the remaining mixture, CaCl 2 and MNase were added and incubation was continued at room temperature. At different times, one-third of the digestion mixture was removed and the digestion stopped as described. Lanes 2 and 7, 75 ng of supercoiled DNA and DNA molecular weight marker, respectively. I, II and III indicate the supercoiled, relaxed and linear form of pUC18, respectively. The positions of the mono-, di-, tri-, tetra- and pentanucleosome bands are indicated (arrows).
    Figure Legend Snippet: Topological and enzymatic characterization of the minichromosomes. ( A ) Time course of chromatin assembly. Supercoiled pMMTV plasmid (1.5 μg) was assembled under standard conditions. At the indicated times, aliquots containing 100 ng of DNA were removed and processed. Samples were co-electrophoresed with topoisomerase-I-relaxed plasmid (lane 1) in a 1% agarose 1× TBE gel at 60 V for 14 h and then stained with ethidium bromide. ( B ) Two-dimensional electrophoresis. Assembled minichromosome (100 ng) was deproteinized and co-electrophoresed with the standard DNA topoisomer ladder (reference pattern) and the relaxed DNA. To obtain a good matching, the deproteinized minichromosome was first loaded, electrophoresed for 90 min and then, the standard ladder was loaded in the same well. The relaxed DNA was loaded at the same time as the standard but several wells to the right. The gel was electrophoresed and processed as described in Materials and methods. The linking-number change in the minichromosomes was determined by counting in the topoisomer ladder the number of spots from the relaxed DNA (0). N p , N m and N r are the nicked form of the reference pattern, minichromosomes and relaxed DNA, respectively. ( C ) Micrococcal nuclease digestion of assembled minichromosomes. Relaxed pUC18, 1.5 μg (as in lane 1), was incubated with yScS116 and added core histones under standard conditions. After 6 h, 75 ng was removed and deproteinized for topological analysis (lane 3). To the remaining mixture, CaCl 2 and MNase were added and incubation was continued at room temperature. At different times, one-third of the digestion mixture was removed and the digestion stopped as described. Lanes 2 and 7, 75 ng of supercoiled DNA and DNA molecular weight marker, respectively. I, II and III indicate the supercoiled, relaxed and linear form of pUC18, respectively. The positions of the mono-, di-, tri-, tetra- and pentanucleosome bands are indicated (arrows).

    Techniques Used: Plasmid Preparation, Staining, Electrophoresis, Incubation, Molecular Weight, Marker

    8) Product Images from "Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *"

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.032953

    Resistance of the rad51 Y388H and rad51 G393D presynaptic filaments to Srs2. A , the schematic of the topoisomerase I-linked assay is shown in  panel I. Panel II  shows the results obtained when presynaptic filaments of Rad51, rad51 A320V, rad51 Y388H, or
    Figure Legend Snippet: Resistance of the rad51 Y388H and rad51 G393D presynaptic filaments to Srs2. A , the schematic of the topoisomerase I-linked assay is shown in panel I. Panel II shows the results obtained when presynaptic filaments of Rad51, rad51 A320V, rad51 Y388H, or

    Techniques Used:

    9) Product Images from "Promotion and Regulation of Homologous Recombination by DNA Helicases"

    Article Title: Promotion and Regulation of Homologous Recombination by DNA Helicases

    Journal: Methods (San Diego, Calif.)

    doi: 10.1016/j.ymeth.2010.02.009

    Topoisomerase I-linked assay. Reaction scheme for detecting Rad51 presynaptic filament disruption. “Hel.” stands for the helicase of interest. Topo I stands for calf-thymus topoisomerase I. PK stands for proteinase K.
    Figure Legend Snippet: Topoisomerase I-linked assay. Reaction scheme for detecting Rad51 presynaptic filament disruption. “Hel.” stands for the helicase of interest. Topo I stands for calf-thymus topoisomerase I. PK stands for proteinase K.

    Techniques Used:

    10) Product Images from "Inter-sigmulon communication through topological promoter coupling"

    Article Title: Inter-sigmulon communication through topological promoter coupling

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw639

    Topological promoter stimulation is reduced by enhanced binding of σ 70 -RNAP and accelerated open-complex kinetics. ( A ) Luciferase reporter gene assay of  P. putida  KT2440:: dmpR -Tel harbouring different Pr- luxAB  (−265 to +215) transcriptional reporter plasmids bearing the wild-type (black) or a mutated (grey, as under Figure   1B ) divergent Po promoter. Growth (circles) and luciferase activity profiles (squares) are of LB-cultured cells grown in the absence (open symbols) or presence (closed symbols) of 2 mM of 2-methylphenol. Values are the average of triplicate determinations ± SE. Where not discernible, SE are within the size of the symbol. ( B ) Single-round  in vitro  transcription assays with 10 nM supercoiled or topoisomerase I-treated plasmid template DNA carrying different (−265 to +8) Pr derivatives. Reactions contained 25 nM σ 70 -RNAP, 100 nM ΔA2-His-DmpR in the presence (+) or absence (−) of 25 nM σ 54 -RNAP. The autoradiographs show differential exposures of the transcripts from the three promoters to accommodate different promoter strengths and in each case are representative of three or more independent experiments.
    Figure Legend Snippet: Topological promoter stimulation is reduced by enhanced binding of σ 70 -RNAP and accelerated open-complex kinetics. ( A ) Luciferase reporter gene assay of P. putida KT2440:: dmpR -Tel harbouring different Pr- luxAB (−265 to +215) transcriptional reporter plasmids bearing the wild-type (black) or a mutated (grey, as under Figure 1B ) divergent Po promoter. Growth (circles) and luciferase activity profiles (squares) are of LB-cultured cells grown in the absence (open symbols) or presence (closed symbols) of 2 mM of 2-methylphenol. Values are the average of triplicate determinations ± SE. Where not discernible, SE are within the size of the symbol. ( B ) Single-round in vitro transcription assays with 10 nM supercoiled or topoisomerase I-treated plasmid template DNA carrying different (−265 to +8) Pr derivatives. Reactions contained 25 nM σ 70 -RNAP, 100 nM ΔA2-His-DmpR in the presence (+) or absence (−) of 25 nM σ 54 -RNAP. The autoradiographs show differential exposures of the transcripts from the three promoters to accommodate different promoter strengths and in each case are representative of three or more independent experiments.

    Techniques Used: Binding Assay, Luciferase, Reporter Gene Assay, Activity Assay, Cell Culture, In Vitro, Plasmid Preparation

    Related Articles

    Recombinant:

    Article Title: Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions *
    Article Snippet: Anti-FLAG antibody (M2, Sigma) was used for detecting recombinant FLAG-SpCdc18. .. The supercoiled and relaxed pARS1 plasmids used in Figs. and were generated by incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of 60 units of topoisomerase I (Invitrogen).

    Article Title: Ciprofloxacin Dimers Target Gyrase in Streptococcus pneumoniae
    Article Snippet: Recombinant S. pneumoniae GyrA, GyrB, ParC, ParE, S81F GyrA, and S79F ParC proteins were overexpressed and purified to > 95% homogeneity as described previously ( , ). .. Relaxed pBR322 DNA was prepared as described previously by incubating the plasmid with calf thymus topoisomerase I (Invitrogen, Paisley, Scotland) ( ).

    Agarose Gel Electrophoresis:

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *
    Article Snippet: Topologically relaxed ϕX174 dsDNA (15 μ m nucleotides) and 3 units of calf thymus topoisomerase I (Invitrogen) were then added in 0.7 and 0.4 μl, respectively, followed by an 8-min incubation. .. The reaction mixtures were deproteinized with SDS (0.5%) and proteinase K (0.5 mg/ml) for 5 min at 37 °C, before being resolved on 0.9% agarose gel run in TAE buffer (30 m m Tris acetate, pH 7.4, 0.5 m m EDTA).

    Article Title: Promotion and Regulation of Homologous Recombination by DNA Helicases
    Article Snippet: The product of this reaction, an underwound DNA species called form U, is resolved from other DNA species by agarose gel electrophoresis and then revealed by ethidium bromide staining. .. The reaction protocol is detailed below: For the preparation of topologically relaxed DNA, supercoiled ϕX174 DNA (New England Biolabs; 80 μg) is generated by its incubation with calf thymus topoisomerase I (Invitrogen; 20 U) for 90 min at 37°C in 500 μl of buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, and 1 mM DTT.

    Article Title: Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract
    Article Snippet: Supercoiled pUC18 (100 ng) was incubated with 0.1 U of calf thymus topoisomerase I (GIBCO) at 37°C for 30 min in a final volume of 10 μl. .. After pelleting and washing, the DNA was dissolved in a few microlitres of 20 μg/ml DNase-free RNaseA and loaded onto a 1% agarose gel in 1× TBE.

    In Vitro:

    Article Title: Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract
    Article Snippet: Paragraph title: In vitro chromatin assembly ... Supercoiled pUC18 (100 ng) was incubated with 0.1 U of calf thymus topoisomerase I (GIBCO) at 37°C for 30 min in a final volume of 10 μl.

    Ligation:

    Article Title: Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions *
    Article Snippet: Topoisomerase I Assay —The 3737-bp pARS1 plasmid containing the 1153-bp ars1 origin DNA sequence was generated by removing a 2967-bp PflFI-BamHI DNA fragment containing the LEU2 gene from pRC20 ( ) and ligation of the product. .. The supercoiled and relaxed pARS1 plasmids used in Figs. and were generated by incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of 60 units of topoisomerase I (Invitrogen).

    Mutagenesis:

    Article Title: Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption
    Article Snippet: Srs2 or mutant srs2 (60 or 80 nM, as indicated) and RPA (1 µM) were added, followed by a 4 min incubation. .. Topologically relaxed ϕX174 DNA (12.5 µM nucleotides) and 2.5 U calf thymus topoisomerase I (Invitrogen) were then incorporated to complete the reaction.

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *
    Article Snippet: To measure the disruption of the presynaptic filament by Srs2, Rad51 or rad51 mutant (2 μ m ) was incubated with ϕX174 ssDNA (8.5 μ m nucleotides) for 5 min with an ATP regenerating system (20 m m creatine phosphate and 20 μg/ml creatine kinase) in 8.1 μl of buffer, followed by the incorporation of RPA (1 μ m , added in 0.5 μl) and Srs2 (35 or 50 n m , added in 0.7 μl) and a 5-min incubation. .. Topologically relaxed ϕX174 dsDNA (15 μ m nucleotides) and 3 units of calf thymus topoisomerase I (Invitrogen) were then added in 0.7 and 0.4 μl, respectively, followed by an 8-min incubation.

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *
    Article Snippet: .. To measure DNA topology change induced by Rad51 or the rad51 mutants, the indicated amount of the wild type or mutant protein was incubated with topologically relaxed ϕX174 dsDNA (15 μ m nucleotides) for 5 min at 37 °C in 8.1 μl of buffer (35 m m Tris-HCl, pH 7.2, 100 m m KCl, 1 m m DTT, 2 m m ATP, 5 m m MgCl2 , 0.1 mg/ml bovine serum albumin), followed by the incorporation of 3 units of calf thymus topoisomerase I (Invitrogen; added in 0.4 μl) and a 20-min incubation. .. To measure the disruption of the presynaptic filament by Srs2, Rad51 or rad51 mutant (2 μ m ) was incubated with ϕX174 ssDNA (8.5 μ m nucleotides) for 5 min with an ATP regenerating system (20 m m creatine phosphate and 20 μg/ml creatine kinase) in 8.1 μl of buffer, followed by the incorporation of RPA (1 μ m , added in 0.5 μl) and Srs2 (35 or 50 n m , added in 0.7 μl) and a 5-min incubation.

    Protease Inhibitor:

    Article Title: Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions *
    Article Snippet: The binding assays were performed by incubating 50 fmol (2 n m ) of purified SpORC with 12.5 fmol (0.5 n m ) of radiolabeled ars1 DNA in 25 μl of B buffer with 0.1 mg/ml bovine serum albumin, 1 m m ATP, and 1 × protease inhibitor mixture for 30 min at 30 °C in the absence or presence of supercoiled and relaxed pARS1 plasmid competitor DNA. .. The supercoiled and relaxed pARS1 plasmids used in Figs. and were generated by incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of 60 units of topoisomerase I (Invitrogen).

    Synthesized:

    Article Title: Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions *
    Article Snippet: For competition nitrocellulose filter binding experiments, a 1153-bp DNA fragment containing the ars1 origin was synthesized and radiolabeled with [α-32 P]dATP by PCR. .. The supercoiled and relaxed pARS1 plasmids used in Figs. and were generated by incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of 60 units of topoisomerase I (Invitrogen).

    Sequencing:

    Article Title: DNA-dependent SUMO modification of PARP-1
    Article Snippet: Histone octamers were assembled on a 11 kbp long DNA molecule consisting of a tandemly repeated 187 bp long high-affinity histone octamer binding sequence . .. Relaxation of a supercoiled plasmid (5 μg) was brought about by 5 U of topoisomerase I (Invitrogen) for 3 h at 37 °C in 500 μL of 50 mM Tris–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2 , 0.1 mM EDTA, 30 mg ml−1 BSA and 0.5 mM DTT.

    Article Title: Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions *
    Article Snippet: Topoisomerase I Assay —The 3737-bp pARS1 plasmid containing the 1153-bp ars1 origin DNA sequence was generated by removing a 2967-bp PflFI-BamHI DNA fragment containing the LEU2 gene from pRC20 ( ) and ligation of the product. .. The supercoiled and relaxed pARS1 plasmids used in Figs. and were generated by incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of 60 units of topoisomerase I (Invitrogen).

    Incubation:

    Article Title: DNA-dependent SUMO modification of PARP-1
    Article Snippet: Digested and nicked plasmids (11 μg) were prepared by incubation with 110 U of the relevant enzyme (New England Biolabs) for 3 h at 37 °C (or 65 °C for nicking) in 120 μL of the appropriate buffer. .. Relaxation of a supercoiled plasmid (5 μg) was brought about by 5 U of topoisomerase I (Invitrogen) for 3 h at 37 °C in 500 μL of 50 mM Tris–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2 , 0.1 mM EDTA, 30 mg ml−1 BSA and 0.5 mM DTT.

    Article Title: Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption
    Article Snippet: Srs2 or mutant srs2 (60 or 80 nM, as indicated) and RPA (1 µM) were added, followed by a 4 min incubation. .. Topologically relaxed ϕX174 DNA (12.5 µM nucleotides) and 2.5 U calf thymus topoisomerase I (Invitrogen) were then incorporated to complete the reaction.

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *
    Article Snippet: .. Topologically relaxed ϕX174 dsDNA (15 μ m nucleotides) and 3 units of calf thymus topoisomerase I (Invitrogen) were then added in 0.7 and 0.4 μl, respectively, followed by an 8-min incubation. .. The reaction mixtures were deproteinized with SDS (0.5%) and proteinase K (0.5 mg/ml) for 5 min at 37 °C, before being resolved on 0.9% agarose gel run in TAE buffer (30 m m Tris acetate, pH 7.4, 0.5 m m EDTA).

    Article Title: Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions *
    Article Snippet: The supercoiled and relaxed pARS1 plasmids used in Figs. and were generated by incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of 60 units of topoisomerase I (Invitrogen). .. The reactions were stopped and deproteinized by addition of 5.5 μl of stop buffer (6% SDS, 30% glycerol, 10 m m EDTA, bromphenol blue, and xylene cyanol FF) and incubation at 42 °C for 10 min.

    Article Title: Promotion and Regulation of Homologous Recombination by DNA Helicases
    Article Snippet: .. The reaction protocol is detailed below: For the preparation of topologically relaxed DNA, supercoiled ϕX174 DNA (New England Biolabs; 80 μg) is generated by its incubation with calf thymus topoisomerase I (Invitrogen; 20 U) for 90 min at 37°C in 500 μl of buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, and 1 mM DTT. .. Reaction mixtures are checked for complete relaxation of the DNA by agarose gel electrophoresis and ethidium bromide staining.

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *
    Article Snippet: .. To measure DNA topology change induced by Rad51 or the rad51 mutants, the indicated amount of the wild type or mutant protein was incubated with topologically relaxed ϕX174 dsDNA (15 μ m nucleotides) for 5 min at 37 °C in 8.1 μl of buffer (35 m m Tris-HCl, pH 7.2, 100 m m KCl, 1 m m DTT, 2 m m ATP, 5 m m MgCl2 , 0.1 mg/ml bovine serum albumin), followed by the incorporation of 3 units of calf thymus topoisomerase I (Invitrogen; added in 0.4 μl) and a 20-min incubation. .. To measure the disruption of the presynaptic filament by Srs2, Rad51 or rad51 mutant (2 μ m ) was incubated with ϕX174 ssDNA (8.5 μ m nucleotides) for 5 min with an ATP regenerating system (20 m m creatine phosphate and 20 μg/ml creatine kinase) in 8.1 μl of buffer, followed by the incorporation of RPA (1 μ m , added in 0.5 μl) and Srs2 (35 or 50 n m , added in 0.7 μl) and a 5-min incubation.

    Article Title: Construction of a Z-DNA-specific restriction endonuclease
    Article Snippet: .. Twenty units of topoisomerase I (Life Technologies) was added to each reaction and incubated for 90 min at 37°C. ..

    Article Title: Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract
    Article Snippet: .. Supercoiled pUC18 (100 ng) was incubated with 0.1 U of calf thymus topoisomerase I (GIBCO) at 37°C for 30 min in a final volume of 10 μl. .. Meanwhile, the chromatin assembly reaction was made by mixing 20 μl of yeast extract with all the reagents in extraction buffer until the following final concentrations: 500–800 ng of purified core histones (histones/DNA mass ratio = 5 to 8), 20 mM HEPES–KOH, pH 7.5, 5 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol, 10 mM β-glycerophosphate; 10% glycerol, 40 mM disodium creatin phosphate, 3 mM ATP, 1 ng/μl creatin phosphokinase, 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin and 3 mM Mg2 Cl in a final volume of 34 μl.

    Purification:

    Article Title: DNA-dependent SUMO modification of PARP-1
    Article Snippet: Relaxation of a supercoiled plasmid (5 μg) was brought about by 5 U of topoisomerase I (Invitrogen) for 3 h at 37 °C in 500 μL of 50 mM Tris–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2 , 0.1 mM EDTA, 30 mg ml−1 BSA and 0.5 mM DTT. .. These DNA molecules were cleaned up with Qiagen's QIAquick PCR Purification Kit.

    Article Title: Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions *
    Article Snippet: The binding assays were performed by incubating 50 fmol (2 n m ) of purified SpORC with 12.5 fmol (0.5 n m ) of radiolabeled ars1 DNA in 25 μl of B buffer with 0.1 mg/ml bovine serum albumin, 1 m m ATP, and 1 × protease inhibitor mixture for 30 min at 30 °C in the absence or presence of supercoiled and relaxed pARS1 plasmid competitor DNA. .. The supercoiled and relaxed pARS1 plasmids used in Figs. and were generated by incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of 60 units of topoisomerase I (Invitrogen).

    Article Title: Promotion and Regulation of Homologous Recombination by DNA Helicases
    Article Snippet: The reaction protocol is detailed below: For the preparation of topologically relaxed DNA, supercoiled ϕX174 DNA (New England Biolabs; 80 μg) is generated by its incubation with calf thymus topoisomerase I (Invitrogen; 20 U) for 90 min at 37°C in 500 μl of buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, and 1 mM DTT. .. The relaxed DNA is purified by use of a PCR clean-up kit (Qiagen) and stored in TE buffer (20 mM Tris-HCl, pH 7.5, with 0.2 mM EDTA).

    Article Title: Ciprofloxacin Dimers Target Gyrase in Streptococcus pneumoniae
    Article Snippet: Recombinant S. pneumoniae GyrA, GyrB, ParC, ParE, S81F GyrA, and S79F ParC proteins were overexpressed and purified to > 95% homogeneity as described previously ( , ). .. Relaxed pBR322 DNA was prepared as described previously by incubating the plasmid with calf thymus topoisomerase I (Invitrogen, Paisley, Scotland) ( ).

    Article Title: Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract
    Article Snippet: Supercoiled pUC18 (100 ng) was incubated with 0.1 U of calf thymus topoisomerase I (GIBCO) at 37°C for 30 min in a final volume of 10 μl. .. Meanwhile, the chromatin assembly reaction was made by mixing 20 μl of yeast extract with all the reagents in extraction buffer until the following final concentrations: 500–800 ng of purified core histones (histones/DNA mass ratio = 5 to 8), 20 mM HEPES–KOH, pH 7.5, 5 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol, 10 mM β-glycerophosphate; 10% glycerol, 40 mM disodium creatin phosphate, 3 mM ATP, 1 ng/μl creatin phosphokinase, 10 μg/ml phenyl-methylsulphonyl fluoride, 2 μg/ml leupeptin, 2 μg/ml pepstatin and 3 mM Mg2 Cl in a final volume of 34 μl.

    Electrophoresis:

    Article Title: Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption
    Article Snippet: Topologically relaxed ϕX174 DNA (12.5 µM nucleotides) and 2.5 U calf thymus topoisomerase I (Invitrogen) were then incorporated to complete the reaction. .. After 8 min of incubation, the reaction mixtures were deproteinized and then subject to electrophoresis in 0.9% agarose gels.

    Concentration Assay:

    Article Title: Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules
    Article Snippet: Enzymes All enzymes were from New England Biolabs, with the exception of wheat germ topoisomerase I (Promega in early experiments, later Inspiralis), calf thymus topoisomerase I (Invitrogen), and nicking endonuclease Nb.Mva1269 I (Fermentas), which was preferred to its isoschizomer Nb.BsmI for its optimal digestion temperature of 37°C, as opposed to 65°C for Nb.BsmI. .. Note: care was taken not to overdigest with Nb.BtsI, as this nicking enzyme tended to show non-specific star activity when used at high concentration (data not shown).

    Generated:

    Article Title: Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions *
    Article Snippet: .. The supercoiled and relaxed pARS1 plasmids used in Figs. and were generated by incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of 60 units of topoisomerase I (Invitrogen). ..

    Article Title: Promotion and Regulation of Homologous Recombination by DNA Helicases
    Article Snippet: .. The reaction protocol is detailed below: For the preparation of topologically relaxed DNA, supercoiled ϕX174 DNA (New England Biolabs; 80 μg) is generated by its incubation with calf thymus topoisomerase I (Invitrogen; 20 U) for 90 min at 37°C in 500 μl of buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, and 1 mM DTT. .. Reaction mixtures are checked for complete relaxation of the DNA by agarose gel electrophoresis and ethidium bromide staining.

    other:

    Article Title: Inter-sigmulon communication through topological promoter coupling
    Article Snippet: Topoisomerase I-treatment of plasmids Ten microgram of supercoiled transcription templates were treated during 1 h at 37°C with 2 μl of calf thymus topoisomerase I (6 U/μl, Invitrogen) in buffer containing 50 mM Tris–HCl pH 7.5, 50 mM KCl, 10 mM MgCl2 , 0.5 mM DTT, 0.1 mM EDTA, 30 μg/ml bovine serun albumin (BSA).

    Article Title: Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome
    Article Snippet: DNA treatment For enzymatic treatment of DNA, samples were diluted into the appropriate buffer provided by each manufacturer and treated with the indicated units of enzyme at the following temperatures and times: topoisomerase I (Invitrogen; 20 U at 37°C for 1 h); topoisomerase II (USB Affymetrix; 40 U at 37°C for 1 h); topoisomerase IV (Inspiralis; 20 U at 37°C for 1 h); DNA gyrase (New England Biolabs; 10 U at 37°C for 1 h); BglII (Fermentas; 4 U at 37°C for 30 min); S1 nuclease (Promega; 0.9 U at 37°C for 30 min); E scherichia coli exonuclease I (New England Biolabs; 20 U at 37°C for 30 min); RNase A (Ambion; 2 µl at 24°C for 1 h, used to generate data in and Supplementary Figures S1 and S2 ); RNase H (Fermentas; 15 U at 37°C for 30 min).

    Activity Assay:

    Article Title: Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules
    Article Snippet: Enzymes All enzymes were from New England Biolabs, with the exception of wheat germ topoisomerase I (Promega in early experiments, later Inspiralis), calf thymus topoisomerase I (Invitrogen), and nicking endonuclease Nb.Mva1269 I (Fermentas), which was preferred to its isoschizomer Nb.BsmI for its optimal digestion temperature of 37°C, as opposed to 65°C for Nb.BsmI. .. Note: care was taken not to overdigest with Nb.BtsI, as this nicking enzyme tended to show non-specific star activity when used at high concentration (data not shown).

    Polymerase Chain Reaction:

    Article Title: DNA-dependent SUMO modification of PARP-1
    Article Snippet: Relaxation of a supercoiled plasmid (5 μg) was brought about by 5 U of topoisomerase I (Invitrogen) for 3 h at 37 °C in 500 μL of 50 mM Tris–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2 , 0.1 mM EDTA, 30 mg ml−1 BSA and 0.5 mM DTT. .. These DNA molecules were cleaned up with Qiagen's QIAquick PCR Purification Kit.

    Article Title: Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions *
    Article Snippet: For competition nitrocellulose filter binding experiments, a 1153-bp DNA fragment containing the ars1 origin was synthesized and radiolabeled with [α-32 P]dATP by PCR. .. The supercoiled and relaxed pARS1 plasmids used in Figs. and were generated by incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of 60 units of topoisomerase I (Invitrogen).

    Article Title: Promotion and Regulation of Homologous Recombination by DNA Helicases
    Article Snippet: The reaction protocol is detailed below: For the preparation of topologically relaxed DNA, supercoiled ϕX174 DNA (New England Biolabs; 80 μg) is generated by its incubation with calf thymus topoisomerase I (Invitrogen; 20 U) for 90 min at 37°C in 500 μl of buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, and 1 mM DTT. .. The relaxed DNA is purified by use of a PCR clean-up kit (Qiagen) and stored in TE buffer (20 mM Tris-HCl, pH 7.5, with 0.2 mM EDTA).

    Staining:

    Article Title: Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption
    Article Snippet: Topologically relaxed ϕX174 DNA (12.5 µM nucleotides) and 2.5 U calf thymus topoisomerase I (Invitrogen) were then incorporated to complete the reaction. .. The DNA species were stained with ethidium bromide.

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *
    Article Snippet: Topologically relaxed ϕX174 dsDNA (15 μ m nucleotides) and 3 units of calf thymus topoisomerase I (Invitrogen) were then added in 0.7 and 0.4 μl, respectively, followed by an 8-min incubation. .. DNA species were stained with ethidium bromide, and the results were recorded in a gel documentation station (Bio-Rad).

    Article Title: Promotion and Regulation of Homologous Recombination by DNA Helicases
    Article Snippet: The product of this reaction, an underwound DNA species called form U, is resolved from other DNA species by agarose gel electrophoresis and then revealed by ethidium bromide staining. .. The reaction protocol is detailed below: For the preparation of topologically relaxed DNA, supercoiled ϕX174 DNA (New England Biolabs; 80 μg) is generated by its incubation with calf thymus topoisomerase I (Invitrogen; 20 U) for 90 min at 37°C in 500 μl of buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, and 1 mM DTT.

    Binding Assay:

    Article Title: DNA-dependent SUMO modification of PARP-1
    Article Snippet: Histone octamers were assembled on a 11 kbp long DNA molecule consisting of a tandemly repeated 187 bp long high-affinity histone octamer binding sequence . .. Relaxation of a supercoiled plasmid (5 μg) was brought about by 5 U of topoisomerase I (Invitrogen) for 3 h at 37 °C in 500 μL of 50 mM Tris–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2 , 0.1 mM EDTA, 30 mg ml−1 BSA and 0.5 mM DTT.

    Article Title: Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions *
    Article Snippet: The binding assays were performed by incubating 50 fmol (2 n m ) of purified SpORC with 12.5 fmol (0.5 n m ) of radiolabeled ars1 DNA in 25 μl of B buffer with 0.1 mg/ml bovine serum albumin, 1 m m ATP, and 1 × protease inhibitor mixture for 30 min at 30 °C in the absence or presence of supercoiled and relaxed pARS1 plasmid competitor DNA. .. The supercoiled and relaxed pARS1 plasmids used in Figs. and were generated by incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of 60 units of topoisomerase I (Invitrogen).

    Article Title: Promotion and Regulation of Homologous Recombination by DNA Helicases
    Article Snippet: Since Rad51 binding induces lengthening of the DNA trap, the level of anti-recombination function can be conveniently monitored as a DNA linking number change upon treatment of the duplex with topoisomerase I ( ; [ ]). .. The reaction protocol is detailed below: For the preparation of topologically relaxed DNA, supercoiled ϕX174 DNA (New England Biolabs; 80 μg) is generated by its incubation with calf thymus topoisomerase I (Invitrogen; 20 U) for 90 min at 37°C in 500 μl of buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, and 1 mM DTT.

    Recombinase Polymerase Amplification:

    Article Title: Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption
    Article Snippet: Srs2 or mutant srs2 (60 or 80 nM, as indicated) and RPA (1 µM) were added, followed by a 4 min incubation. .. Topologically relaxed ϕX174 DNA (12.5 µM nucleotides) and 2.5 U calf thymus topoisomerase I (Invitrogen) were then incorporated to complete the reaction.

    Article Title: Regulation of Rad51 Recombinase Presynaptic Filament Assembly via Interactions with the Rad52 Mediator and the Srs2 Anti-recombinase *
    Article Snippet: To measure the disruption of the presynaptic filament by Srs2, Rad51 or rad51 mutant (2 μ m ) was incubated with ϕX174 ssDNA (8.5 μ m nucleotides) for 5 min with an ATP regenerating system (20 m m creatine phosphate and 20 μg/ml creatine kinase) in 8.1 μl of buffer, followed by the incorporation of RPA (1 μ m , added in 0.5 μl) and Srs2 (35 or 50 n m , added in 0.7 μl) and a 5-min incubation. .. Topologically relaxed ϕX174 dsDNA (15 μ m nucleotides) and 3 units of calf thymus topoisomerase I (Invitrogen) were then added in 0.7 and 0.4 μl, respectively, followed by an 8-min incubation.

    Article Title: Promotion and Regulation of Homologous Recombination by DNA Helicases
    Article Snippet: The presynaptic filament disruptive action of the helicase protein can be enhanced by RPA, which prevents the reformation of the presynaptic filament. .. The reaction protocol is detailed below: For the preparation of topologically relaxed DNA, supercoiled ϕX174 DNA (New England Biolabs; 80 μg) is generated by its incubation with calf thymus topoisomerase I (Invitrogen; 20 U) for 90 min at 37°C in 500 μl of buffer containing 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2 , 100 mM NaCl, and 1 mM DTT.

    Plasmid Preparation:

    Article Title: DNA-dependent SUMO modification of PARP-1
    Article Snippet: .. Relaxation of a supercoiled plasmid (5 μg) was brought about by 5 U of topoisomerase I (Invitrogen) for 3 h at 37 °C in 500 μL of 50 mM Tris–HCl, pH 7.5, 50 mM KCl, 10 mM MgCl2 , 0.1 mM EDTA, 30 mg ml−1 BSA and 0.5 mM DTT. .. These DNA molecules were cleaned up with Qiagen's QIAquick PCR Purification Kit.

    Article Title: Multiple Mechanisms Contribute to Schizosaccharomyces pombe Origin Recognition Complex-DNA Interactions *
    Article Snippet: Topoisomerase I Assay —The 3737-bp pARS1 plasmid containing the 1153-bp ars1 origin DNA sequence was generated by removing a 2967-bp PflFI-BamHI DNA fragment containing the LEU2 gene from pRC20 ( ) and ligation of the product. .. The supercoiled and relaxed pARS1 plasmids used in Figs. and were generated by incubating 5 μg of pARS1 for 2 h at 37 °C in the presence or absence of 60 units of topoisomerase I (Invitrogen).

    Article Title: Ciprofloxacin Dimers Target Gyrase in Streptococcus pneumoniae
    Article Snippet: .. Relaxed pBR322 DNA was prepared as described previously by incubating the plasmid with calf thymus topoisomerase I (Invitrogen, Paisley, Scotland) ( ). .. Kinetoplast DNA (kDNA) from Crithidia fasciculata was purchased from Topogen Inc., Columbus, Ohio.

    Article Title: Transcriptionally competent chromatin assembled with exogenous histones in a yeast whole cell extract
    Article Snippet: Chromatin assembly was monitored following the supercoiling of a topoisomerase-I-relaxed plasmid. .. Supercoiled pUC18 (100 ng) was incubated with 0.1 U of calf thymus topoisomerase I (GIBCO) at 37°C for 30 min in a final volume of 10 μl.

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  • 93
    Thermo Fisher gene exp top2a hs01032137 m1
    Sensitivity of neurofibroma and MPNST cell lines to doxorubucin. ( A ) An in vitro cytotoxicity assay (SRB assay) was used to determine IC 50 values (ng/ml) for doxorubucin of neurofibroma and MPNST cell lines after a 48h exposure to the drug. Graphs show cell viability as a function of doxorubucin concentration. Depicted is the average viability (n = 4) of a representative experiment. ( B ) Listing of calculated IC 50 values and correlation plot, with <t>TOP2A</t> protein expression levels on the Y-axis and IC 50 values for doxorubicin on the X-axis. Pearson correlation coefficient is depicted in the graph.
    Gene Exp Top2a Hs01032137 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp top2a hs01032137 m1/product/Thermo Fisher
    Average 93 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    gene exp top2a hs01032137 m1 - by Bioz Stars, 2020-04
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    85
    Thermo Fisher gene exp topors mm00506480 m1
    Deletion analysis of the S1CD-interacting domain of <t>Topors.</t> ( A ) Model of murine Topors protein domains, with the sequence corresponding to the S1CD-interacting prey 36 sequence identified by a black box. ( B ) Deletional analysis of the S1CD-binding sequence of Topors. Yeast with either LexA-S1CD or LexA-Lamin bait plasmid were transformed with either prey plasmid for the expression of complete S1CD binding sequence (Topors 398–516), or with prey plasmid expressing deletion mutants of Topors 398–516. After selection of co-transformants, interacting bait-prey pairs were identified by assay of β-galactosidase activity. Yeast two-hybrid analysis of Topors deletion mutants identifies residues 493–510 as critical for the interaction.
    Gene Exp Topors Mm00506480 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp topors mm00506480 m1/product/Thermo Fisher
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    85
    Thermo Fisher gene exp top2a hs03063307 m1
    Effects of <t>TOP2A,</t> PTK7, and CHEK2 knockdown in ASCs and liposarcoma cell lines. A, Cell proliferation following shRNA knockdown relative to SCR at day 6 after infection. B, Apoptosis at day 6 after infection, assessed by the percentage of cells staining
    Gene Exp Top2a Hs03063307 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sensitivity of neurofibroma and MPNST cell lines to doxorubucin. ( A ) An in vitro cytotoxicity assay (SRB assay) was used to determine IC 50 values (ng/ml) for doxorubucin of neurofibroma and MPNST cell lines after a 48h exposure to the drug. Graphs show cell viability as a function of doxorubucin concentration. Depicted is the average viability (n = 4) of a representative experiment. ( B ) Listing of calculated IC 50 values and correlation plot, with TOP2A protein expression levels on the Y-axis and IC 50 values for doxorubicin on the X-axis. Pearson correlation coefficient is depicted in the graph.

    Journal: PLoS ONE

    Article Title: Expression and inhibition of BRD4, EZH2 and TOP2A in neurofibromas and malignant peripheral nerve sheath tumors

    doi: 10.1371/journal.pone.0183155

    Figure Lengend Snippet: Sensitivity of neurofibroma and MPNST cell lines to doxorubucin. ( A ) An in vitro cytotoxicity assay (SRB assay) was used to determine IC 50 values (ng/ml) for doxorubucin of neurofibroma and MPNST cell lines after a 48h exposure to the drug. Graphs show cell viability as a function of doxorubucin concentration. Depicted is the average viability (n = 4) of a representative experiment. ( B ) Listing of calculated IC 50 values and correlation plot, with TOP2A protein expression levels on the Y-axis and IC 50 values for doxorubicin on the X-axis. Pearson correlation coefficient is depicted in the graph.

    Article Snippet: The following assays were used EZH2 (Hs01016789_m1), TOP2A (Hs01032137_m1), BRD4 (Hs04188087_m1).

    Techniques: In Vitro, Cytotoxicity Assay, Sulforhodamine B Assay, Concentration Assay, Expressing

    Expression level of TOP2A in human neurofibroma and MPNST samples and cell lines. ( A ) qRT-PCR was used to determine mRNA levels of TOP2A in paired plexiform neurofibroma (NF, blue, n = 9) and MPNST (red, n = 9) formalin-fixed paraffin-embedded tumor samples, each pair being derived from the same NF1 patient. Asterisk indicates P

    Journal: PLoS ONE

    Article Title: Expression and inhibition of BRD4, EZH2 and TOP2A in neurofibromas and malignant peripheral nerve sheath tumors

    doi: 10.1371/journal.pone.0183155

    Figure Lengend Snippet: Expression level of TOP2A in human neurofibroma and MPNST samples and cell lines. ( A ) qRT-PCR was used to determine mRNA levels of TOP2A in paired plexiform neurofibroma (NF, blue, n = 9) and MPNST (red, n = 9) formalin-fixed paraffin-embedded tumor samples, each pair being derived from the same NF1 patient. Asterisk indicates P

    Article Snippet: The following assays were used EZH2 (Hs01016789_m1), TOP2A (Hs01032137_m1), BRD4 (Hs04188087_m1).

    Techniques: Expressing, Quantitative RT-PCR, Formalin-fixed Paraffin-Embedded, Derivative Assay

    Deletion analysis of the S1CD-interacting domain of Topors. ( A ) Model of murine Topors protein domains, with the sequence corresponding to the S1CD-interacting prey 36 sequence identified by a black box. ( B ) Deletional analysis of the S1CD-binding sequence of Topors. Yeast with either LexA-S1CD or LexA-Lamin bait plasmid were transformed with either prey plasmid for the expression of complete S1CD binding sequence (Topors 398–516), or with prey plasmid expressing deletion mutants of Topors 398–516. After selection of co-transformants, interacting bait-prey pairs were identified by assay of β-galactosidase activity. Yeast two-hybrid analysis of Topors deletion mutants identifies residues 493–510 as critical for the interaction.

    Journal: PLoS ONE

    Article Title: Inhibition of PDGF-B Induction and Cell Growth by Syndecan-1 Involves the Ubiquitin and SUMO-1 Ligase, Topors

    doi: 10.1371/journal.pone.0043701

    Figure Lengend Snippet: Deletion analysis of the S1CD-interacting domain of Topors. ( A ) Model of murine Topors protein domains, with the sequence corresponding to the S1CD-interacting prey 36 sequence identified by a black box. ( B ) Deletional analysis of the S1CD-binding sequence of Topors. Yeast with either LexA-S1CD or LexA-Lamin bait plasmid were transformed with either prey plasmid for the expression of complete S1CD binding sequence (Topors 398–516), or with prey plasmid expressing deletion mutants of Topors 398–516. After selection of co-transformants, interacting bait-prey pairs were identified by assay of β-galactosidase activity. Yeast two-hybrid analysis of Topors deletion mutants identifies residues 493–510 as critical for the interaction.

    Article Snippet: Separate samples were used to extract RNA for qPCR for Topors that was done using the Applied Biosystems Taqman probe for Topors (Assay# Mm00506480_m1).

    Techniques: Sequencing, Binding Assay, Plasmid Preparation, Transformation Assay, Expressing, Selection, Activity Assay

    Co-immune precipitation of Sdc-1 and Topors from lysates of NMuMG cells. ( A ) Total cell lysates prepared from NMuMG cells were immune precipitated (IP) with non-immune rat IgG (lane 1) or rat anti-Sdc-1 antibody (lane 2 and 3) and then western blotted for Sdc-1 (lanes 1–2), and after stripping, re-probed with anti-Topors (lanes 3). Total cell lysate (lane 4) was included as a control for anti-Topors immunoreactivity. Arrows mark major co-precipitated Topors bands (110 and 175 kDa). ( B ) Non-immune chicken IgY-preadsorbed total cell lysates of NMuMG cells were immunoprecipitated with chicken anti-Topors, and immune precipitates were heparinase- and chondroitin ABC lyase-digested prior to SDS-PAGE and western blotting. Lysate (lane 1) and Topors immunoprecipitates (lane 2 and 3) that were probed on western blots for Topors (lanes 1 and 2) had Topors bands (110 and 175 kDa bands, double arrow), or for Sdc-1 (lane 3), which gave a band of ∼85 kDa (large arrow, lane 3 and 4), similar in size to a band present in a Sdc-1-positive control (lane 4), prepared by ion-exchange chromatography from the medium of NMuMG cultures. Lower MW heavy bands represent chicken IgY (NChIgY) from the immune precipitate (arrow). Lanes 1–3 are from the same blot with lane 3 representing a stripped and re-probed lane of the Topors immune precipitate run in parallel to that shown in lane 2. Lane 4 represents an adjacent lane from the same blot, at a longer exposure time.

    Journal: PLoS ONE

    Article Title: Inhibition of PDGF-B Induction and Cell Growth by Syndecan-1 Involves the Ubiquitin and SUMO-1 Ligase, Topors

    doi: 10.1371/journal.pone.0043701

    Figure Lengend Snippet: Co-immune precipitation of Sdc-1 and Topors from lysates of NMuMG cells. ( A ) Total cell lysates prepared from NMuMG cells were immune precipitated (IP) with non-immune rat IgG (lane 1) or rat anti-Sdc-1 antibody (lane 2 and 3) and then western blotted for Sdc-1 (lanes 1–2), and after stripping, re-probed with anti-Topors (lanes 3). Total cell lysate (lane 4) was included as a control for anti-Topors immunoreactivity. Arrows mark major co-precipitated Topors bands (110 and 175 kDa). ( B ) Non-immune chicken IgY-preadsorbed total cell lysates of NMuMG cells were immunoprecipitated with chicken anti-Topors, and immune precipitates were heparinase- and chondroitin ABC lyase-digested prior to SDS-PAGE and western blotting. Lysate (lane 1) and Topors immunoprecipitates (lane 2 and 3) that were probed on western blots for Topors (lanes 1 and 2) had Topors bands (110 and 175 kDa bands, double arrow), or for Sdc-1 (lane 3), which gave a band of ∼85 kDa (large arrow, lane 3 and 4), similar in size to a band present in a Sdc-1-positive control (lane 4), prepared by ion-exchange chromatography from the medium of NMuMG cultures. Lower MW heavy bands represent chicken IgY (NChIgY) from the immune precipitate (arrow). Lanes 1–3 are from the same blot with lane 3 representing a stripped and re-probed lane of the Topors immune precipitate run in parallel to that shown in lane 2. Lane 4 represents an adjacent lane from the same blot, at a longer exposure time.

    Article Snippet: Separate samples were used to extract RNA for qPCR for Topors that was done using the Applied Biosystems Taqman probe for Topors (Assay# Mm00506480_m1).

    Techniques: Western Blot, Stripping Membranes, Immunoprecipitation, SDS Page, Positive Control, Ion Exchange Chromatography

    Affinity capture and western blotting of Topors. ( A ) Western blot of NMuMG cell detergent lysate (supernate) and detergent-insoluble material (pellet) probed with chicken anti-Topors antibody. Two major bands (∼110 and ∼175 kDa (arrowheads)) are apparent. The larger band is prevalent in detergent-insoluble fractions, which contain relatively more histone proteins (lower blot), consistent with enhanced presence of nuclear proteins. ( B ) Capture of endogenous Topors from cell lysates by GST-S1CD. GST-S1CD or GST alone were adsorbed to glutathione-agarose beads and incubated with detergent lysates of 3T3 cells. Anti-Topors-reactive bands captured by GST-S1CD were detected in lysates of 3T3 cells that overexpress Topors (first lane, arrows), and were similar in size to the major bands seen in western blots of whole cell lysates. Similar bands were not captured by GST alone (lane 2), nor are they present in the GST-S1CD reagent (lane 3). Non-specific bands are seen at ∼55 kDa. ( C ) Co-precipitation of Topors with polyhistidine-tagged Sdc-1 isolated by metal-ion affinity chromatography. Metal ion-affinity gel bound material was isolated from detergent extracts of cells that express polyhistidine-tagged Sdc-1. Parallel samples were run on SDS-PAGE, either directly or after heparinase and chondroitinase digestion and transferred to nitrocellulose. Parallel blots were probed with chicken anti-Topors or, for digested samples, with anti Sdc-1.

    Journal: PLoS ONE

    Article Title: Inhibition of PDGF-B Induction and Cell Growth by Syndecan-1 Involves the Ubiquitin and SUMO-1 Ligase, Topors

    doi: 10.1371/journal.pone.0043701

    Figure Lengend Snippet: Affinity capture and western blotting of Topors. ( A ) Western blot of NMuMG cell detergent lysate (supernate) and detergent-insoluble material (pellet) probed with chicken anti-Topors antibody. Two major bands (∼110 and ∼175 kDa (arrowheads)) are apparent. The larger band is prevalent in detergent-insoluble fractions, which contain relatively more histone proteins (lower blot), consistent with enhanced presence of nuclear proteins. ( B ) Capture of endogenous Topors from cell lysates by GST-S1CD. GST-S1CD or GST alone were adsorbed to glutathione-agarose beads and incubated with detergent lysates of 3T3 cells. Anti-Topors-reactive bands captured by GST-S1CD were detected in lysates of 3T3 cells that overexpress Topors (first lane, arrows), and were similar in size to the major bands seen in western blots of whole cell lysates. Similar bands were not captured by GST alone (lane 2), nor are they present in the GST-S1CD reagent (lane 3). Non-specific bands are seen at ∼55 kDa. ( C ) Co-precipitation of Topors with polyhistidine-tagged Sdc-1 isolated by metal-ion affinity chromatography. Metal ion-affinity gel bound material was isolated from detergent extracts of cells that express polyhistidine-tagged Sdc-1. Parallel samples were run on SDS-PAGE, either directly or after heparinase and chondroitinase digestion and transferred to nitrocellulose. Parallel blots were probed with chicken anti-Topors or, for digested samples, with anti Sdc-1.

    Article Snippet: Separate samples were used to extract RNA for qPCR for Topors that was done using the Applied Biosystems Taqman probe for Topors (Assay# Mm00506480_m1).

    Techniques: Western Blot, Incubation, Isolation, Affinity Chromatography, SDS Page

    The effect of siRNA-mediated Topors knockdown on cell proliferation and PDGF-B chain expression by wild-type and Sdc-1 null SMCs. ( A ) The effect of siRNA to Topors (closed bars) or a scrambled control (open bars) on [ 3 H]-thymidine incorporation mediated by thrombin in wild-type and Sdc-1 null SMCs. Values are the mean ± SEM of four experiments expressed as the fold of wild-type scrambled siRNA control. ( B ) The same data as in section (A) except that data is expressed as the fold of its appropriate control (either the scrambled control or the siRNA to Topors of either wild-type or Sdc-1 null). ( C ) The effect of siRNA to Topors (closed bars) or a scrambled control (open bars) on PDGF-B chain mRNA mediated by thrombin in wild-type and Sdc-1 null SMCs. Values are the mean ± SEM of four experiments for thrombin expressed as the fold of wild-type scrambled siRNA control. * P

    Journal: PLoS ONE

    Article Title: Inhibition of PDGF-B Induction and Cell Growth by Syndecan-1 Involves the Ubiquitin and SUMO-1 Ligase, Topors

    doi: 10.1371/journal.pone.0043701

    Figure Lengend Snippet: The effect of siRNA-mediated Topors knockdown on cell proliferation and PDGF-B chain expression by wild-type and Sdc-1 null SMCs. ( A ) The effect of siRNA to Topors (closed bars) or a scrambled control (open bars) on [ 3 H]-thymidine incorporation mediated by thrombin in wild-type and Sdc-1 null SMCs. Values are the mean ± SEM of four experiments expressed as the fold of wild-type scrambled siRNA control. ( B ) The same data as in section (A) except that data is expressed as the fold of its appropriate control (either the scrambled control or the siRNA to Topors of either wild-type or Sdc-1 null). ( C ) The effect of siRNA to Topors (closed bars) or a scrambled control (open bars) on PDGF-B chain mRNA mediated by thrombin in wild-type and Sdc-1 null SMCs. Values are the mean ± SEM of four experiments for thrombin expressed as the fold of wild-type scrambled siRNA control. * P

    Article Snippet: Separate samples were used to extract RNA for qPCR for Topors that was done using the Applied Biosystems Taqman probe for Topors (Assay# Mm00506480_m1).

    Techniques: Expressing

    Immunolocalization of Topors and Sdc-1. ( A ) Immunofluorescent staining of confluent NMuMG cells demonstrates that Topors is present in a punctate pattern in nuclei (e.g., arrowhead), consistent with previous reports of nuclear localization. In addition, Topors immunoreactivity is present at cell boundaries (arrows) as well as heavily present in the cytoplasm of cells undergoing mitosis (open arrow). Bar = 20 μm. ( B ) Immunofluorescent images of a single field of confluent NMuMG cells immunostained for mouse Sdc-1 (green, left) and Topors (red, right), with a merged image presented in the middle panel. Bar = 20 μm. ( C ) Immunofluorescent staining of dorsal epidermis of an e13.5 mouse embryo for Sdc-1 (red, left) and Topors (green, right). The merged imaged (center) includes a nuclear stain (DAPI, blue). Bar = 50 μm.

    Journal: PLoS ONE

    Article Title: Inhibition of PDGF-B Induction and Cell Growth by Syndecan-1 Involves the Ubiquitin and SUMO-1 Ligase, Topors

    doi: 10.1371/journal.pone.0043701

    Figure Lengend Snippet: Immunolocalization of Topors and Sdc-1. ( A ) Immunofluorescent staining of confluent NMuMG cells demonstrates that Topors is present in a punctate pattern in nuclei (e.g., arrowhead), consistent with previous reports of nuclear localization. In addition, Topors immunoreactivity is present at cell boundaries (arrows) as well as heavily present in the cytoplasm of cells undergoing mitosis (open arrow). Bar = 20 μm. ( B ) Immunofluorescent images of a single field of confluent NMuMG cells immunostained for mouse Sdc-1 (green, left) and Topors (red, right), with a merged image presented in the middle panel. Bar = 20 μm. ( C ) Immunofluorescent staining of dorsal epidermis of an e13.5 mouse embryo for Sdc-1 (red, left) and Topors (green, right). The merged imaged (center) includes a nuclear stain (DAPI, blue). Bar = 50 μm.

    Article Snippet: Separate samples were used to extract RNA for qPCR for Topors that was done using the Applied Biosystems Taqman probe for Topors (Assay# Mm00506480_m1).

    Techniques: Staining

    Effects of TOP2A, PTK7, and CHEK2 knockdown in ASCs and liposarcoma cell lines. A, Cell proliferation following shRNA knockdown relative to SCR at day 6 after infection. B, Apoptosis at day 6 after infection, assessed by the percentage of cells staining

    Journal: Cancer research

    Article Title: Expression Profiling of Liposarcoma Yields a Multigene Predictor of Patient Outcome and Identifies Genes that Contribute to Liposarcomagenesis

    doi: 10.1158/0008-5472.CAN-10-3588

    Figure Lengend Snippet: Effects of TOP2A, PTK7, and CHEK2 knockdown in ASCs and liposarcoma cell lines. A, Cell proliferation following shRNA knockdown relative to SCR at day 6 after infection. B, Apoptosis at day 6 after infection, assessed by the percentage of cells staining

    Article Snippet: TaqMan Gene Expression Assays were used according to the manufacturer’s protocol to detect TOP2A (Hs03063307_m1), PTK7 (Hs00177173_m1), CHEK1 (Hs00176236_m1), and 18s rRNA (Hs99999901_s1).

    Techniques: shRNA, Infection, Staining

    Kaplan-Meir analysis of distant-recurrence–free survival in the training set according to gene expression. A, TOP2A expression; B, PTK7 expression; C, CHEK1 expression.

    Journal: Cancer research

    Article Title: Expression Profiling of Liposarcoma Yields a Multigene Predictor of Patient Outcome and Identifies Genes that Contribute to Liposarcomagenesis

    doi: 10.1158/0008-5472.CAN-10-3588

    Figure Lengend Snippet: Kaplan-Meir analysis of distant-recurrence–free survival in the training set according to gene expression. A, TOP2A expression; B, PTK7 expression; C, CHEK1 expression.

    Article Snippet: TaqMan Gene Expression Assays were used according to the manufacturer’s protocol to detect TOP2A (Hs03063307_m1), PTK7 (Hs00177173_m1), CHEK1 (Hs00176236_m1), and 18s rRNA (Hs99999901_s1).

    Techniques: Expressing