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    HinP1I
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    HinP1I 2 000 units
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    Restriction Enzymes
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    New England Biolabs hinp1i
    HinP1I
    HinP1I 2 000 units
    https://www.bioz.com/result/hinp1i/product/New England Biolabs
    Average 95 stars, based on 17 article reviews
    Price from $9.99 to $1999.99
    hinp1i - by Bioz Stars, 2020-02
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    1) Product Images from "BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism"

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq567

    Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.
    Figure Legend Snippet: Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Binding Assay

    2) Product Images from "BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism"

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq567

    Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.
    Figure Legend Snippet: Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Binding Assay

    3) Product Images from "BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism"

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq567

    Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.
    Figure Legend Snippet: Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Binding Assay

    4) Product Images from "BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism"

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq567

    Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.
    Figure Legend Snippet: Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Binding Assay

    5) Product Images from "BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism"

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq567

    Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.
    Figure Legend Snippet: Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Binding Assay

    6) Product Images from "BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism"

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq567

    Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.
    Figure Legend Snippet: Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Binding Assay

    7) Product Images from "BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism"

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq567

    Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.
    Figure Legend Snippet: Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.

    Techniques Used: Plasmid Preparation, Agarose Gel Electrophoresis, Binding Assay

    8) Product Images from "Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes"

    Article Title: Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes

    Journal: Journal of Virology

    doi: 10.1128/JVI.00736-17

    EBV Rta expression increases DNA methylation and decreases CTCF binding in the promoter regions of MYC , CCND1 , and JUN . (A, left) Schematic diagrams of methylation-sensitive restriction enzyme sites, CTCF binding sites, and Rta binding sites in each target promoter region. These regions contain no EcoRI site, thus EcoRI served as an input control for AciI, HpaII, and HinP1I. The MYC gene body without Rta and CTCF binding sites served as a negative control (N.C.). Lengths of promoters are illustrated to scale. (Right) CpG methylation levels in the cellular promoters of 293TetLuc and 293TetER cells. Cellular DNAs of paired untreated and doxycycline (Dox)-treated (12 and 24 h) cells were extracted and subjected to restriction enzyme digestions. DNA fragments protected by each methylation-sensitive enzyme were quantified by real-time PCR. Fold changes of each restriction enzyme assessment denote the relative CpG methylation levels in the Dox-treated cells compared to their untreated counterparts. Error bars depict the means ± SD from four independent experiments. Student's t test was used to evaluate the significant difference between the indicated data set. ***, P
    Figure Legend Snippet: EBV Rta expression increases DNA methylation and decreases CTCF binding in the promoter regions of MYC , CCND1 , and JUN . (A, left) Schematic diagrams of methylation-sensitive restriction enzyme sites, CTCF binding sites, and Rta binding sites in each target promoter region. These regions contain no EcoRI site, thus EcoRI served as an input control for AciI, HpaII, and HinP1I. The MYC gene body without Rta and CTCF binding sites served as a negative control (N.C.). Lengths of promoters are illustrated to scale. (Right) CpG methylation levels in the cellular promoters of 293TetLuc and 293TetER cells. Cellular DNAs of paired untreated and doxycycline (Dox)-treated (12 and 24 h) cells were extracted and subjected to restriction enzyme digestions. DNA fragments protected by each methylation-sensitive enzyme were quantified by real-time PCR. Fold changes of each restriction enzyme assessment denote the relative CpG methylation levels in the Dox-treated cells compared to their untreated counterparts. Error bars depict the means ± SD from four independent experiments. Student's t test was used to evaluate the significant difference between the indicated data set. ***, P

    Techniques Used: Expressing, DNA Methylation Assay, Binding Assay, Methylation, Negative Control, CpG Methylation Assay, Real-time Polymerase Chain Reaction

    9) Product Images from "Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells"

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx026

    Schematic representation of CGI-seq. TEST: single cell being studied. Methylation control (MC): cells that are used to define the detectable CGIs or any other accessible methylated regions (AMRs), sharing the same genome as the TEST. For CGI-seq analysis, the genomic DNA is classified into three classes of sequences: Me-CGIs (representing methylated CGIs, and more generally, Me-AMRs) indicated as a red line, Um-CGIs (unmethylated CGIs, and generally Um-AMRs) indicated as a blue line and undetectable regions indicated as a black line. Green lines are amplified sequences. Solid dot: Me-CpG sites. Hollow dot: Um-CpG sites. The intact gDNA is released from cell (step 1) and digested with RE1 (TEST) or no digestion (MC) (step 2), followed by MDA amplification (step 3). The amplification product is then digested by RE2 (step 4), in which the short fragments are converted t‘o a library and subjected to massive sequencing (step 5). The reads are aligned to the genome (step 6) and the methylation status of the AMRs (particularly CGIs) is determined (‘Materials and Methods’ section). The AMRs detected in both the TEST and the MC are called Me-CGIs (or Me-AMRs). The AMRs uniquely detected in the MC but not in the TEST are called Um-CGIs (or Um-AMRs) of the TEST. RE1 (four MREs in combination: Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI(BssHII), FastDigest enzyme from Thermal Scientific Inc.) distinguishes Me-CGIs (Me-AMRs) from Um-CGIs (Um-AMRs). MDA selectively depletes Um-CGIs (Um-AMRs) of the TEST and amplifies Me-CGIs (Me-AMRs). RE2 (either BstUI alone, or a combination of 3 separate REs (ABH: AciI, BstUI and Hinp1I from NEB)) generates fragments specifically enriching CGI sequences (and other CG-rich regions) from non-CGI sequences.
    Figure Legend Snippet: Schematic representation of CGI-seq. TEST: single cell being studied. Methylation control (MC): cells that are used to define the detectable CGIs or any other accessible methylated regions (AMRs), sharing the same genome as the TEST. For CGI-seq analysis, the genomic DNA is classified into three classes of sequences: Me-CGIs (representing methylated CGIs, and more generally, Me-AMRs) indicated as a red line, Um-CGIs (unmethylated CGIs, and generally Um-AMRs) indicated as a blue line and undetectable regions indicated as a black line. Green lines are amplified sequences. Solid dot: Me-CpG sites. Hollow dot: Um-CpG sites. The intact gDNA is released from cell (step 1) and digested with RE1 (TEST) or no digestion (MC) (step 2), followed by MDA amplification (step 3). The amplification product is then digested by RE2 (step 4), in which the short fragments are converted t‘o a library and subjected to massive sequencing (step 5). The reads are aligned to the genome (step 6) and the methylation status of the AMRs (particularly CGIs) is determined (‘Materials and Methods’ section). The AMRs detected in both the TEST and the MC are called Me-CGIs (or Me-AMRs). The AMRs uniquely detected in the MC but not in the TEST are called Um-CGIs (or Um-AMRs) of the TEST. RE1 (four MREs in combination: Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI(BssHII), FastDigest enzyme from Thermal Scientific Inc.) distinguishes Me-CGIs (Me-AMRs) from Um-CGIs (Um-AMRs). MDA selectively depletes Um-CGIs (Um-AMRs) of the TEST and amplifies Me-CGIs (Me-AMRs). RE2 (either BstUI alone, or a combination of 3 separate REs (ABH: AciI, BstUI and Hinp1I from NEB)) generates fragments specifically enriching CGI sequences (and other CG-rich regions) from non-CGI sequences.

    Techniques Used: Methylation, Amplification, Multiple Displacement Amplification, Sequencing

    10) Product Images from "Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells"

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx026

    Schematic representation of CGI-seq. TEST: single cell being studied. Methylation control (MC): cells that are used to define the detectable CGIs or any other accessible methylated regions (AMRs), sharing the same genome as the TEST. For CGI-seq analysis, the genomic DNA is classified into three classes of sequences: Me-CGIs (representing methylated CGIs, and more generally, Me-AMRs) indicated as a red line, Um-CGIs (unmethylated CGIs, and generally Um-AMRs) indicated as a blue line and undetectable regions indicated as a black line. Green lines are amplified sequences. Solid dot: Me-CpG sites. Hollow dot: Um-CpG sites. The intact gDNA is released from cell (step 1) and digested with RE1 (TEST) or no digestion (MC) (step 2), followed by MDA amplification (step 3). The amplification product is then digested by RE2 (step 4), in which the short fragments are converted t‘o a library and subjected to massive sequencing (step 5). The reads are aligned to the genome (step 6) and the methylation status of the AMRs (particularly CGIs) is determined (‘Materials and Methods’ section). The AMRs detected in both the TEST and the MC are called Me-CGIs (or Me-AMRs). The AMRs uniquely detected in the MC but not in the TEST are called Um-CGIs (or Um-AMRs) of the TEST. RE1 (four MREs in combination: Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI(BssHII), FastDigest enzyme from Thermal Scientific Inc.) distinguishes Me-CGIs (Me-AMRs) from Um-CGIs (Um-AMRs). MDA selectively depletes Um-CGIs (Um-AMRs) of the TEST and amplifies Me-CGIs (Me-AMRs). RE2 (either BstUI alone, or a combination of 3 separate REs (ABH: AciI, BstUI and Hinp1I from NEB)) generates fragments specifically enriching CGI sequences (and other CG-rich regions) from non-CGI sequences.
    Figure Legend Snippet: Schematic representation of CGI-seq. TEST: single cell being studied. Methylation control (MC): cells that are used to define the detectable CGIs or any other accessible methylated regions (AMRs), sharing the same genome as the TEST. For CGI-seq analysis, the genomic DNA is classified into three classes of sequences: Me-CGIs (representing methylated CGIs, and more generally, Me-AMRs) indicated as a red line, Um-CGIs (unmethylated CGIs, and generally Um-AMRs) indicated as a blue line and undetectable regions indicated as a black line. Green lines are amplified sequences. Solid dot: Me-CpG sites. Hollow dot: Um-CpG sites. The intact gDNA is released from cell (step 1) and digested with RE1 (TEST) or no digestion (MC) (step 2), followed by MDA amplification (step 3). The amplification product is then digested by RE2 (step 4), in which the short fragments are converted t‘o a library and subjected to massive sequencing (step 5). The reads are aligned to the genome (step 6) and the methylation status of the AMRs (particularly CGIs) is determined (‘Materials and Methods’ section). The AMRs detected in both the TEST and the MC are called Me-CGIs (or Me-AMRs). The AMRs uniquely detected in the MC but not in the TEST are called Um-CGIs (or Um-AMRs) of the TEST. RE1 (four MREs in combination: Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI(BssHII), FastDigest enzyme from Thermal Scientific Inc.) distinguishes Me-CGIs (Me-AMRs) from Um-CGIs (Um-AMRs). MDA selectively depletes Um-CGIs (Um-AMRs) of the TEST and amplifies Me-CGIs (Me-AMRs). RE2 (either BstUI alone, or a combination of 3 separate REs (ABH: AciI, BstUI and Hinp1I from NEB)) generates fragments specifically enriching CGI sequences (and other CG-rich regions) from non-CGI sequences.

    Techniques Used: Methylation, Amplification, Multiple Displacement Amplification, Sequencing

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    Article Title: DXZ4 chromatin adopts an opposing conformation to that of the surrounding chromosome and acquires a novel inactive X-specific role involving CTCF and antisense transcripts
    Article Snippet: The 1.1 kb product was TA cloned into pCR2.1 (Invitrogen) prior to sequence verification, excision with HindIII and XbaI (NEB), and cloning into pGEM3 (Promega). .. In vitro CpG methylation of DNA was achieved using M.SssI CpG methyltransferase (NEB) and methylation assessed using the methyl-sensitive restriction endonuclease Hinp1I (NEB).

    Article Title: Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and "Nonbacterial" Prostatitis
    Article Snippet: Paragraph title: Cloning and RFLP analysis. ... For RFLP analysis, DNA was digested with Msp I and Hin P1I in NEBuffer 2 (New England Biolabs, Beverly, Mass.) and 0.01% Triton X-100.

    Amplification:

    Article Title: A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples
    Article Snippet: The digestion was performed for 2h at 37°C by 10 U of HinP1-I (New England Biolabs) and 10U of MseI (New England Biolabs) restriction enzymes or only by 10U of MseI (New England Biolabs). .. The first round of PCR amplification was performed with primers annealing to the anchors and covers 20 cycles, or 45 cycles in case only a single PCR was used.

    Article Title: Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and "Nonbacterial" Prostatitis
    Article Snippet: Ninety-six-well minipreps ( ) were carried out to isolate plasmids from individual clones for restriction fragment length polymorphism (RFLP) analysis and sequencing. rDNA inserts from pCR2.1 vector clones were amplified by PCR with vector primers equidistant from the 5′ and 3′ ends of the insert. .. For RFLP analysis, DNA was digested with Msp I and Hin P1I in NEBuffer 2 (New England Biolabs, Beverly, Mass.) and 0.01% Triton X-100.

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: .. Library construction for reduced representation sequencing To enrich the sequences for CGIs and other CpG-rich regions, 1.5 μg of amplified DNA from each sample was divided into three equal amounts and digested by one of the three RE2 reagents respectively: AciI, BstUI and Hinp1I (NEB), called ABH collectively. .. The condition of incubation is 3 h at 60°C for BstUI and at 37°C for AciI and Hinp1I.

    Stable Transfection:

    Article Title: YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas
    Article Snippet: Methylation was assayed with the methyl-sensitive restriction endonuclease HinP1I (New England Biolabs). .. Nuclear extracts from HeLa cells stably overexpressing Flag-epitope tagged YY1 ( ) and purification of bacterially expressed non-tagged YY1 protein ( ) have been described previously.

    Synthesized:

    Article Title: YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas
    Article Snippet: Methylation was assayed with the methyl-sensitive restriction endonuclease HinP1I (New England Biolabs). .. Oligonucleotides modified with 5-methyl Cytosine were synthesized directly with the same sequence as DXZ4-YY1 above; only the three C's in the CpG context were methylated for the forward and reverse sequence (Eurofins MWG Operon).

    TA Cloning:

    Article Title: Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and "Nonbacterial" Prostatitis
    Article Snippet: PCR products were gel purified and cloned with the TOPO TA cloning kit pCR2.1-TOPO vector (Invitrogen Corporation, Carlsbad, Calif.) by following the manufacturer’s recommendations. .. For RFLP analysis, DNA was digested with Msp I and Hin P1I in NEBuffer 2 (New England Biolabs, Beverly, Mass.) and 0.01% Triton X-100.

    Construct:

    Article Title: Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling
    Article Snippet: The two pRNA constructs used in this work are designated as A/b′ and br_B/a′ ( and ). .. The 118-nucleotide (nt) A/b′ was generated by in vitro run-off transcription using a linearized double-stranded DNA template that contains a T7 RNA polymerase promoter followed by the RNA sequence, with the 5′ terminus of the DNA antisense strand mutated to 5′-GCGC-3′ to allow linearization of the plasmid using the Hinp1I restriction endonuclease (cleaving 5′…G/CGC…3′, New England Biolabs, Inc.).

    Electrophoresis:

    Article Title: DXZ4 chromatin adopts an opposing conformation to that of the surrounding chromosome and acquires a novel inactive X-specific role involving CTCF and antisense transcripts
    Article Snippet: Paragraph title: Electrophoresis mobility shift assay ... In vitro CpG methylation of DNA was achieved using M.SssI CpG methyltransferase (NEB) and methylation assessed using the methyl-sensitive restriction endonuclease Hinp1I (NEB).

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism
    Article Snippet: Digestion of pC194 with HinP1I (New England Biolabs) and BsuRI (Fermentas) was tested using buffers recommended by the manufacturers. .. Plasmid isoforms were separated by electrophoresis in 1% agarose gel at low voltage (1.25 V/cm) and stained with ethidium bromide after the run.

    Article Title: YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas
    Article Snippet: Paragraph title: Electrophoresis mobility shift assays ... Methylation was assayed with the methyl-sensitive restriction endonuclease HinP1I (New England Biolabs).

    Incubation:

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism
    Article Snippet: DNA cleavage analysis For kinetic analysis, supercoiled pC194 plasmid DNA (2.84 nM) was incubated in 33 mM Tris–acetate pH 7.9, 10 mM Mg–acetate, 66 mM K–acetate, 0.1 mg/ml BSA (Fermentas Tango buffer) with His-tagged BspRI endonuclease (0.0054 pM) at 37°C. .. Digestion of pC194 with HinP1I (New England Biolabs) and BsuRI (Fermentas) was tested using buffers recommended by the manufacturers.

    Article Title: YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas
    Article Snippet: HeLa whole-cell extracts were incubated with the32 P-labeled double-stranded oligomers on ice for 30 min in binding buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM DTT, 5% glycerol), in the presence of 1 µg dIdC and 1 µg of non-specific double-stranded DNA oligomers. .. Methylation was assayed with the methyl-sensitive restriction endonuclease HinP1I (New England Biolabs).

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: .. The condition of incubation is 3 h at 60°C for BstUI and at 37°C for AciI and Hinp1I. ..

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: Library construction for reduced representation sequencing To enrich the sequences for CGIs and other CpG-rich regions, 1.5 μg of amplified DNA from each sample was divided into three equal amounts and digested by one of the three RE2 reagents respectively: AciI, BstUI and Hinp1I (NEB), called ABH collectively. .. The condition of incubation is 3 h at 60°C for BstUI and at 37°C for AciI and Hinp1I.

    Activity Assay:

    Article Title: Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes
    Article Snippet: .. For each sample, 5 μg DNA was digested by AciI, HpaII, and HinP1I (NEB Inc.) at 37°C for 30 min, followed by heat inactivation to eliminate enzyme activity. .. EcoRI (NEB Inc.), which is insensitive to CpG methylation, was run in parallel to serve as a DNA input control.

    Modification:

    Article Title: YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas
    Article Snippet: Methylation was assayed with the methyl-sensitive restriction endonuclease HinP1I (New England Biolabs). .. Oligonucleotides modified with 5-methyl Cytosine were synthesized directly with the same sequence as DXZ4-YY1 above; only the three C's in the CpG context were methylated for the forward and reverse sequence (Eurofins MWG Operon).

    Ligation:

    Article Title: A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples
    Article Snippet: The digestion was performed for 2h at 37°C by 10 U of HinP1-I (New England Biolabs) and 10U of MseI (New England Biolabs) restriction enzymes or only by 10U of MseI (New England Biolabs). .. Ligation of MSE and HINP anchors was performed as described .

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: Library construction for reduced representation sequencing To enrich the sequences for CGIs and other CpG-rich regions, 1.5 μg of amplified DNA from each sample was divided into three equal amounts and digested by one of the three RE2 reagents respectively: AciI, BstUI and Hinp1I (NEB), called ABH collectively. .. After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min.

    Generated:

    Article Title: Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling
    Article Snippet: .. The 118-nucleotide (nt) A/b′ was generated by in vitro run-off transcription using a linearized double-stranded DNA template that contains a T7 RNA polymerase promoter followed by the RNA sequence, with the 5′ terminus of the DNA antisense strand mutated to 5′-GCGC-3′ to allow linearization of the plasmid using the Hinp1I restriction endonuclease (cleaving 5′…G/CGC…3′, New England Biolabs, Inc.). .. The transcribed RNA was purified by denaturing polyacrylamide gel electrophoresis (PAGE), then quantified and stored as previously described.

    other:

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism
    Article Snippet: Conversion of supercoiled pC194 into the linear form by HinP1I was accompanied by the appearance of the open circular intermediate in a very similar fashion as for BspRI ( A), which is consistent with both enzymes acting as a monomer.

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism
    Article Snippet: Of the Type IIP REases that have been shown by crystallographic evidence to be monomeric, only HinP1I has been analyzed with regard to the cleavage mechanism.

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism
    Article Snippet: HinP1I and BsuRI were used at 0.016 and 0.05 U/µl concentrations, respectively.

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism
    Article Snippet: Since pC194 contains a single site also for HinP1I, we could test the cleavage kinetics of HinP1I with this more informative substrate.

    CpG Methylation Assay:

    Article Title: DXZ4 chromatin adopts an opposing conformation to that of the surrounding chromosome and acquires a novel inactive X-specific role involving CTCF and antisense transcripts
    Article Snippet: .. In vitro CpG methylation of DNA was achieved using M.SssI CpG methyltransferase (NEB) and methylation assessed using the methyl-sensitive restriction endonuclease Hinp1I (NEB). .. ChIP was performed essentially as described ( ) except chromatin was sheared using a Bioruptor (Diagenode) with the following conditions: cell density of ∼1 × 107 cells/mL, sonicated at 4°C using 12 cycles of 30 sec on, 30 sec off at maximum power.

    Article Title: Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes
    Article Snippet: For each sample, 5 μg DNA was digested by AciI, HpaII, and HinP1I (NEB Inc.) at 37°C for 30 min, followed by heat inactivation to eliminate enzyme activity. .. EcoRI (NEB Inc.), which is insensitive to CpG methylation, was run in parallel to serve as a DNA input control.

    Imaging:

    Article Title: Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and "Nonbacterial" Prostatitis
    Article Snippet: For RFLP analysis, DNA was digested with Msp I and Hin P1I in NEBuffer 2 (New England Biolabs, Beverly, Mass.) and 0.01% Triton X-100. .. The digested DNA was separated on a 3% MetaPhor gel (FMC Bioproducts, Rockland, Maine) in 1× TBE (Tris-borate-EDTA) for about 2.5 h at 50 V. Ethidium bromide-stained gels were visualized with a NucleoVision digital imaging system (NucleoTech Corporation, San Carlos, Calif.).

    Polymerase Chain Reaction:

    Article Title: A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples
    Article Snippet: The digestion was performed for 2h at 37°C by 10 U of HinP1-I (New England Biolabs) and 10U of MseI (New England Biolabs) restriction enzymes or only by 10U of MseI (New England Biolabs). .. The first round of PCR amplification was performed with primers annealing to the anchors and covers 20 cycles, or 45 cycles in case only a single PCR was used.

    Article Title: Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and "Nonbacterial" Prostatitis
    Article Snippet: Ninety-six-well minipreps ( ) were carried out to isolate plasmids from individual clones for restriction fragment length polymorphism (RFLP) analysis and sequencing. rDNA inserts from pCR2.1 vector clones were amplified by PCR with vector primers equidistant from the 5′ and 3′ ends of the insert. .. For RFLP analysis, DNA was digested with Msp I and Hin P1I in NEBuffer 2 (New England Biolabs, Beverly, Mass.) and 0.01% Triton X-100.

    Binding Assay:

    Article Title: DXZ4 chromatin adopts an opposing conformation to that of the surrounding chromosome and acquires a novel inactive X-specific role involving CTCF and antisense transcripts
    Article Snippet: Binding was performed at room temperature for 30 min using 1 μL of corresponding TNT protein with DIG-DNA in 1× phosphate buffered saline supplemented with 5 mM MgCl2 , 0.1 mM ZnSO4 , 1 mM DTT, 0.1% NP40, and 10% glycerol. .. In vitro CpG methylation of DNA was achieved using M.SssI CpG methyltransferase (NEB) and methylation assessed using the methyl-sensitive restriction endonuclease Hinp1I (NEB).

    Article Title: YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas
    Article Snippet: We assayed competition by adding cold oligomers and performed supershifts by adding specific anti-YY1, anti-YY2, or non-specific antibody (anti-GAPDH) to the binding reactions, as indicated. .. Methylation was assayed with the methyl-sensitive restriction endonuclease HinP1I (New England Biolabs).

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: Library construction for reduced representation sequencing To enrich the sequences for CGIs and other CpG-rich regions, 1.5 μg of amplified DNA from each sample was divided into three equal amounts and digested by one of the three RE2 reagents respectively: AciI, BstUI and Hinp1I (NEB), called ABH collectively. .. After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min.

    Methylation:

    Article Title: DXZ4 chromatin adopts an opposing conformation to that of the surrounding chromosome and acquires a novel inactive X-specific role involving CTCF and antisense transcripts
    Article Snippet: .. In vitro CpG methylation of DNA was achieved using M.SssI CpG methyltransferase (NEB) and methylation assessed using the methyl-sensitive restriction endonuclease Hinp1I (NEB). .. ChIP was performed essentially as described ( ) except chromatin was sheared using a Bioruptor (Diagenode) with the following conditions: cell density of ∼1 × 107 cells/mL, sonicated at 4°C using 12 cycles of 30 sec on, 30 sec off at maximum power.

    Article Title: Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes
    Article Snippet: Paragraph title: DNA methylation analysis by using methylation-sensitive restriction enzymes. ... For each sample, 5 μg DNA was digested by AciI, HpaII, and HinP1I (NEB Inc.) at 37°C for 30 min, followed by heat inactivation to eliminate enzyme activity.

    Article Title: YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas
    Article Snippet: .. Methylation was assayed with the methyl-sensitive restriction endonuclease HinP1I (New England Biolabs). .. Both methylated and non-methylated DXZ4-YY1Ls were then used in mobility-shift assays as described above.

    Purification:

    Article Title: Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and "Nonbacterial" Prostatitis
    Article Snippet: PCR products were gel purified and cloned with the TOPO TA cloning kit pCR2.1-TOPO vector (Invitrogen Corporation, Carlsbad, Calif.) by following the manufacturer’s recommendations. .. For RFLP analysis, DNA was digested with Msp I and Hin P1I in NEBuffer 2 (New England Biolabs, Beverly, Mass.) and 0.01% Triton X-100.

    Article Title: Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling
    Article Snippet: The 118-nucleotide (nt) A/b′ was generated by in vitro run-off transcription using a linearized double-stranded DNA template that contains a T7 RNA polymerase promoter followed by the RNA sequence, with the 5′ terminus of the DNA antisense strand mutated to 5′-GCGC-3′ to allow linearization of the plasmid using the Hinp1I restriction endonuclease (cleaving 5′…G/CGC…3′, New England Biolabs, Inc.). .. The transcribed RNA was purified by denaturing polyacrylamide gel electrophoresis (PAGE), then quantified and stored as previously described.

    Article Title: YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas
    Article Snippet: Methylation was assayed with the methyl-sensitive restriction endonuclease HinP1I (New England Biolabs). .. Nuclear extracts from HeLa cells stably overexpressing Flag-epitope tagged YY1 ( ) and purification of bacterially expressed non-tagged YY1 protein ( ) have been described previously.

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: Library construction for reduced representation sequencing To enrich the sequences for CGIs and other CpG-rich regions, 1.5 μg of amplified DNA from each sample was divided into three equal amounts and digested by one of the three RE2 reagents respectively: AciI, BstUI and Hinp1I (NEB), called ABH collectively. .. After deactivation of the enzymes with the binding buffer of the purification kit DNA clean & concentrator kit (Zymo Research), the digested DNA samples were pooled for purification and then subjected to the reaction for End-repair and A-addition using NEBNext Ultra End Repair/dA-Tailing Module at 20°C for 1 h and then 65°C for 30 min, followed by Illumina sequencing adaptor ligation using NEBNext Ultra Ligation Module at 20°C for 30 min.

    Sequencing:

    Article Title: DXZ4 chromatin adopts an opposing conformation to that of the surrounding chromosome and acquires a novel inactive X-specific role involving CTCF and antisense transcripts
    Article Snippet: The 1.1 kb product was TA cloned into pCR2.1 (Invitrogen) prior to sequence verification, excision with HindIII and XbaI (NEB), and cloning into pGEM3 (Promega). .. In vitro CpG methylation of DNA was achieved using M.SssI CpG methyltransferase (NEB) and methylation assessed using the methyl-sensitive restriction endonuclease Hinp1I (NEB).

    Article Title: Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and "Nonbacterial" Prostatitis
    Article Snippet: We used RFLP analysis to estimate the diversity of bacteria in the samples from patients with prostatitis and to identify unique clones for sequence analysis. .. For RFLP analysis, DNA was digested with Msp I and Hin P1I in NEBuffer 2 (New England Biolabs, Beverly, Mass.) and 0.01% Triton X-100.

    Article Title: Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling
    Article Snippet: .. The 118-nucleotide (nt) A/b′ was generated by in vitro run-off transcription using a linearized double-stranded DNA template that contains a T7 RNA polymerase promoter followed by the RNA sequence, with the 5′ terminus of the DNA antisense strand mutated to 5′-GCGC-3′ to allow linearization of the plasmid using the Hinp1I restriction endonuclease (cleaving 5′…G/CGC…3′, New England Biolabs, Inc.). .. The transcribed RNA was purified by denaturing polyacrylamide gel electrophoresis (PAGE), then quantified and stored as previously described.

    Article Title: YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas
    Article Snippet: Using double-stranded DXZ4-YY1L as a template, we methylated the C's of CpG dinucleotides in the sequence with M.SssI according to the manufacturers’ recommendations (New England Biolabs). .. Methylation was assayed with the methyl-sensitive restriction endonuclease HinP1I (New England Biolabs).

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells
    Article Snippet: .. Library construction for reduced representation sequencing To enrich the sequences for CGIs and other CpG-rich regions, 1.5 μg of amplified DNA from each sample was divided into three equal amounts and digested by one of the three RE2 reagents respectively: AciI, BstUI and Hinp1I (NEB), called ABH collectively. .. The condition of incubation is 3 h at 60°C for BstUI and at 37°C for AciI and Hinp1I.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling
    Article Snippet: The 118-nucleotide (nt) A/b′ was generated by in vitro run-off transcription using a linearized double-stranded DNA template that contains a T7 RNA polymerase promoter followed by the RNA sequence, with the 5′ terminus of the DNA antisense strand mutated to 5′-GCGC-3′ to allow linearization of the plasmid using the Hinp1I restriction endonuclease (cleaving 5′…G/CGC…3′, New England Biolabs, Inc.). .. The transcribed RNA was purified by denaturing polyacrylamide gel electrophoresis (PAGE), then quantified and stored as previously described.

    Activated Clotting Time Assay:

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism
    Article Snippet: .. This assignment is supported by the similar time-course of cleavage detected for HinP1I, an enzyme shown by structural evidence to act as a monomer ( A). ..

    Chromatin Immunoprecipitation:

    Article Title: Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes
    Article Snippet: For each sample, 5 μg DNA was digested by AciI, HpaII, and HinP1I (NEB Inc.) at 37°C for 30 min, followed by heat inactivation to eliminate enzyme activity. .. One-hundredth of the digested DNA was subjected to real-time PCR analysis using the same primer sets as those described for the ChIP experiments.

    Plasmid Preparation:

    Article Title: Prevalence of Corynebacterial 16S rRNA Sequences in Patients with Bacterial and "Nonbacterial" Prostatitis
    Article Snippet: Ninety-six-well minipreps ( ) were carried out to isolate plasmids from individual clones for restriction fragment length polymorphism (RFLP) analysis and sequencing. rDNA inserts from pCR2.1 vector clones were amplified by PCR with vector primers equidistant from the 5′ and 3′ ends of the insert. .. For RFLP analysis, DNA was digested with Msp I and Hin P1I in NEBuffer 2 (New England Biolabs, Beverly, Mass.) and 0.01% Triton X-100.

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism
    Article Snippet: DNA cleavage analysis For kinetic analysis, supercoiled pC194 plasmid DNA (2.84 nM) was incubated in 33 mM Tris–acetate pH 7.9, 10 mM Mg–acetate, 66 mM K–acetate, 0.1 mg/ml BSA (Fermentas Tango buffer) with His-tagged BspRI endonuclease (0.0054 pM) at 37°C. .. Digestion of pC194 with HinP1I (New England Biolabs) and BsuRI (Fermentas) was tested using buffers recommended by the manufacturers.

    Article Title: Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling
    Article Snippet: .. The 118-nucleotide (nt) A/b′ was generated by in vitro run-off transcription using a linearized double-stranded DNA template that contains a T7 RNA polymerase promoter followed by the RNA sequence, with the 5′ terminus of the DNA antisense strand mutated to 5′-GCGC-3′ to allow linearization of the plasmid using the Hinp1I restriction endonuclease (cleaving 5′…G/CGC…3′, New England Biolabs, Inc.). .. The transcribed RNA was purified by denaturing polyacrylamide gel electrophoresis (PAGE), then quantified and stored as previously described.

    Real-time Polymerase Chain Reaction:

    Article Title: Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes
    Article Snippet: For each sample, 5 μg DNA was digested by AciI, HpaII, and HinP1I (NEB Inc.) at 37°C for 30 min, followed by heat inactivation to eliminate enzyme activity. .. One-hundredth of the digested DNA was subjected to real-time PCR analysis using the same primer sets as those described for the ChIP experiments.

    Agarose Gel Electrophoresis:

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism
    Article Snippet: Digestion of pC194 with HinP1I (New England Biolabs) and BsuRI (Fermentas) was tested using buffers recommended by the manufacturers. .. Plasmid isoforms were separated by electrophoresis in 1% agarose gel at low voltage (1.25 V/cm) and stained with ethidium bromide after the run.

    In Vitro:

    Article Title: DXZ4 chromatin adopts an opposing conformation to that of the surrounding chromosome and acquires a novel inactive X-specific role involving CTCF and antisense transcripts
    Article Snippet: .. In vitro CpG methylation of DNA was achieved using M.SssI CpG methyltransferase (NEB) and methylation assessed using the methyl-sensitive restriction endonuclease Hinp1I (NEB). .. ChIP was performed essentially as described ( ) except chromatin was sheared using a Bioruptor (Diagenode) with the following conditions: cell density of ∼1 × 107 cells/mL, sonicated at 4°C using 12 cycles of 30 sec on, 30 sec off at maximum power.

    Article Title: Global Structure of a Three-Way Junction in a Phi29 Packaging RNA Dimer Determined Using Site-Directed Spin Labeling
    Article Snippet: .. The 118-nucleotide (nt) A/b′ was generated by in vitro run-off transcription using a linearized double-stranded DNA template that contains a T7 RNA polymerase promoter followed by the RNA sequence, with the 5′ terminus of the DNA antisense strand mutated to 5′-GCGC-3′ to allow linearization of the plasmid using the Hinp1I restriction endonuclease (cleaving 5′…G/CGC…3′, New England Biolabs, Inc.). .. The transcribed RNA was purified by denaturing polyacrylamide gel electrophoresis (PAGE), then quantified and stored as previously described.

    Ethanol Precipitation:

    Article Title: A Sensitive Assay for Virus Discovery in Respiratory Clinical Samples
    Article Snippet: After the RT reaction, second strand synthesis was performed with 5 U Klenow frament (3′ - 5′ exo-) (Westburg) and 7.5 U of RNase H (Amersham) followed by a phenol chloroform extraction and ethanol precipitation. .. The digestion was performed for 2h at 37°C by 10 U of HinP1-I (New England Biolabs) and 10U of MseI (New England Biolabs) restriction enzymes or only by 10U of MseI (New England Biolabs).

    DNA Methylation Assay:

    Article Title: Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes
    Article Snippet: Paragraph title: DNA methylation analysis by using methylation-sensitive restriction enzymes. ... For each sample, 5 μg DNA was digested by AciI, HpaII, and HinP1I (NEB Inc.) at 37°C for 30 min, followed by heat inactivation to eliminate enzyme activity.

    Mobility Shift:

    Article Title: DXZ4 chromatin adopts an opposing conformation to that of the surrounding chromosome and acquires a novel inactive X-specific role involving CTCF and antisense transcripts
    Article Snippet: Paragraph title: Electrophoresis mobility shift assay ... In vitro CpG methylation of DNA was achieved using M.SssI CpG methyltransferase (NEB) and methylation assessed using the methyl-sensitive restriction endonuclease Hinp1I (NEB).

    Article Title: YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas
    Article Snippet: Paragraph title: Electrophoresis mobility shift assays ... Methylation was assayed with the methyl-sensitive restriction endonuclease HinP1I (New England Biolabs).

    FLAG-tag:

    Article Title: YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas
    Article Snippet: Methylation was assayed with the methyl-sensitive restriction endonuclease HinP1I (New England Biolabs). .. Nuclear extracts from HeLa cells stably overexpressing Flag-epitope tagged YY1 ( ) and purification of bacterially expressed non-tagged YY1 protein ( ) have been described previously.

    Staining:

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism
    Article Snippet: Digestion of pC194 with HinP1I (New England Biolabs) and BsuRI (Fermentas) was tested using buffers recommended by the manufacturers. .. Plasmid isoforms were separated by electrophoresis in 1% agarose gel at low voltage (1.25 V/cm) and stained with ethidium bromide after the run.

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    New England Biolabs hinp1i
    Digestion of the single-site plasmid pC194 with BspRI, <t>HinP1I</t> and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.
    Hinp1i, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 17 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.

    Journal: Nucleic Acids Research

    Article Title: BspRI restriction endonuclease: cloning, expression in Escherichia coli and sequential cleavage mechanism

    doi: 10.1093/nar/gkq567

    Figure Lengend Snippet: Digestion of the single-site plasmid pC194 with BspRI, HinP1I and BsuRI endonucleases. ( A ) Agarose gel electrophoresis of the digestion products. Reaction times are shown above the lanes. Digestions were performed as described in ‘Materials and Methods’ section. Sc, l and oc denote supercoiled, linear and open circular forms, respectively. ( B ) Proposed kinetic scheme for DNA cleavage by BspRI endonuclease. Step 1, equilibrium binding to uncut DNA; step 2, nicking of the first strand; step 3, equilibrium binding to nicked DNA, BspRI facing the nicked strand; step 4, equilibrium binding to nicked DNA, BspRI facing the intact strand; step 5, transposition of BspRI from the nicked strand to the uncut strand; step 6, cleaving the second strand. ( C ) Time course of digestion of supercoiled plasmid DNA with BspRI. pC194 plasmid DNA was digested for the indicated times and the plasmid forms were quantitated as described in ‘Materials and Methods’ section. Experimental data are denoted by symbols (circle, supercoiled; diamond, nicked; square, linear). Lines represent results of the simulation (dashed–dotted, supercoiled; solid, nicked; dashed, linear). Error bars indicate the standard error of the data calculated from four parallel experiments.

    Article Snippet: Conversion of supercoiled pC194 into the linear form by HinP1I was accompanied by the appearance of the open circular intermediate in a very similar fashion as for BspRI ( A), which is consistent with both enzymes acting as a monomer.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Binding Assay