genejet plasmid miniprep kit  (Thermo Fisher)


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    Name:
    GeneJET Plasmid Miniprep Kit
    Description:
    Thermo Scientific GeneJET Plasmid Miniprep Kit utilizes an exclusive silica based membrane technology in the form of a convenient spin column The kit recovers up to 20 µg of high copy plasmid DNA per isolation procedure Highlights• Efficient high yields of up to 20 µg of high quality plasmid DNA• Fast procedure takes less than 14 minutes• Convenient no phenol chloroform extraction or alcohol precipitation required• Pure purified DNA is immediately ready to useApplications• Fast isolation of high purity plasmid DNA suitable for all conventional molecular biology procedures including • FastDigest or conventional restriction digestion• Automated fluorescent and radioactive sequencing• PCR• In vitro transcription• TransformationPrincipleA bacterial culture is harvested and lysed The lysate is then cleared by centrifugation and applied on the silica column to selectively bind DNA molecules at a high salt concentration The adsorbed DNA is washed to remove contaminants and the pure plasmid DNA is eluted in a small volume of elution buffer or water The purified DNA is ready for immediate use in all molecular biology procedures such as fast and conventional digestion with restriction enzymes PCR in vitro transcription transformation and automated sequencing Components Resuspension Solution Lysis Solution Neutralization Solution Wash Solution concentrated RNase A Solution Elution Buffer Spin Columns Collection Tubes Detailed ProtocolRelated ProductsGeneJET Plasmid Miniprep KitRNase AElution Buffer for GeneJET Plasmid Miniprep KitLysis Solution for GeneJET Plasmid Miniprep KitResuspension Solution for GeneJET Plasmid Miniprep KitNeutralization Solution for GeneJET Plasmid Miniprep KitWash Solution for GeneJET Plasmid Miniprep KitBinding Buffer for GeneJET Plasmid Miniprep Kit
    Catalog Number:
    k0502
    Price:
    None
    Applications:
    DNA & RNA Purification & Analysis|DNA Extraction|Miniprep|Plasmid DNA Purification
    Category:
    Kits and Assays
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    Structured Review

    Thermo Fisher genejet plasmid miniprep kit
    Thermo Scientific GeneJET Plasmid Miniprep Kit utilizes an exclusive silica based membrane technology in the form of a convenient spin column The kit recovers up to 20 µg of high copy plasmid DNA per isolation procedure Highlights• Efficient high yields of up to 20 µg of high quality plasmid DNA• Fast procedure takes less than 14 minutes• Convenient no phenol chloroform extraction or alcohol precipitation required• Pure purified DNA is immediately ready to useApplications• Fast isolation of high purity plasmid DNA suitable for all conventional molecular biology procedures including • FastDigest or conventional restriction digestion• Automated fluorescent and radioactive sequencing• PCR• In vitro transcription• TransformationPrincipleA bacterial culture is harvested and lysed The lysate is then cleared by centrifugation and applied on the silica column to selectively bind DNA molecules at a high salt concentration The adsorbed DNA is washed to remove contaminants and the pure plasmid DNA is eluted in a small volume of elution buffer or water The purified DNA is ready for immediate use in all molecular biology procedures such as fast and conventional digestion with restriction enzymes PCR in vitro transcription transformation and automated sequencing Components Resuspension Solution Lysis Solution Neutralization Solution Wash Solution concentrated RNase A Solution Elution Buffer Spin Columns Collection Tubes Detailed ProtocolRelated ProductsGeneJET Plasmid Miniprep KitRNase AElution Buffer for GeneJET Plasmid Miniprep KitLysis Solution for GeneJET Plasmid Miniprep KitResuspension Solution for GeneJET Plasmid Miniprep KitNeutralization Solution for GeneJET Plasmid Miniprep KitWash Solution for GeneJET Plasmid Miniprep KitBinding Buffer for GeneJET Plasmid Miniprep Kit
    https://www.bioz.com/result/genejet plasmid miniprep kit/product/Thermo Fisher
    Average 99 stars, based on 871 article reviews
    Price from $9.99 to $1999.99
    genejet plasmid miniprep kit - by Bioz Stars, 2020-08
    99/100 stars

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    Related Articles

    DNA Extraction:

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity
    Article Snippet: .. DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania). ..

    Clone Assay:

    Article Title: The characterization of the circadian clock in the olive fly Bactrocera oleae (Diptera: Tephritidae) reveals a Drosophila-like organization
    Article Snippet: .. Positive clones were identified by colony PCR screening and purified plasmids (GeneJET Plasmid Miniprep Kit, Thermo Fisher Scientific) were sent to sequencing (GATC Biotech). ..

    Article Title: Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development
    Article Snippet: .. Positive clones were selected on agar plates with tetracycline (5 μg ml−1 ), and plasmids were isolated with the GeneJet Plamid Miniprep Kit (Thermo Scientific). .. Transformation of Agrobacterium tumefaciens (strain C58C1-RS with the helper plasmid pCH32) was done with the heat-thaw method as described [ ].

    Isolation:

    Article Title: Overexpression of PaNAC03, a stress induced NAC gene family transcription factor in Norway spruce leads to reduced flavonol biosynthesis and aberrant embryo development
    Article Snippet: .. Positive clones were selected on agar plates with tetracycline (5 μg ml−1 ), and plasmids were isolated with the GeneJet Plamid Miniprep Kit (Thermo Scientific). .. Transformation of Agrobacterium tumefaciens (strain C58C1-RS with the helper plasmid pCH32) was done with the heat-thaw method as described [ ].

    Purification:

    Article Title: Developmental progression of equine immunoglobulin heavy chain variable region diversity
    Article Snippet: .. Individual colonies were expanded in LB broth with ampicillin, and plasmid DNA was purified with the GeneJET plasmid miniprep kit (Thermo Scientific). .. For reverse transcription, cDNA synthesis reactions contained 5.5 mM MgCl2 , 0.5 mM dNTPs, 2.5 µM oligo d(T) (Applied Biosystems, Foster City, CA), 0.4 U RNasin Ribonuclease Inhibitor (Promega, Madison, WI), and 1 U Moloney Murine Leukemia Virus Reverse Transcriptase (MuLV RT), and 1X M-MuLV RT buffer (Applied Biosystems).

    Article Title: The characterization of the circadian clock in the olive fly Bactrocera oleae (Diptera: Tephritidae) reveals a Drosophila-like organization
    Article Snippet: .. Positive clones were identified by colony PCR screening and purified plasmids (GeneJET Plasmid Miniprep Kit, Thermo Fisher Scientific) were sent to sequencing (GATC Biotech). ..

    Polymerase Chain Reaction:

    Article Title: The characterization of the circadian clock in the olive fly Bactrocera oleae (Diptera: Tephritidae) reveals a Drosophila-like organization
    Article Snippet: .. Positive clones were identified by colony PCR screening and purified plasmids (GeneJET Plasmid Miniprep Kit, Thermo Fisher Scientific) were sent to sequencing (GATC Biotech). ..

    Sequencing:

    Article Title: The characterization of the circadian clock in the olive fly Bactrocera oleae (Diptera: Tephritidae) reveals a Drosophila-like organization
    Article Snippet: .. Positive clones were identified by colony PCR screening and purified plasmids (GeneJET Plasmid Miniprep Kit, Thermo Fisher Scientific) were sent to sequencing (GATC Biotech). ..

    Plasmid Preparation:

    Article Title: Anti-FGFR1 aptamer-tagged superparamagnetic conjugates for anticancer hyperthermia therapy
    Article Snippet: .. Plate-picked single colonies were used to isolate single aptamer clone-containing plasmids (prepared with GeneJET Plasmid Miniprep Kit; Thermo Fisher Scientific), which were subsequently sequenced (LGC Genomics GmbH, Berlin, Germany). .. Sequences were analyzed using Finch TV software and aligned with the MUSCLE alignment algorithm at Analysis Tool Web Services from European Molecular Biology Laboratory–European Bioinformatics Institute.,

    Article Title: Developmental progression of equine immunoglobulin heavy chain variable region diversity
    Article Snippet: .. Individual colonies were expanded in LB broth with ampicillin, and plasmid DNA was purified with the GeneJET plasmid miniprep kit (Thermo Scientific). .. For reverse transcription, cDNA synthesis reactions contained 5.5 mM MgCl2 , 0.5 mM dNTPs, 2.5 µM oligo d(T) (Applied Biosystems, Foster City, CA), 0.4 U RNasin Ribonuclease Inhibitor (Promega, Madison, WI), and 1 U Moloney Murine Leukemia Virus Reverse Transcriptase (MuLV RT), and 1X M-MuLV RT buffer (Applied Biosystems).

    Article Title: The characterization of the circadian clock in the olive fly Bactrocera oleae (Diptera: Tephritidae) reveals a Drosophila-like organization
    Article Snippet: .. Positive clones were identified by colony PCR screening and purified plasmids (GeneJET Plasmid Miniprep Kit, Thermo Fisher Scientific) were sent to sequencing (GATC Biotech). ..

    Article Title: Antibiotic Inducibility of the mexXY Multidrug Efflux Operon of Pseudomonas aeruginosa: Involvement of the MexZ Anti-Repressor ArmZ
    Article Snippet: .. Plasmid DNA was extracted from E. coli using the Fermentas GeneJET Plasmid Miniprep Kit or the Qiagen Plasmid Midi Kit according to protocols provided by the manufacturers. .. Chromosomal DNA was extracted from P. aeruginosa using the Qiagen DNeasy Blood & Tissue Kit according to a protocol provided by the manufacturer.

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity
    Article Snippet: .. DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania). ..

    Article Title: Detection of Cryptosporidium parvum Oocysts on Fresh Produce Using DNA Aptamers
    Article Snippet: .. Plasmids were extracted using a GeneJET plasmid miniprep kit (Cat. # K0502, Thermo Scientific / Fisher Scientific, Canada), according to the supplied protocol. .. Plasmids with the insert were amplified using an M13 forward primer (5′-GTA AAA CGA CGG CCA GT-3′) and M13 reverse primer (5′-AGC GGA TAA CAA TTT CAC ACA GG-3′), according to the following protocol.

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    Thermo Fisher single aptamer clone containing plasmids
    <t>Aptamer-targeted</t> hyperthermia compared to crude unwashed nanoparticles. Notes: Control experiments for targeted hyperthermia study on USO2-R1 cells. Nontreated cells (washed only; first column) were used as 100% viability control. Samples: columns 2–4 represent different negative controls, while column 5 (+ nanoparticles, + magnetic field) represents a positive control, where Dspio nanoparticles were added to the cells and were not washed before magnetic field application – it shows the maximum killing effect in this setup. The last column shows aptamer-targeted nanoparticles-induced hyperthermia effect. Statistics: ** and *** denote statistically significant difference between the sample and nontreated control (first column) at multiplicity adjusted P -values
    Single Aptamer Clone Containing Plasmids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/single aptamer clone containing plasmids/product/Thermo Fisher
    Average 99 stars, based on 81 article reviews
    Price from $9.99 to $1999.99
    single aptamer clone containing plasmids - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    96
    Thermo Fisher dna cleanup micro kit
    <t>DNA</t> gyrase behavior depends on a DNA substrate gyrase binding score. ( A ) EMSA analysis of DNA gyrase binding with 133 bp fragments; fragments scores are showed above the pictures; molar gyrase/DNA ratio is indicated under the lanes. SC—scrambled consensus, C—consensus. ( B ) Time-course of gyrase supercoiling of <t>pUC19</t> plasmids harboring indicated 133 bp fragments; RE—relaxed plasmid, NSC—negatively supercoiled plasmid. 1—scrambled consensus, 2—Mu SGS, 3—consensus. ( C ) Time-course of ATP-independent relaxation of pUC19 plasmids harbouring indicated 133 bp fragments. ( D ) Time-course of ATP-dependent relaxation of the same plasmids by truncated GyrA59 2 GyrB 2 gyrase lacking CTD.
    Dna Cleanup Micro Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna cleanup micro kit/product/Thermo Fisher
    Average 96 stars, based on 24 article reviews
    Price from $9.99 to $1999.99
    dna cleanup micro kit - by Bioz Stars, 2020-08
    96/100 stars
      Buy from Supplier

    Image Search Results


    Aptamer-targeted hyperthermia compared to crude unwashed nanoparticles. Notes: Control experiments for targeted hyperthermia study on USO2-R1 cells. Nontreated cells (washed only; first column) were used as 100% viability control. Samples: columns 2–4 represent different negative controls, while column 5 (+ nanoparticles, + magnetic field) represents a positive control, where Dspio nanoparticles were added to the cells and were not washed before magnetic field application – it shows the maximum killing effect in this setup. The last column shows aptamer-targeted nanoparticles-induced hyperthermia effect. Statistics: ** and *** denote statistically significant difference between the sample and nontreated control (first column) at multiplicity adjusted P -values

    Journal: International Journal of Nanomedicine

    Article Title: Anti-FGFR1 aptamer-tagged superparamagnetic conjugates for anticancer hyperthermia therapy

    doi: 10.2147/IJN.S125231

    Figure Lengend Snippet: Aptamer-targeted hyperthermia compared to crude unwashed nanoparticles. Notes: Control experiments for targeted hyperthermia study on USO2-R1 cells. Nontreated cells (washed only; first column) were used as 100% viability control. Samples: columns 2–4 represent different negative controls, while column 5 (+ nanoparticles, + magnetic field) represents a positive control, where Dspio nanoparticles were added to the cells and were not washed before magnetic field application – it shows the maximum killing effect in this setup. The last column shows aptamer-targeted nanoparticles-induced hyperthermia effect. Statistics: ** and *** denote statistically significant difference between the sample and nontreated control (first column) at multiplicity adjusted P -values

    Article Snippet: Plate-picked single colonies were used to isolate single aptamer clone-containing plasmids (prepared with GeneJET Plasmid Miniprep Kit; Thermo Fisher Scientific), which were subsequently sequenced (LGC Genomics GmbH, Berlin, Germany).

    Techniques: Positive Control

    Aptamer A11 predicted structure. Note: The sequence of A11 aptamer and its two-dimensional structure prediction by NUPACK software, showing distinct stem-loop structure involving both the random region and the primer regions.

    Journal: International Journal of Nanomedicine

    Article Title: Anti-FGFR1 aptamer-tagged superparamagnetic conjugates for anticancer hyperthermia therapy

    doi: 10.2147/IJN.S125231

    Figure Lengend Snippet: Aptamer A11 predicted structure. Note: The sequence of A11 aptamer and its two-dimensional structure prediction by NUPACK software, showing distinct stem-loop structure involving both the random region and the primer regions.

    Article Snippet: Plate-picked single colonies were used to isolate single aptamer clone-containing plasmids (prepared with GeneJET Plasmid Miniprep Kit; Thermo Fisher Scientific), which were subsequently sequenced (LGC Genomics GmbH, Berlin, Germany).

    Techniques: Sequencing, Software

    Schematic model of targeted hyperthermia. Note: The figure shows schematically aptamer-tagged dextran-coated superparamagnetic conjugates (called for brevity aptamer superparamagnetic conjugates or ASCs) specifically binding target-receptors on cell surface and exhibiting hyperthermia upon alternating-current magnetic field (ACMF) induction. Abbreviation: FGFR1, fibroblast growth factor receptor type-1.

    Journal: International Journal of Nanomedicine

    Article Title: Anti-FGFR1 aptamer-tagged superparamagnetic conjugates for anticancer hyperthermia therapy

    doi: 10.2147/IJN.S125231

    Figure Lengend Snippet: Schematic model of targeted hyperthermia. Note: The figure shows schematically aptamer-tagged dextran-coated superparamagnetic conjugates (called for brevity aptamer superparamagnetic conjugates or ASCs) specifically binding target-receptors on cell surface and exhibiting hyperthermia upon alternating-current magnetic field (ACMF) induction. Abbreviation: FGFR1, fibroblast growth factor receptor type-1.

    Article Snippet: Plate-picked single colonies were used to isolate single aptamer clone-containing plasmids (prepared with GeneJET Plasmid Miniprep Kit; Thermo Fisher Scientific), which were subsequently sequenced (LGC Genomics GmbH, Berlin, Germany).

    Techniques: Binding Assay

    Fluorescence microscopy images of aptamer A11-G18T binding to FGFR1-expressing cells. Notes: U2OS (upper row), U2OS-R1 (middle row) and NIH-3T3 (lower row) cells were incubated with fluorescein-labeled A11-G18T anti-FGFR1 aptamer, as examined by fluorescence microscopy. Columns from the left: blue DAPI staining of nuclei, red phalloidin staining of actin, green fluorescein signal (staining of surface FGFR1) and a merged picture. Scale bars correspond to 50 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; Phalloidin, fluorescent-labeled phalloidin; AF, FITC-labeled A11-G18T anti-FGFR1 aptamer; FGFR1, fibroblast growth factor receptor type-1; FITC, fluorescein isothiocyanate.

    Journal: International Journal of Nanomedicine

    Article Title: Anti-FGFR1 aptamer-tagged superparamagnetic conjugates for anticancer hyperthermia therapy

    doi: 10.2147/IJN.S125231

    Figure Lengend Snippet: Fluorescence microscopy images of aptamer A11-G18T binding to FGFR1-expressing cells. Notes: U2OS (upper row), U2OS-R1 (middle row) and NIH-3T3 (lower row) cells were incubated with fluorescein-labeled A11-G18T anti-FGFR1 aptamer, as examined by fluorescence microscopy. Columns from the left: blue DAPI staining of nuclei, red phalloidin staining of actin, green fluorescein signal (staining of surface FGFR1) and a merged picture. Scale bars correspond to 50 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; Phalloidin, fluorescent-labeled phalloidin; AF, FITC-labeled A11-G18T anti-FGFR1 aptamer; FGFR1, fibroblast growth factor receptor type-1; FITC, fluorescein isothiocyanate.

    Article Snippet: Plate-picked single colonies were used to isolate single aptamer clone-containing plasmids (prepared with GeneJET Plasmid Miniprep Kit; Thermo Fisher Scientific), which were subsequently sequenced (LGC Genomics GmbH, Berlin, Germany).

    Techniques: Fluorescence, Microscopy, Binding Assay, Expressing, Incubation, Labeling, Staining

    Aptamer-targeted hyperthermia compared to unselected oligonucleotide library. Notes: Aptamer-targeted hyperthermia study on U2OS-R1 cells. All samples were washed prior to the magnetic field treatment as described in the Methods section. Nontreated cells (first column) were used as 100% viability control. Samples: columns 2–4 represent different negative controls: column 3 (- nanoparticles, + A11-G18T, + magnetic field) validates the effect of free A11-G18T aptamer without the nanoparticles, while column 4 (+ nanoparticles, + Lib, + magnetic field) employs conjugates prepared with unselected randomized oligonucleotide library (Lib) instead of the selected aptamer. The last column shows aptamer-targeted nanoparticles-induced hyperthermia effect. Statistics: **statistically significant difference between the sample and the nontreated control (first column) at multiplicity adjusted P -value

    Journal: International Journal of Nanomedicine

    Article Title: Anti-FGFR1 aptamer-tagged superparamagnetic conjugates for anticancer hyperthermia therapy

    doi: 10.2147/IJN.S125231

    Figure Lengend Snippet: Aptamer-targeted hyperthermia compared to unselected oligonucleotide library. Notes: Aptamer-targeted hyperthermia study on U2OS-R1 cells. All samples were washed prior to the magnetic field treatment as described in the Methods section. Nontreated cells (first column) were used as 100% viability control. Samples: columns 2–4 represent different negative controls: column 3 (- nanoparticles, + A11-G18T, + magnetic field) validates the effect of free A11-G18T aptamer without the nanoparticles, while column 4 (+ nanoparticles, + Lib, + magnetic field) employs conjugates prepared with unselected randomized oligonucleotide library (Lib) instead of the selected aptamer. The last column shows aptamer-targeted nanoparticles-induced hyperthermia effect. Statistics: **statistically significant difference between the sample and the nontreated control (first column) at multiplicity adjusted P -value

    Article Snippet: Plate-picked single colonies were used to isolate single aptamer clone-containing plasmids (prepared with GeneJET Plasmid Miniprep Kit; Thermo Fisher Scientific), which were subsequently sequenced (LGC Genomics GmbH, Berlin, Germany).

    Techniques:

    ) was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM of the selected effector and 25% (v/v) DMSO at 65 °C. The pictures on the right side of the figure’s panels show the theoretical arrangement of the DNA bands in an agarose gel after digesting the given substrate DNA with TthHB27I under standard conditions. a Cleavage pattern of non-methylated 1789 bp PCR fragment DNA. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder, lane K, untreated DNA; lane 1, cleavage reaction in the absence of effector and DMSO; lane 2, in the presence of DMSO only; lane 3, in the presence of SAM only; lane 4, in the presence of SAM and DMSO; lane 5, in the presence of SIN only; lane 6, in the presence of SIN and DMSO. b The same as ( a ), but with previously methylated 1789 bp PCR fragment. c The same as ( a ), but with 1850 bp PCR fragment without TthHB27I cognate recognition sequence

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: ) was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM of the selected effector and 25% (v/v) DMSO at 65 °C. The pictures on the right side of the figure’s panels show the theoretical arrangement of the DNA bands in an agarose gel after digesting the given substrate DNA with TthHB27I under standard conditions. a Cleavage pattern of non-methylated 1789 bp PCR fragment DNA. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder, lane K, untreated DNA; lane 1, cleavage reaction in the absence of effector and DMSO; lane 2, in the presence of DMSO only; lane 3, in the presence of SAM only; lane 4, in the presence of SAM and DMSO; lane 5, in the presence of SIN only; lane 6, in the presence of SIN and DMSO. b The same as ( a ), but with previously methylated 1789 bp PCR fragment. c The same as ( a ), but with 1850 bp PCR fragment without TthHB27I cognate recognition sequence

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Agarose Gel Electrophoresis, Methylation, Polymerase Chain Reaction, Sequencing

    Cleavage of λ DNA under TthHB27I specificity relaxation conditions. a Cleavage pattern of λ DNA digested with TthHB27I under various conditions. 0.5 μg of DNA substrate was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM SAM and/or 25% (v/v) DMSO for 4 h at 65 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, digestion in the presence of SAM; lane 2, digestion in the presence of DMSO; lane 3, digestion in the presence of both SAM and DMSO. b The same as ( a ), but SIN was used instead of SAM. c Distribution of insert lengths in 200 clones randomly selected from TthHB27I/DMSO/SAM-generated λ DNA library. d The same as ( c ), but for TthHB27I/DMSO/SIN-generated λ DNA library

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: Cleavage of λ DNA under TthHB27I specificity relaxation conditions. a Cleavage pattern of λ DNA digested with TthHB27I under various conditions. 0.5 μg of DNA substrate was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM SAM and/or 25% (v/v) DMSO for 4 h at 65 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, digestion in the presence of SAM; lane 2, digestion in the presence of DMSO; lane 3, digestion in the presence of both SAM and DMSO. b The same as ( a ), but SIN was used instead of SAM. c Distribution of insert lengths in 200 clones randomly selected from TthHB27I/DMSO/SAM-generated λ DNA library. d The same as ( c ), but for TthHB27I/DMSO/SIN-generated λ DNA library

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Clone Assay, Generated

    ) was incubated with 2 U of TthHB27I for 1 h at 65 °C in REase buffer with increasing amount of DMSO. Reaction mixtures were precipitated and electrophoresed in 1.3% agarose/TBE gels. The pictures on the right side of the figure show the theoretical arrangement of the DNA bands in an agarose gel after digesting the substrate DNA with TthHB27I under standard conditions. a Digestions performed only with the addition of DMSO, without any cofactor. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested PCR fragment; lane 1, 0% ( v /v) DMSO present in the reaction mixture; lane 2, with 5% (v/v) DMSO; lane 3, with 10% (v/v) DMSO; lane 4, with 15% (v/v) DMSO; lane 5, with 20% (v/v) DMSO; lane 6, with 25% (v/v) DMSO; lane 7, with 30% (v/v) DMSO. As in ( a ), but in the presence of 100 μM SAM ( b ); 100 μM SIN ( c ); 100 μM SAC ( d ); 100 μM SAH ( e ); 100 μM ATP ( f )

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: ) was incubated with 2 U of TthHB27I for 1 h at 65 °C in REase buffer with increasing amount of DMSO. Reaction mixtures were precipitated and electrophoresed in 1.3% agarose/TBE gels. The pictures on the right side of the figure show the theoretical arrangement of the DNA bands in an agarose gel after digesting the substrate DNA with TthHB27I under standard conditions. a Digestions performed only with the addition of DMSO, without any cofactor. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested PCR fragment; lane 1, 0% ( v /v) DMSO present in the reaction mixture; lane 2, with 5% (v/v) DMSO; lane 3, with 10% (v/v) DMSO; lane 4, with 15% (v/v) DMSO; lane 5, with 20% (v/v) DMSO; lane 6, with 25% (v/v) DMSO; lane 7, with 30% (v/v) DMSO. As in ( a ), but in the presence of 100 μM SAM ( b ); 100 μM SIN ( c ); 100 μM SAC ( d ); 100 μM SAH ( e ); 100 μM ATP ( f )

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Incubation, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Controlling of partial cleavage of genomic DNAs by TthHB27I under cofactor/analogue-induced ‘star’ activity conditions. E. coli and λ genomic DNAs were digested with TthHB27I under cofactor/analogue-induced ‘star’ conditions, while varying: temperature, incubation time and TthHB27I amount. 0.5 μg of DNAs were digested with TthHB27I in REase buffer supplemented with 100 μM SIN and 25% (v/v) DMSO. a Cleavage control by reaction temperature. Reactions conducted with E. coli DNA digested with 2 U of TthHB27I for 1 h at temperatures from 30 °C to 80 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction at 30 °C; lane 2, 35 °C; lane 3, 40 °C; lane 4, 45 °C; lane 5, 50 °C; lane 6, 55 °C; lane 7, 60 °C; lane 8, 65 °C; lane 9, 70 °C; lane 10, 75 °C; lane 11, 80 °C. b The same as ( a ), except that λ DNA was used. c Cleavage control by reaction time. E. coli DNA was digested at 65 °C at times ranging from 7.5 min to 16 h. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction conducted for 7.5 min; lane 2, 15 min; lane 3, 30 min; lane 4, 1 h; lane 5, 2 h; lane 6, 4 h; lane 7, 8 h; lane 8, 16 h. d The same as ( c ), except that λ DNA was used. e Cleavage control by the enzyme amount. E. coli DNA was digested at 65 °C with TthHB27I. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, DNA digested with 8 U TthHB27I; lane 2, 4 U; lane 3, 2 U; lane 4, 1 U; lane 5, 0.5 U; lane 6, 0.25 U; lane 7, 0.12 U; lane 8, 0.06 U. f The same as ( e ), except that λ DNA was used

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: Controlling of partial cleavage of genomic DNAs by TthHB27I under cofactor/analogue-induced ‘star’ activity conditions. E. coli and λ genomic DNAs were digested with TthHB27I under cofactor/analogue-induced ‘star’ conditions, while varying: temperature, incubation time and TthHB27I amount. 0.5 μg of DNAs were digested with TthHB27I in REase buffer supplemented with 100 μM SIN and 25% (v/v) DMSO. a Cleavage control by reaction temperature. Reactions conducted with E. coli DNA digested with 2 U of TthHB27I for 1 h at temperatures from 30 °C to 80 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction at 30 °C; lane 2, 35 °C; lane 3, 40 °C; lane 4, 45 °C; lane 5, 50 °C; lane 6, 55 °C; lane 7, 60 °C; lane 8, 65 °C; lane 9, 70 °C; lane 10, 75 °C; lane 11, 80 °C. b The same as ( a ), except that λ DNA was used. c Cleavage control by reaction time. E. coli DNA was digested at 65 °C at times ranging from 7.5 min to 16 h. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction conducted for 7.5 min; lane 2, 15 min; lane 3, 30 min; lane 4, 1 h; lane 5, 2 h; lane 6, 4 h; lane 7, 8 h; lane 8, 16 h. d The same as ( c ), except that λ DNA was used. e Cleavage control by the enzyme amount. E. coli DNA was digested at 65 °C with TthHB27I. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, DNA digested with 8 U TthHB27I; lane 2, 4 U; lane 3, 2 U; lane 4, 1 U; lane 5, 0.5 U; lane 6, 0.25 U; lane 7, 0.12 U; lane 8, 0.06 U. f The same as ( e ), except that λ DNA was used

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Activity Assay, Incubation

    TthHB27I cofactor/analogue-induced ‘star’ activity towards methylated or non-methylated substrate DNA. 0.5 μg of methylated or non-methylated PCR DNA fragment (see Additional file 1 ) was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM of the selected effector and 25% (v/v) DMSO at 65 °C. The pictures on the right side of the figure’s panels show the theoretical arrangement of the DNA bands in an agarose gel after digesting the given substrate DNA with TthHB27I under standard conditions. a Cleavage pattern of non-methylated 1789 bp PCR fragment DNA. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder, lane K, untreated DNA; lane 1, cleavage reaction in the absence of effector and DMSO; lane 2, in the presence of DMSO only; lane 3, in the presence of SAM only; lane 4, in the presence of SAM and DMSO; lane 5, in the presence of SIN only; lane 6, in the presence of SIN and DMSO. b The same as ( a ), but with previously methylated 1789 bp PCR fragment. c The same as ( a ), but with 1850 bp PCR fragment without TthHB27I cognate recognition sequence

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: TthHB27I cofactor/analogue-induced ‘star’ activity towards methylated or non-methylated substrate DNA. 0.5 μg of methylated or non-methylated PCR DNA fragment (see Additional file 1 ) was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM of the selected effector and 25% (v/v) DMSO at 65 °C. The pictures on the right side of the figure’s panels show the theoretical arrangement of the DNA bands in an agarose gel after digesting the given substrate DNA with TthHB27I under standard conditions. a Cleavage pattern of non-methylated 1789 bp PCR fragment DNA. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder, lane K, untreated DNA; lane 1, cleavage reaction in the absence of effector and DMSO; lane 2, in the presence of DMSO only; lane 3, in the presence of SAM only; lane 4, in the presence of SAM and DMSO; lane 5, in the presence of SIN only; lane 6, in the presence of SIN and DMSO. b The same as ( a ), but with previously methylated 1789 bp PCR fragment. c The same as ( a ), but with 1850 bp PCR fragment without TthHB27I cognate recognition sequence

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Activity Assay, Methylation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sequencing

    Cleavage of λ DNA under TthHB27I specificity relaxation conditions. a Cleavage pattern of λ DNA digested with TthHB27I under various conditions. 0.5 μg of DNA substrate was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM SAM and/or 25% (v/v) DMSO for 4 h at 65 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, digestion in the presence of SAM; lane 2, digestion in the presence of DMSO; lane 3, digestion in the presence of both SAM and DMSO. b The same as ( a ), but SIN was used instead of SAM. c Distribution of insert lengths in 200 clones randomly selected from TthHB27I/DMSO/SAM-generated λ DNA library. d The same as ( c ), but for TthHB27I/DMSO/SIN-generated λ DNA library

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: Cleavage of λ DNA under TthHB27I specificity relaxation conditions. a Cleavage pattern of λ DNA digested with TthHB27I under various conditions. 0.5 μg of DNA substrate was digested with 2 U of TthHB27I in REase buffer supplemented with 100 μM SAM and/or 25% (v/v) DMSO for 4 h at 65 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, digestion in the presence of SAM; lane 2, digestion in the presence of DMSO; lane 3, digestion in the presence of both SAM and DMSO. b The same as ( a ), but SIN was used instead of SAM. c Distribution of insert lengths in 200 clones randomly selected from TthHB27I/DMSO/SAM-generated λ DNA library. d The same as ( c ), but for TthHB27I/DMSO/SIN-generated λ DNA library

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Clone Assay, Generated

    TthHB27I digestion patterns comparison in the presence of DMSO and cofactor SAM or its analogues. 0.5 μg of 1789 bp PCR DNA substrate (see Additional file 1 ) was incubated with 2 U of TthHB27I for 1 h at 65 °C in REase buffer with increasing amount of DMSO. Reaction mixtures were precipitated and electrophoresed in 1.3% agarose/TBE gels. The pictures on the right side of the figure show the theoretical arrangement of the DNA bands in an agarose gel after digesting the substrate DNA with TthHB27I under standard conditions. a Digestions performed only with the addition of DMSO, without any cofactor. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested PCR fragment; lane 1, 0% ( v /v) DMSO present in the reaction mixture; lane 2, with 5% (v/v) DMSO; lane 3, with 10% (v/v) DMSO; lane 4, with 15% (v/v) DMSO; lane 5, with 20% (v/v) DMSO; lane 6, with 25% (v/v) DMSO; lane 7, with 30% (v/v) DMSO. As in ( a ), but in the presence of 100 μM SAM ( b ); 100 μM SIN ( c ); 100 μM SAC ( d ); 100 μM SAH ( e ); 100 μM ATP ( f )

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: TthHB27I digestion patterns comparison in the presence of DMSO and cofactor SAM or its analogues. 0.5 μg of 1789 bp PCR DNA substrate (see Additional file 1 ) was incubated with 2 U of TthHB27I for 1 h at 65 °C in REase buffer with increasing amount of DMSO. Reaction mixtures were precipitated and electrophoresed in 1.3% agarose/TBE gels. The pictures on the right side of the figure show the theoretical arrangement of the DNA bands in an agarose gel after digesting the substrate DNA with TthHB27I under standard conditions. a Digestions performed only with the addition of DMSO, without any cofactor. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested PCR fragment; lane 1, 0% ( v /v) DMSO present in the reaction mixture; lane 2, with 5% (v/v) DMSO; lane 3, with 10% (v/v) DMSO; lane 4, with 15% (v/v) DMSO; lane 5, with 20% (v/v) DMSO; lane 6, with 25% (v/v) DMSO; lane 7, with 30% (v/v) DMSO. As in ( a ), but in the presence of 100 μM SAM ( b ); 100 μM SIN ( c ); 100 μM SAC ( d ); 100 μM SAH ( e ); 100 μM ATP ( f )

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Polymerase Chain Reaction, Incubation, Agarose Gel Electrophoresis

    Controlling of partial cleavage of genomic DNAs by TthHB27I under cofactor/analogue-induced ‘star’ activity conditions. E. coli and λ genomic DNAs were digested with TthHB27I under cofactor/analogue-induced ‘star’ conditions, while varying: temperature, incubation time and TthHB27I amount. 0.5 μg of DNAs were digested with TthHB27I in REase buffer supplemented with 100 μM SIN and 25% (v/v) DMSO. a Cleavage control by reaction temperature. Reactions conducted with E. coli DNA digested with 2 U of TthHB27I for 1 h at temperatures from 30 °C to 80 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction at 30 °C; lane 2, 35 °C; lane 3, 40 °C; lane 4, 45 °C; lane 5, 50 °C; lane 6, 55 °C; lane 7, 60 °C; lane 8, 65 °C; lane 9, 70 °C; lane 10, 75 °C; lane 11, 80 °C. b The same as ( a ), except that λ DNA was used. c Cleavage control by reaction time. E. coli DNA was digested at 65 °C at times ranging from 7.5 min to 16 h. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction conducted for 7.5 min; lane 2, 15 min; lane 3, 30 min; lane 4, 1 h; lane 5, 2 h; lane 6, 4 h; lane 7, 8 h; lane 8, 16 h. d The same as ( c ), except that λ DNA was used. e Cleavage control by the enzyme amount. E. coli DNA was digested at 65 °C with TthHB27I. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, DNA digested with 8 U TthHB27I; lane 2, 4 U; lane 3, 2 U; lane 4, 1 U; lane 5, 0.5 U; lane 6, 0.25 U; lane 7, 0.12 U; lane 8, 0.06 U. f The same as ( e ), except that λ DNA was used

    Journal: BMC Genomics

    Article Title: Randomized DNA libraries construction tool: a new 3-bp ‘frequent cutter’ TthHB27I/sinefungin endonuclease with chemically-induced specificity

    doi: 10.1186/s12864-018-4748-0

    Figure Lengend Snippet: Controlling of partial cleavage of genomic DNAs by TthHB27I under cofactor/analogue-induced ‘star’ activity conditions. E. coli and λ genomic DNAs were digested with TthHB27I under cofactor/analogue-induced ‘star’ conditions, while varying: temperature, incubation time and TthHB27I amount. 0.5 μg of DNAs were digested with TthHB27I in REase buffer supplemented with 100 μM SIN and 25% (v/v) DMSO. a Cleavage control by reaction temperature. Reactions conducted with E. coli DNA digested with 2 U of TthHB27I for 1 h at temperatures from 30 °C to 80 °C. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction at 30 °C; lane 2, 35 °C; lane 3, 40 °C; lane 4, 45 °C; lane 5, 50 °C; lane 6, 55 °C; lane 7, 60 °C; lane 8, 65 °C; lane 9, 70 °C; lane 10, 75 °C; lane 11, 80 °C. b The same as ( a ), except that λ DNA was used. c Cleavage control by reaction time. E. coli DNA was digested at 65 °C at times ranging from 7.5 min to 16 h. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, reaction conducted for 7.5 min; lane 2, 15 min; lane 3, 30 min; lane 4, 1 h; lane 5, 2 h; lane 6, 4 h; lane 7, 8 h; lane 8, 16 h. d The same as ( c ), except that λ DNA was used. e Cleavage control by the enzyme amount. E. coli DNA was digested at 65 °C with TthHB27I. Lane M1, GeneRuler 1 kb DNA Ladder; lane M2, 100 bp Plus DNA Ladder; lane K, undigested DNA; lane 1, DNA digested with 8 U TthHB27I; lane 2, 4 U; lane 3, 2 U; lane 4, 1 U; lane 5, 0.5 U; lane 6, 0.25 U; lane 7, 0.12 U; lane 8, 0.06 U. f The same as ( e ), except that λ DNA was used

    Article Snippet: DNA isolation kit (GeneJet Plasmid Miniprep Kit), DNA markers (GeneRuler 1 kb DNA Ladder, 100 bp Plus DNA Ladder), SmaI REase, FastAP Thermosensitive Alkaline Phosphatase, T4 DNA Polymerase and T4 DNA Ligase were from Thermo Fisher Scientific/Fermentas (Vilnius, USA/Lithuania).

    Techniques: Activity Assay, Incubation

    DNA gyrase behavior depends on a DNA substrate gyrase binding score. ( A ) EMSA analysis of DNA gyrase binding with 133 bp fragments; fragments scores are showed above the pictures; molar gyrase/DNA ratio is indicated under the lanes. SC—scrambled consensus, C—consensus. ( B ) Time-course of gyrase supercoiling of pUC19 plasmids harboring indicated 133 bp fragments; RE—relaxed plasmid, NSC—negatively supercoiled plasmid. 1—scrambled consensus, 2—Mu SGS, 3—consensus. ( C ) Time-course of ATP-independent relaxation of pUC19 plasmids harbouring indicated 133 bp fragments. ( D ) Time-course of ATP-dependent relaxation of the same plasmids by truncated GyrA59 2 GyrB 2 gyrase lacking CTD.

    Journal: Nucleic Acids Research

    Article Title: Single-nucleotide-resolution mapping of DNA gyrase cleavage sites across the Escherichia coli genome

    doi: 10.1093/nar/gky1222

    Figure Lengend Snippet: DNA gyrase behavior depends on a DNA substrate gyrase binding score. ( A ) EMSA analysis of DNA gyrase binding with 133 bp fragments; fragments scores are showed above the pictures; molar gyrase/DNA ratio is indicated under the lanes. SC—scrambled consensus, C—consensus. ( B ) Time-course of gyrase supercoiling of pUC19 plasmids harboring indicated 133 bp fragments; RE—relaxed plasmid, NSC—negatively supercoiled plasmid. 1—scrambled consensus, 2—Mu SGS, 3—consensus. ( C ) Time-course of ATP-independent relaxation of pUC19 plasmids harbouring indicated 133 bp fragments. ( D ) Time-course of ATP-dependent relaxation of the same plasmids by truncated GyrA59 2 GyrB 2 gyrase lacking CTD.

    Article Snippet: For EMSA and competition assays DNA fragments were PCR amplified using pUC19_for and pUC19_rev primers ( ) and purified with Gel Extraction and DNA Cleanup Micro Kit (Thermo Scientific).

    Techniques: Binding Assay, Plasmid Preparation