methyl sensitive restriction endonuclease hinp1i  (New England Biolabs)


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    Name:
    HinP1I
    Description:
    HinP1I 2 000 units
    Catalog Number:
    r0124s
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    69
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    2 000 units
    Category:
    Restriction Enzymes
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    New England Biolabs methyl sensitive restriction endonuclease hinp1i
    HinP1I
    HinP1I 2 000 units
    https://www.bioz.com/result/methyl sensitive restriction endonuclease hinp1i/product/New England Biolabs
    Average 90 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    methyl sensitive restriction endonuclease hinp1i - by Bioz Stars, 2020-04
    90/100 stars

    Images

    1) Product Images from "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 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

    Journal: Genome Research

    doi: 10.1101/gr.075713.107

    CTCF binds to DXZ4 DNA in vitro independent of flanking CpG methylation. ( A ) A single 3-kb DXZ4 monomer is represented by 1–3000 bp scale, under which are shown the 20 different fragments used in the original EMSA analysis (see Supplemental Fig. 2). The asterisk indicates fragment-6, the mobility of which is retarded in the presence of CTCF protein. ( B ) Confirmation of CTCF specificity by 100× excess competition with unlabeled 6-DNA and supershift ( upper arrowhead and arrow, respectively). ( C ) Refining CTCF to 6c only in the presence of the CTCF zinc fingers region (C-ZF) and full-length CTCF (C-FL) as indicated by the arrowheads, but not by luciferase ( L ). ( D ) Map of fragment 6c. The 142-bp fragment 6c is represented by the horizontal line, with the location of the Hinp1I site (GCGC) indicated. Arrowheads below ). ( E ) In vitro methylation of fragment 6c by M.SssI CpG methyltransferase blocks digestion by the methyl-sensitive Hinp1I restriction endonuclease. ( F ) CTCF binding to CpG methylated fragment 6c (Me-6c).
    Figure Legend Snippet: CTCF binds to DXZ4 DNA in vitro independent of flanking CpG methylation. ( A ) A single 3-kb DXZ4 monomer is represented by 1–3000 bp scale, under which are shown the 20 different fragments used in the original EMSA analysis (see Supplemental Fig. 2). The asterisk indicates fragment-6, the mobility of which is retarded in the presence of CTCF protein. ( B ) Confirmation of CTCF specificity by 100× excess competition with unlabeled 6-DNA and supershift ( upper arrowhead and arrow, respectively). ( C ) Refining CTCF to 6c only in the presence of the CTCF zinc fingers region (C-ZF) and full-length CTCF (C-FL) as indicated by the arrowheads, but not by luciferase ( L ). ( D ) Map of fragment 6c. The 142-bp fragment 6c is represented by the horizontal line, with the location of the Hinp1I site (GCGC) indicated. Arrowheads below ). ( E ) In vitro methylation of fragment 6c by M.SssI CpG methyltransferase blocks digestion by the methyl-sensitive Hinp1I restriction endonuclease. ( F ) CTCF binding to CpG methylated fragment 6c (Me-6c).

    Techniques Used: In Vitro, CpG Methylation Assay, Refining, Zinc-Fingers, Luciferase, Methylation, Binding Assay

    2) Product Images from "YY1 associates with the macrosatellite DXZ4 on the inactive X chromosome and binds with CTCF to a hypomethylated form in some male carcinomas"

    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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr964

    YY1 electrophoresis mobility-shift assays (EMSA). ( A ) Alignment of the candidate YY1-binding sequence of the macrosatellite DXZ4 (top) with two other well characterized YY1 binding consensus sequences. The core CAT element is highlighted. Below the alignments is the synthesized mutant (Mut.) sequence that no longer contains the CAT motif that was used in the EMSA. ( B ) Phosphoimager output of a dried EMSA gel. Radioactive bands appear dark. Samples loaded in each lane are labeled across the top and include specific competition (SC) and non-specific competition (NSC). Supershift analysis is shown in the last three lanes to the right with two independent YY1 antibodies and a non-specific control. The locations of the shift (S) and supershift (SS) are indicated by the arrowheads to the right of the gel. Free probe (FP) is indicated at the bottom of the gel. ( C ) Supershift analysis with anti-YY1 and anti-YY2. ( D ) Extended DNA sequence flanking the YY1 core CAT element (highlighted in black). CpG dinucleotides are highlighted by the dots below the sequence, and the location of the HinP1I site is highlighted in gray (GCGC). ( E ) Agarose gel (4.0%) analysis showing results of HinP1I digestions of the DNA sequence shown in (C), each of which was first treated or not with M.SssI, which methylates CpG dinucleotides in vitro . Presence or absence of either enzyme in the procedure is indicated above the corresponding lanes of the gel by the plus or minus symbol. DNA size is indicated to the right by a 10-bp ladder. ( F ) Phosphoimager output of a dried EMSA gel with the DNA sequence shown in (C). Radioactive bands appear dark. Target DNA sequence that has not (left two samples) or has been (right two samples) treated with M.SsspI before analysis is indicated above the gel by plus and minus symbols. Inclusion of whole-cell extract is indicated below. The location of the shift is indicated by the small black right-facing arrows. ( G ) Methylation insensitivity and YY1 specificity EMSA. EMSA shows shift with HeLa nuclear extract (Extract), a molecular weight shift for Flag-epitope-tagged YY1 from nuclear extract from HeLa cells overexpressing YY1-Flag (Extract Flag-YY1), and shift with purified non-tagged YY1 from bacteria (Purified YY1). Analyses are shown side by side, non-methylated double-stranded oligonucleotides to the left and CpG-methylated ones to the right. The arrows to the right of the gel indicate the Flag shift (top) and non-tagged shift (bottom).
    Figure Legend Snippet: YY1 electrophoresis mobility-shift assays (EMSA). ( A ) Alignment of the candidate YY1-binding sequence of the macrosatellite DXZ4 (top) with two other well characterized YY1 binding consensus sequences. The core CAT element is highlighted. Below the alignments is the synthesized mutant (Mut.) sequence that no longer contains the CAT motif that was used in the EMSA. ( B ) Phosphoimager output of a dried EMSA gel. Radioactive bands appear dark. Samples loaded in each lane are labeled across the top and include specific competition (SC) and non-specific competition (NSC). Supershift analysis is shown in the last three lanes to the right with two independent YY1 antibodies and a non-specific control. The locations of the shift (S) and supershift (SS) are indicated by the arrowheads to the right of the gel. Free probe (FP) is indicated at the bottom of the gel. ( C ) Supershift analysis with anti-YY1 and anti-YY2. ( D ) Extended DNA sequence flanking the YY1 core CAT element (highlighted in black). CpG dinucleotides are highlighted by the dots below the sequence, and the location of the HinP1I site is highlighted in gray (GCGC). ( E ) Agarose gel (4.0%) analysis showing results of HinP1I digestions of the DNA sequence shown in (C), each of which was first treated or not with M.SssI, which methylates CpG dinucleotides in vitro . Presence or absence of either enzyme in the procedure is indicated above the corresponding lanes of the gel by the plus or minus symbol. DNA size is indicated to the right by a 10-bp ladder. ( F ) Phosphoimager output of a dried EMSA gel with the DNA sequence shown in (C). Radioactive bands appear dark. Target DNA sequence that has not (left two samples) or has been (right two samples) treated with M.SsspI before analysis is indicated above the gel by plus and minus symbols. Inclusion of whole-cell extract is indicated below. The location of the shift is indicated by the small black right-facing arrows. ( G ) Methylation insensitivity and YY1 specificity EMSA. EMSA shows shift with HeLa nuclear extract (Extract), a molecular weight shift for Flag-epitope-tagged YY1 from nuclear extract from HeLa cells overexpressing YY1-Flag (Extract Flag-YY1), and shift with purified non-tagged YY1 from bacteria (Purified YY1). Analyses are shown side by side, non-methylated double-stranded oligonucleotides to the left and CpG-methylated ones to the right. The arrows to the right of the gel indicate the Flag shift (top) and non-tagged shift (bottom).

    Techniques Used: Electrophoresis, Mobility Shift, Binding Assay, Sequencing, Synthesized, Mutagenesis, Labeling, Agarose Gel Electrophoresis, In Vitro, Methylation, Molecular Weight, FLAG-tag, Purification

    Related Articles

    Clone 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: 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.

    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.

    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.

    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).

    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 .

    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).

    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: 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.

    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: 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).

    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.

    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).

    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.

    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: 99/100, based on 19 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

    CTCF binds to DXZ4 DNA in vitro independent of flanking CpG methylation. ( A ) A single 3-kb DXZ4 monomer is represented by 1–3000 bp scale, under which are shown the 20 different fragments used in the original EMSA analysis (see Supplemental Fig. 2). The asterisk indicates fragment-6, the mobility of which is retarded in the presence of CTCF protein. ( B ) Confirmation of CTCF specificity by 100× excess competition with unlabeled 6-DNA and supershift ( upper arrowhead and arrow, respectively). ( C ) Refining CTCF to 6c only in the presence of the CTCF zinc fingers region (C-ZF) and full-length CTCF (C-FL) as indicated by the arrowheads, but not by luciferase ( L ). ( D ) Map of fragment 6c. The 142-bp fragment 6c is represented by the horizontal line, with the location of the Hinp1I site (GCGC) indicated. Arrowheads below ). ( E ) In vitro methylation of fragment 6c by M.SssI CpG methyltransferase blocks digestion by the methyl-sensitive Hinp1I restriction endonuclease. ( F ) CTCF binding to CpG methylated fragment 6c (Me-6c).

    Journal: Genome Research

    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

    doi: 10.1101/gr.075713.107

    Figure Lengend Snippet: CTCF binds to DXZ4 DNA in vitro independent of flanking CpG methylation. ( A ) A single 3-kb DXZ4 monomer is represented by 1–3000 bp scale, under which are shown the 20 different fragments used in the original EMSA analysis (see Supplemental Fig. 2). The asterisk indicates fragment-6, the mobility of which is retarded in the presence of CTCF protein. ( B ) Confirmation of CTCF specificity by 100× excess competition with unlabeled 6-DNA and supershift ( upper arrowhead and arrow, respectively). ( C ) Refining CTCF to 6c only in the presence of the CTCF zinc fingers region (C-ZF) and full-length CTCF (C-FL) as indicated by the arrowheads, but not by luciferase ( L ). ( D ) Map of fragment 6c. The 142-bp fragment 6c is represented by the horizontal line, with the location of the Hinp1I site (GCGC) indicated. Arrowheads below ). ( E ) In vitro methylation of fragment 6c by M.SssI CpG methyltransferase blocks digestion by the methyl-sensitive Hinp1I restriction endonuclease. ( F ) CTCF binding to CpG methylated fragment 6c (Me-6c).

    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).

    Techniques: In Vitro, CpG Methylation Assay, Refining, Zinc-Fingers, Luciferase, Methylation, Binding Assay