microccocal nuclease  (New England Biolabs)


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    Structured Review

    New England Biolabs microccocal nuclease
    Association of Vpr with chromatin correlates with the formation of nuclear foci. A ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT) or an empty plasmid used as negative control. Forty-eight hours after transfection, cells were harvested and lysed with 0.5% Triton X-100. The soluble fraction was used as input control (Soluble). Insoluble debris containing chromatin was treated with <t>microccocal</t> nuclease (+MNase) or with buffer alone (−MNase). The resulting solubilized fractions and input controls were resolved by SDS-PAGE and analyzed by western blot. Specific monoclonal antibodies were used to detect GAPDH (cytoplasmic marker) and HA-Vpr. Histone 3 (chromatin marker) and VPRBP were detected using rabbit polyclonal antibodies. * Denotes a non-specific band detected with the anti-HA antibody. B ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT), Vpr (Q65R), Vpr (R80A), and Vpr (1–78). Cell extracts were processed and analysed as in A). C ) HeLa cells were first transfected with scrambled siRNA or siRNA targeting VPRBP. Twenty-four hours after transfection, cells were transfected with a plasmid expressing HA-Vpr (WT) or an empty plasmid as negative control. Cell extracts were processed and analyzed as in A).
    Microccocal Nuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest"

    Article Title: Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001080

    Association of Vpr with chromatin correlates with the formation of nuclear foci. A ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT) or an empty plasmid used as negative control. Forty-eight hours after transfection, cells were harvested and lysed with 0.5% Triton X-100. The soluble fraction was used as input control (Soluble). Insoluble debris containing chromatin was treated with microccocal nuclease (+MNase) or with buffer alone (−MNase). The resulting solubilized fractions and input controls were resolved by SDS-PAGE and analyzed by western blot. Specific monoclonal antibodies were used to detect GAPDH (cytoplasmic marker) and HA-Vpr. Histone 3 (chromatin marker) and VPRBP were detected using rabbit polyclonal antibodies. * Denotes a non-specific band detected with the anti-HA antibody. B ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT), Vpr (Q65R), Vpr (R80A), and Vpr (1–78). Cell extracts were processed and analysed as in A). C ) HeLa cells were first transfected with scrambled siRNA or siRNA targeting VPRBP. Twenty-four hours after transfection, cells were transfected with a plasmid expressing HA-Vpr (WT) or an empty plasmid as negative control. Cell extracts were processed and analyzed as in A).
    Figure Legend Snippet: Association of Vpr with chromatin correlates with the formation of nuclear foci. A ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT) or an empty plasmid used as negative control. Forty-eight hours after transfection, cells were harvested and lysed with 0.5% Triton X-100. The soluble fraction was used as input control (Soluble). Insoluble debris containing chromatin was treated with microccocal nuclease (+MNase) or with buffer alone (−MNase). The resulting solubilized fractions and input controls were resolved by SDS-PAGE and analyzed by western blot. Specific monoclonal antibodies were used to detect GAPDH (cytoplasmic marker) and HA-Vpr. Histone 3 (chromatin marker) and VPRBP were detected using rabbit polyclonal antibodies. * Denotes a non-specific band detected with the anti-HA antibody. B ) HeLa cells were transfected with plasmids expressing HA-tagged Vpr (WT), Vpr (Q65R), Vpr (R80A), and Vpr (1–78). Cell extracts were processed and analysed as in A). C ) HeLa cells were first transfected with scrambled siRNA or siRNA targeting VPRBP. Twenty-four hours after transfection, cells were transfected with a plasmid expressing HA-Vpr (WT) or an empty plasmid as negative control. Cell extracts were processed and analyzed as in A).

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Negative Control, SDS Page, Western Blot, Marker

    Related Articles

    Centrifugation:

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
    Article Snippet: Chromatin immunoprecipitation Chromatin from 5×106 cells per antibody that had been cross-linked with 0.5% formaldehyde for 10 min was isolated by sonication with a Branson sonifier (10 pulses of 10″ at setting 4) in Lysis buffer (50 mM Hepes-KOH, pH 7.5, 1% TritonX-100, 0.1% SDS) and centrifugation for 10′ at 21,000×g . .. To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA.

    Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism
    Article Snippet: For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract. .. After clarification of the lysate by three successive centrifugation steps at 21K x g for 10 min, the lysate was pre-cleared and the ORF57-RNA complexes were immunoprecipitated with protein A Dynabeads (Invitrogen).

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
    Article Snippet: Parasites were lysed with 200 strokes in a prechilled Dounce homogenizer and nuclei collected by centrifugation, cleaned through a 0.34 M sucrose cushion, and resuspended in 1 ml of prechilled buffer A supplemented with 2 mM CaCl2 plus protease inhibitors. .. Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0.

    Article Title: Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest
    Article Snippet: Insoluble cell debris, including chromatin, was pelleted by centrifugation (2500g for 10 minutes). .. Pellets were washed once with nuclease buffer (50 mM Tris pH 8.0, 5 mM CaCl2 , and 100 µg/ml BSA), split in two, and resuspended in nuclease buffer alone or nuclease buffer containing 200 U/ml microccocal nuclease (New England Biolabs, Ipswich, MA, USA).

    Filtration:

    Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
    Article Snippet: Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment. .. After filtration, the qualified tags (in fastq format) were aligned to the mouse genome (mm9) with bowtie2 (2.0.6) .

    Electrophoresis:

    Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
    Article Snippet: .. Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment. .. Co-purified DNA and whole cell extraction (WCE) input genomic DNA were subject to library construction, cluster generation and next-generation sequencing (Illumina Genome Analyzer II and HiSeq 2000).

    Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps
    Article Snippet: NET digestion by nucleases In a first set of experiments, 4 μg of purified λDNA (Invitrogen) was treated with 4 U/mL DNase I (Sigma Aldrich), 4 U/mL MNase (New England BioLabs, France), or 4 U/mL Alu I (New England BioLabs) for 20 min at 37°C. .. Electrophoresis was run at 100 V for 30 min and DNA was visualized with an ultraviolet transilluminator (MiniBIS-Pro, DNR Bio-imaging Systems).

    Incubation:

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
    Article Snippet: To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. Digested chromatin was diluted 3-fold with High Salt ChIP buffer (10 mM Tris-HCl, pH 8, 400 mM NaCl, 1% TritonX-100, 2 mM EDTA, Complete protease inhibitors w/o EDTA (Roche)) to yield 600 μl total volume and incubated overnight at 4°C with either 5 μl of anti-H3 (39163, Active Motif, concentration is not known), 4 μg of anti-H2A.Z (ab4174; Abcam), 1 μg of anti-H3K4me1 (ab8895; Abcam), 1 μg of anti-H3K4me3 (ab8580; Abcam) or 1 μg of H3K27ac (ab4729; Abcam).

    Article Title: CAL1 is the Drosophila CENP-A assembly factor
    Article Snippet: To assemble CENP-A nucleosomes, Drosophila CENP-A101–225 –H4–Cal11–160 (3.8 µM), Alexa Fluor 488–labeled H2A–H2B (2.5 µM), and 147-bp Widom 601 DNA (0.72 µM) were mixed and incubated for 1 h under low salt buffer conditions. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

    Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism
    Article Snippet: Next, CaCl2 was added to 5 mM with 30 U of RQ1 DNase (Promega) and incubated for 15 min at 25°C. .. For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract.

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
    Article Snippet: Briefly, schizont‐stage parasites of WT or WT + PfH3p‐HA were isolated from infected erythrocytes by saponin lysis, resuspended in 1 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 ) supplemented with protease inhibitors (Complete, Roche), and incubated for 30 min at 4°C. .. Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0.

    Article Title: Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest
    Article Snippet: Pellets were washed once with nuclease buffer (50 mM Tris pH 8.0, 5 mM CaCl2 , and 100 µg/ml BSA), split in two, and resuspended in nuclease buffer alone or nuclease buffer containing 200 U/ml microccocal nuclease (New England Biolabs, Ipswich, MA, USA). .. Pellets were incubated for 30 minutes on ice and then centrifuged at 12000g for 10 minutes.

    Gel Extraction:

    Article Title: Organization of DNA in a bacterial nucleoid
    Article Snippet: RNAs and proteins were removed from the sample by intensive treatment first with RNase A and then with proteinase K. Resultant samples were run in 1.2 % agarose gel and the band containing the shortest DNA digestion fragments (around 100 bp) was cut out for subsequent DNA purification with QIAquick Gel Extraction Kit (Qiagen) and sequencing. .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Modification:

    Article Title: Ectopic T Cell Receptor-? Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once
    Article Snippet: Nucleosomes were prepared by incubating with 500 units of MNase (NEB) for 10 minutes at 37°C. .. To 100 µl of the sheared chromatin was successively added: 1 µl protease inhibitor cocktail, 20 µl ChIP buffer 1, 15 µl of protein G coupled magnetic beads (all from Active Motif), 25 µl of 10 mg/ml Salmon Sperm DNA (Invitrogen), 1 µl of 10 mg/ml BSA (NEB), 36 µl deionized water and 3 µl of 1 mg/ml an antibody recognizing the trimethyl-lysine 4 modification on histone H3 (anti-H3K4me3) (Abcam).

    Western Blot:

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
    Article Snippet: Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp. .. Flag-RBPJ was precipitated using Flag-M2 agarose beads (Sigma) and washed immunoprecipates were subjected to western blotting using Abs to Flag or HistoneH3 (sc-8654) from Santa Cruz.

    Transfection:

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
    Article Snippet: To quantify RBPJ binding to nucleosomes, Flag-RBPJ was transfected into 293 cells. .. Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Concentration Assay:

    Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure
    Article Snippet: Reactions were carried out in an initial volume of 10 μl with 10 nM nucleosomes and 0–40 U/ml of MNase (New England Biolabs) in 20 mM Tris, pH 8.0, 0.5 mM CaCl2 at 37°C to prevent H3(T118ph) nucleosome disassembly. .. After 20 min, reactions were quenched with 15 mM EDTA final concentration and resolved by PAGE.

    Article Title: Organization of DNA in a bacterial nucleoid
    Article Snippet: To quench the reactions 12 μl of 10 % solution of phenol in ethanol and EGTA to final concentration 25 mM were added on ice to each aliquot. .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
    Article Snippet: To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. Digested chromatin was diluted 3-fold with High Salt ChIP buffer (10 mM Tris-HCl, pH 8, 400 mM NaCl, 1% TritonX-100, 2 mM EDTA, Complete protease inhibitors w/o EDTA (Roche)) to yield 600 μl total volume and incubated overnight at 4°C with either 5 μl of anti-H3 (39163, Active Motif, concentration is not known), 4 μg of anti-H2A.Z (ab4174; Abcam), 1 μg of anti-H3K4me1 (ab8895; Abcam), 1 μg of anti-H3K4me3 (ab8580; Abcam) or 1 μg of H3K27ac (ab4729; Abcam).

    Protease Inhibitor:

    Article Title: Ectopic T Cell Receptor-? Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once
    Article Snippet: The cells were washed twice with 1× PBS and the cell pellets were re-suspended in 1 ml 1× micrococcal nuclease (MNase) buffer (NEB) containing 2.5 µl protease inhibitor cocktail (Sigma) and 1 µl 100 mM PMSF. .. Nucleosomes were prepared by incubating with 500 units of MNase (NEB) for 10 minutes at 37°C.

    Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism
    Article Snippet: The composition of the SDS lysis buffer was 0.5% SDS, 50mM Tris pH 6.8, 1mM EDTA, 0.125 mg/ml Heparin, 1mM DTT, 1mM PMSF and 1X protease inhibitor (Calbiochem), while RIPA correction buffer was 1.25% NP40, 0.625% sodium deoxycholate, 62.5 mM Tris pH 8.0, 2.25 mM EDTA, 187.5 mM NaCl, 0.125 mg/ml Heparin, 1mM DTT, 1mM PMSF, with 1X protease inhibitors. .. For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract.

    Footprinting:

    Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure
    Article Snippet: Paragraph title: MNase footprinting ... Reactions were carried out in an initial volume of 10 μl with 10 nM nucleosomes and 0–40 U/ml of MNase (New England Biolabs) in 20 mM Tris, pH 8.0, 0.5 mM CaCl2 at 37°C to prevent H3(T118ph) nucleosome disassembly.

    Infection:

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
    Article Snippet: Briefly, schizont‐stage parasites of WT or WT + PfH3p‐HA were isolated from infected erythrocytes by saponin lysis, resuspended in 1 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 ) supplemented with protease inhibitors (Complete, Roche), and incubated for 30 min at 4°C. .. Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0.

    Imaging:

    Article Title: CAL1 is the Drosophila CENP-A assembly factor
    Article Snippet: The nucleosomes were analyzed by 6% native PAGE and stained by SYBR gold; the incorporation of H2A–H2B was confirmed by fluorescence imaging of the gel for Alexa Fluor 488. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

    Polymerase Chain Reaction:

    Article Title: Organization of DNA in a bacterial nucleoid
    Article Snippet: The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C. .. DNA fragments with sizes comparable to those from in vivo digestion were purified directly from the reaction with QIAquick PCR Purification Kit (Qiagen).

    Sonication:

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
    Article Snippet: For ChIP-PCR and for RNA Pol II and H3K9ac ChIP-seq, the MNAse digestion step was replaced with a sonication step in 0.12% SDS followed by 5× dilution with concentrated ChIP buffer. .. Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Article Title: Drosophila PAF1 modulates PIWI/piRNA silencing capacity
    Article Snippet: Pellets were then thawed in 500ul MNase Lysis Buffer (20 mM Tris-HCl pH8, 50 mM NaCl, 5mM CaCl2, 0.1 % Triton X-100, 10% glycerol, 1mM DTT, 1X Protease Inhibitors), warmed to 37C, and treated with 2ul of MNase (NEB, 2000gel units/ul) for 10 min before stopping with 10ul of 250mM EDTA, 250mM EGTA, and 10ul of 10% SDS. .. Ice-chilled, MNase-digested chromatin was further solubilized with sonication on a Q125 Sonicator, 5 sec ON/OFF cycle, for 1.5 min, at 35% amplitude.

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
    Article Snippet: Chromatin immunoprecipitation Chromatin from 5×106 cells per antibody that had been cross-linked with 0.5% formaldehyde for 10 min was isolated by sonication with a Branson sonifier (10 pulses of 10″ at setting 4) in Lysis buffer (50 mM Hepes-KOH, pH 7.5, 1% TritonX-100, 0.1% SDS) and centrifugation for 10′ at 21,000×g . .. To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA.

    Binding Assay:

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
    Article Snippet: To quantify RBPJ binding to nucleosomes, Flag-RBPJ was transfected into 293 cells. .. Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
    Article Snippet: .. To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. Digested chromatin was diluted 3-fold with High Salt ChIP buffer (10 mM Tris-HCl, pH 8, 400 mM NaCl, 1% TritonX-100, 2 mM EDTA, Complete protease inhibitors w/o EDTA (Roche)) to yield 600 μl total volume and incubated overnight at 4°C with either 5 μl of anti-H3 (39163, Active Motif, concentration is not known), 4 μg of anti-H2A.Z (ab4174; Abcam), 1 μg of anti-H3K4me1 (ab8895; Abcam), 1 μg of anti-H3K4me3 (ab8580; Abcam) or 1 μg of H3K27ac (ab4729; Abcam).

    Article Title: Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest
    Article Snippet: Paragraph title: Chromatin binding assays ... Pellets were washed once with nuclease buffer (50 mM Tris pH 8.0, 5 mM CaCl2 , and 100 µg/ml BSA), split in two, and resuspended in nuclease buffer alone or nuclease buffer containing 200 U/ml microccocal nuclease (New England Biolabs, Ipswich, MA, USA).

    ChIP-sequencing:

    Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
    Article Snippet: Briefly, approximately 10 million ESCs were used for each ChIP and massive parallel sequencing (ChIP-Seq) experiment. .. Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment.

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
    Article Snippet: Paragraph title: ChIP-seq and ChIP-PCR ... Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Nucleic Acid Electrophoresis:

    Article Title: Drosophila PAF1 modulates PIWI/piRNA silencing capacity
    Article Snippet: Pellets were then thawed in 500ul MNase Lysis Buffer (20 mM Tris-HCl pH8, 50 mM NaCl, 5mM CaCl2, 0.1 % Triton X-100, 10% glycerol, 1mM DTT, 1X Protease Inhibitors), warmed to 37C, and treated with 2ul of MNase (NEB, 2000gel units/ul) for 10 min before stopping with 10ul of 250mM EDTA, 250mM EGTA, and 10ul of 10% SDS. .. Gel electrophoresis confirmed significant digestion of chromatin to ~30% mononucleosomes and a spectrum of polynucleosome fragments.

    In Vivo:

    Article Title: Organization of DNA in a bacterial nucleoid
    Article Snippet: Paragraph title: In vivo MNase digestion ... The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Fluorescence:

    Article Title: CAL1 is the Drosophila CENP-A assembly factor
    Article Snippet: The nucleosomes were analyzed by 6% native PAGE and stained by SYBR gold; the incorporation of H2A–H2B was confirmed by fluorescence imaging of the gel for Alexa Fluor 488. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

    Magnetic Beads:

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
    Article Snippet: To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. 20 μl of Protein A/G magnetic beads (Pierce) were added to the reaction and incubated at 4°C for 2 h. Beads were washed with 280 μl each of TSE buffer (20 mM Tris pH 8.0, 0.1% SDS, 1% TritonX-100, 2 mM EDTA), TSE250 (TSE buffer, 250 mM NaCl) and TSE500 (TSE buffer, 500 mM NaCl), Wash buffer III (10 mM Tris pH 8.5, 0.25 M LiCl, 1% NP-40/Igepal, 1% deoxycholate, 1 mM EDTA) and TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) all containing Complete protease inhibitors.

    Article Title: Ectopic T Cell Receptor-? Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once
    Article Snippet: Nucleosomes were prepared by incubating with 500 units of MNase (NEB) for 10 minutes at 37°C. .. To 100 µl of the sheared chromatin was successively added: 1 µl protease inhibitor cocktail, 20 µl ChIP buffer 1, 15 µl of protein G coupled magnetic beads (all from Active Motif), 25 µl of 10 mg/ml Salmon Sperm DNA (Invitrogen), 1 µl of 10 mg/ml BSA (NEB), 36 µl deionized water and 3 µl of 1 mg/ml an antibody recognizing the trimethyl-lysine 4 modification on histone H3 (anti-H3K4me3) (Abcam).

    Isolation:

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
    Article Snippet: .. To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. Digested chromatin was diluted 3-fold with High Salt ChIP buffer (10 mM Tris-HCl, pH 8, 400 mM NaCl, 1% TritonX-100, 2 mM EDTA, Complete protease inhibitors w/o EDTA (Roche)) to yield 600 μl total volume and incubated overnight at 4°C with either 5 μl of anti-H3 (39163, Active Motif, concentration is not known), 4 μg of anti-H2A.Z (ab4174; Abcam), 1 μg of anti-H3K4me1 (ab8895; Abcam), 1 μg of anti-H3K4me3 (ab8580; Abcam) or 1 μg of H3K27ac (ab4729; Abcam).

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
    Article Snippet: .. Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0. .. After spinning out debris at 18,800 g for 15 min at 4°C, the supernatant was analyzed for the presence of mononucleosomes by extracting DNA and resolving it on a 2% agarose gel stained with ethidium bromide.

    Clarification Assay:

    Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism
    Article Snippet: For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract. .. After clarification of the lysate by three successive centrifugation steps at 21K x g for 10 min, the lysate was pre-cleared and the ORF57-RNA complexes were immunoprecipitated with protein A Dynabeads (Invitrogen).

    Size-exclusion Chromatography:

    Article Title: Drosophila PAF1 modulates PIWI/piRNA silencing capacity
    Article Snippet: Pellets were then thawed in 500ul MNase Lysis Buffer (20 mM Tris-HCl pH8, 50 mM NaCl, 5mM CaCl2, 0.1 % Triton X-100, 10% glycerol, 1mM DTT, 1X Protease Inhibitors), warmed to 37C, and treated with 2ul of MNase (NEB, 2000gel units/ul) for 10 min before stopping with 10ul of 250mM EDTA, 250mM EGTA, and 10ul of 10% SDS. .. Ice-chilled, MNase-digested chromatin was further solubilized with sonication on a Q125 Sonicator, 5 sec ON/OFF cycle, for 1.5 min, at 35% amplitude.

    Purification:

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics
    Article Snippet: Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C. .. DNA was purified and resolved on 6.5% polyacrylamide gel (37.5:1) in 1× TAE buffer (40 mM Tris-OH, 20.6 mM acetic acid, 5 mM sodium acetate, 2 mM EDTA) and stained with SYBR Gold.

    Article Title: Organization of DNA in a bacterial nucleoid
    Article Snippet: .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C. ..

    Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps
    Article Snippet: .. NET digestion by nucleases In a first set of experiments, 4 μg of purified λDNA (Invitrogen) was treated with 4 U/mL DNase I (Sigma Aldrich), 4 U/mL MNase (New England BioLabs, France), or 4 U/mL Alu I (New England BioLabs) for 20 min at 37°C. .. The samples were then loaded on 0.8% agarose gels (w/v) prepared in Tris-acetate-EDTA buffer containing 0.5 μg/mL ethidium bromide (Invitrogen).

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics
    Article Snippet: Micrococcal nuclease digestion Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C. .. DNA was purified and resolved on 6.5% polyacrylamide gel (37.5:1) in 1× TAE buffer (40 mM Tris-OH, 20.6 mM acetic acid, 5 mM sodium acetate, 2 mM EDTA) and stained with SYBR Gold.

    Sequencing:

    Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
    Article Snippet: Briefly, approximately 10 million ESCs were used for each ChIP and massive parallel sequencing (ChIP-Seq) experiment. .. Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment.

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
    Article Snippet: DNA samples for high throughput sequencing were prepared by the UNC genomics core and 50bp or 100 bp paired ends sequencing was performed using the Illumina platform. .. Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Article Title: Organization of DNA in a bacterial nucleoid
    Article Snippet: As control in sequence analysis, we used DNA fragments obtained from in vitro MNase digestion of purified wild type genomic DNA. .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Article Title: CAL1 is the Drosophila CENP-A assembly factor
    Article Snippet: MNase protection assays CENP-A nucleosomes were assembled by Nap1 or CAL11–160 under low salt buffer conditions (10 mM Tris-HCl, pH 7.5, 270 mM NaCl, 2% glycerol, 60 µg/ml BSA, 0.75 mM EDTA, and 0.1 mM DTT) on the 147-bp Widom 601 DNA sequence. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure
    Article Snippet: Reactions were carried out in an initial volume of 10 μl with 10 nM nucleosomes and 0–40 U/ml of MNase (New England Biolabs) in 20 mM Tris, pH 8.0, 0.5 mM CaCl2 at 37°C to prevent H3(T118ph) nucleosome disassembly. .. After 20 min, reactions were quenched with 15 mM EDTA final concentration and resolved by PAGE.

    Lysis:

    Article Title: Drosophila PAF1 modulates PIWI/piRNA silencing capacity
    Article Snippet: .. Pellets were then thawed in 500ul MNase Lysis Buffer (20 mM Tris-HCl pH8, 50 mM NaCl, 5mM CaCl2, 0.1 % Triton X-100, 10% glycerol, 1mM DTT, 1X Protease Inhibitors), warmed to 37C, and treated with 2ul of MNase (NEB, 2000gel units/ul) for 10 min before stopping with 10ul of 250mM EDTA, 250mM EGTA, and 10ul of 10% SDS. .. Ice-chilled, MNase-digested chromatin was further solubilized with sonication on a Q125 Sonicator, 5 sec ON/OFF cycle, for 1.5 min, at 35% amplitude.

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
    Article Snippet: Chromatin immunoprecipitation Chromatin from 5×106 cells per antibody that had been cross-linked with 0.5% formaldehyde for 10 min was isolated by sonication with a Branson sonifier (10 pulses of 10″ at setting 4) in Lysis buffer (50 mM Hepes-KOH, pH 7.5, 1% TritonX-100, 0.1% SDS) and centrifugation for 10′ at 21,000×g . .. To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA.

    Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism
    Article Snippet: The composition of the SDS lysis buffer was 0.5% SDS, 50mM Tris pH 6.8, 1mM EDTA, 0.125 mg/ml Heparin, 1mM DTT, 1mM PMSF and 1X protease inhibitor (Calbiochem), while RIPA correction buffer was 1.25% NP40, 0.625% sodium deoxycholate, 62.5 mM Tris pH 8.0, 2.25 mM EDTA, 187.5 mM NaCl, 0.125 mg/ml Heparin, 1mM DTT, 1mM PMSF, with 1X protease inhibitors. .. For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract.

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
    Article Snippet: Briefly, schizont‐stage parasites of WT or WT + PfH3p‐HA were isolated from infected erythrocytes by saponin lysis, resuspended in 1 ml of buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2 ) supplemented with protease inhibitors (Complete, Roche), and incubated for 30 min at 4°C. .. Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0.

    Article Title: Formation of Mobile Chromatin-Associated Nuclear Foci Containing HIV-1 Vpr and VPRBP Is Critical for the Induction of G2 Cell Cycle Arrest
    Article Snippet: Chromatin binding assays Cells were lysed in triton lysis buffer (50 mM Tris pH 7.5, 150 mM NaCl, 0.5% Triton X-100, and complete protease inhibitors cocktail (Roche) for 15 minutes. .. Pellets were washed once with nuclease buffer (50 mM Tris pH 8.0, 5 mM CaCl2 , and 100 µg/ml BSA), split in two, and resuspended in nuclease buffer alone or nuclease buffer containing 200 U/ml microccocal nuclease (New England Biolabs, Ipswich, MA, USA).

    Chromatin Immunoprecipitation:

    Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
    Article Snippet: .. Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment. .. Co-purified DNA and whole cell extraction (WCE) input genomic DNA were subject to library construction, cluster generation and next-generation sequencing (Illumina Genome Analyzer II and HiSeq 2000).

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
    Article Snippet: Paragraph title: ChIP-seq and ChIP-PCR ... Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Article Title: Drosophila PAF1 modulates PIWI/piRNA silencing capacity
    Article Snippet: Paragraph title: Chromatin immunoprecipitation and qPCR ... Pellets were then thawed in 500ul MNase Lysis Buffer (20 mM Tris-HCl pH8, 50 mM NaCl, 5mM CaCl2, 0.1 % Triton X-100, 10% glycerol, 1mM DTT, 1X Protease Inhibitors), warmed to 37C, and treated with 2ul of MNase (NEB, 2000gel units/ul) for 10 min before stopping with 10ul of 250mM EDTA, 250mM EGTA, and 10ul of 10% SDS.

    Article Title: Nucleosomes Are Stably Evicted from Enhancers but Not Promoters upon Induction of Certain Pro-Inflammatory Genes in Mouse Macrophages
    Article Snippet: .. To increase the resolution of ChIP experiments when detecting histones or histone modifications, and to differentiate nucleosome binding from PolII and TBP binding, the isolated chromatin was digested with 0.5 or 1 U MNase (NEB) for 1 h 30′ in the presence of 0.15 mM CaCl2 , and the digestion reaction was stopped by addition of 20 mM EDTA. .. Digested chromatin was diluted 3-fold with High Salt ChIP buffer (10 mM Tris-HCl, pH 8, 400 mM NaCl, 1% TritonX-100, 2 mM EDTA, Complete protease inhibitors w/o EDTA (Roche)) to yield 600 μl total volume and incubated overnight at 4°C with either 5 μl of anti-H3 (39163, Active Motif, concentration is not known), 4 μg of anti-H2A.Z (ab4174; Abcam), 1 μg of anti-H3K4me1 (ab8895; Abcam), 1 μg of anti-H3K4me3 (ab8580; Abcam) or 1 μg of H3K27ac (ab4729; Abcam).

    Article Title: Ectopic T Cell Receptor-? Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once
    Article Snippet: Paragraph title: Chromatin Immunoprecipitation (ChIP) ... Nucleosomes were prepared by incubating with 500 units of MNase (NEB) for 10 minutes at 37°C.

    Real-time Polymerase Chain Reaction:

    Article Title: Drosophila PAF1 modulates PIWI/piRNA silencing capacity
    Article Snippet: Paragraph title: Chromatin immunoprecipitation and qPCR ... Pellets were then thawed in 500ul MNase Lysis Buffer (20 mM Tris-HCl pH8, 50 mM NaCl, 5mM CaCl2, 0.1 % Triton X-100, 10% glycerol, 1mM DTT, 1X Protease Inhibitors), warmed to 37C, and treated with 2ul of MNase (NEB, 2000gel units/ul) for 10 min before stopping with 10ul of 250mM EDTA, 250mM EGTA, and 10ul of 10% SDS.

    Agarose Gel Electrophoresis:

    Article Title: Organization of DNA in a bacterial nucleoid
    Article Snippet: RNAs and proteins were removed from the sample by intensive treatment first with RNase A and then with proteinase K. Resultant samples were run in 1.2 % agarose gel and the band containing the shortest DNA digestion fragments (around 100 bp) was cut out for subsequent DNA purification with QIAquick Gel Extraction Kit (Qiagen) and sequencing. .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
    Article Snippet: Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0. .. After spinning out debris at 18,800 g for 15 min at 4°C, the supernatant was analyzed for the presence of mononucleosomes by extracting DNA and resolving it on a 2% agarose gel stained with ethidium bromide.

    In Vitro:

    Article Title: Organization of DNA in a bacterial nucleoid
    Article Snippet: As control in sequence analysis, we used DNA fragments obtained from in vitro MNase digestion of purified wild type genomic DNA. .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C.

    Transgenic Assay:

    Article Title: Ectopic T Cell Receptor-? Locus Control Region Activity in B Cells Is Suppressed by Direct Linkage to Two Flanking Genes at Once
    Article Snippet: Chromatin Immunoprecipitation (ChIP) Thymocytes and B cells of analyzed transgenic mouse lines were fixed in 10 ml RPMI medium with 1% formaldehyde at room temperature for 10 minutes. .. Nucleosomes were prepared by incubating with 500 units of MNase (NEB) for 10 minutes at 37°C.

    Next-Generation Sequencing:

    Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
    Article Snippet: Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment. .. Co-purified DNA and whole cell extraction (WCE) input genomic DNA were subject to library construction, cluster generation and next-generation sequencing (Illumina Genome Analyzer II and HiSeq 2000).

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF
    Article Snippet: DNA samples for high throughput sequencing were prepared by the UNC genomics core and 50bp or 100 bp paired ends sequencing was performed using the Illumina platform. .. Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Immunoprecipitation:

    Article Title: HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism
    Article Snippet: Paragraph title: HITS-CLIP lysate preparation and immunoprecipitation ... For RNA digestion, MNase (New England Biolabs) was diluted to 10 gel units/μL in RIPA buffer and 5 μL of this freshly diluted 1:200 stock was added to the extract.

    N-ChIP:

    Article Title: Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells
    Article Snippet: Paragraph title: Native Chromatin immunoprecipitation (N-ChIP) ... Chromatin pellets were briefly digested with MNase (New England BioLabs) and the mononucleosomes were monitored by electrophoresis 0.5ug anti-H2A.X antibody (active motif 39689) was used per ChIP experiment.

    DNA Purification:

    Article Title: Organization of DNA in a bacterial nucleoid
    Article Snippet: .. The wild type genomic DNA was purified using Wizard Genomic DNA Purification Kit (Promega) and treated with MNase (New England Biolabs) at 12 °C. ..

    Staining:

    Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure
    Article Snippet: Reactions were carried out in an initial volume of 10 μl with 10 nM nucleosomes and 0–40 U/ml of MNase (New England Biolabs) in 20 mM Tris, pH 8.0, 0.5 mM CaCl2 at 37°C to prevent H3(T118ph) nucleosome disassembly. .. Gels were stained with SYBR Gold (Invitrogen) and imaged by a Typhoon 8600 variable mode imager (GE Healthcare).

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics
    Article Snippet: Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C. .. DNA was purified and resolved on 6.5% polyacrylamide gel (37.5:1) in 1× TAE buffer (40 mM Tris-OH, 20.6 mM acetic acid, 5 mM sodium acetate, 2 mM EDTA) and stained with SYBR Gold.

    Article Title: CAL1 is the Drosophila CENP-A assembly factor
    Article Snippet: The nucleosomes were analyzed by 6% native PAGE and stained by SYBR gold; the incorporation of H2A–H2B was confirmed by fluorescence imaging of the gel for Alexa Fluor 488. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

    Article Title: Effects of histone H2B ubiquitylation on the nucleosome structure and dynamics
    Article Snippet: Micrococcal nuclease digestion Assembled nucleosome (∼30 ng DNA) were digested with increasing amounts (typically 0.3/1/3/10/30/100 Kunitz units) of MNase (NEB) for 20 min at 26 or 37°C. .. DNA was purified and resolved on 6.5% polyacrylamide gel (37.5:1) in 1× TAE buffer (40 mM Tris-OH, 20.6 mM acetic acid, 5 mM sodium acetate, 2 mM EDTA) and stained with SYBR Gold.

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum
    Article Snippet: Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0. .. After spinning out debris at 18,800 g for 15 min at 4°C, the supernatant was analyzed for the presence of mononucleosomes by extracting DNA and resolving it on a 2% agarose gel stained with ethidium bromide.

    Clear Native PAGE:

    Article Title: CAL1 is the Drosophila CENP-A assembly factor
    Article Snippet: The nucleosomes were analyzed by 6% native PAGE and stained by SYBR gold; the incorporation of H2A–H2B was confirmed by fluorescence imaging of the gel for Alexa Fluor 488. .. MNase digestion of nucleosomes was conducted by incubating nucleosomes (∼60 nM) with 2,000 U/ml MNase (New England Biolabs, Inc.) for 30–120 s. After the reactions were stopped by addition of 20 mM EDTA, the samples were treated with 2% SDS and 0.4 mg/ml proteinase K for 30 min at 55°C.

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    New England Biolabs mnase
    DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL <t>DNase,</t> <t>MNase,</t> or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.
    Mnase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase/product/New England Biolabs
    Average 99 stars, based on 41 article reviews
    Price from $9.99 to $1999.99
    mnase - by Bioz Stars, 2020-02
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    91
    New England Biolabs mnase reaction buffer
    Characterization of the NRS sequences. ( A ) EMSA comparing binding of the <t>DNA</t> binding domain of GR to a GBS sequence flanked by either the control sequence (left) or by NRS2 (right). ( B ) DNase I accessibility assay was performed with populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR ( FKBP5 : control accessible region; IGFBP1 : control inaccessible region; integr. GBS: integrated reporter region). Results are shown as % of input remaining after DNAse I digestion ±SEM (n = 3). ( C ) Nucleosome occupancy was analyzed using micrococcal nuclease <t>(MNase)</t> assays for populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR. Integr. GBS: integrated reporter region. Results are shown as % of input remaining after MNase digestion ±SEM (n = 3).
    Mnase Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mnase reaction buffer/product/New England Biolabs
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mnase reaction buffer - by Bioz Stars, 2020-02
    91/100 stars
      Buy from Supplier

    Image Search Results


    DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

    Journal: Frontiers in Immunology

    Article Title: An Improved Strategy to Recover Large Fragments of Functional Human Neutrophil Extracellular Traps

    doi: 10.3389/fimmu.2013.00166

    Figure Lengend Snippet: DNA nucleases induce NET digestion . (A) Migration profile of pure λDNA after digestion with 4 U/mL DNase, MNase, or Alu-I. (B) Alu-I, DNase, and MNase dose-effects on NET dsDNA obtained after A23187 stimulation of PMN. Incubation with the restriction enzymes lasted 20 min at 37°C. DNA migration took place in 0.8% agarose gel containing ethidium bromide.

    Article Snippet: NET digestion by nucleases In a first set of experiments, 4 μg of purified λDNA (Invitrogen) was treated with 4 U/mL DNase I (Sigma Aldrich), 4 U/mL MNase (New England BioLabs, France), or 4 U/mL Alu I (New England BioLabs) for 20 min at 37°C.

    Techniques: Migration, Incubation, Agarose Gel Electrophoresis

    Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Journal: Nucleic Acids Research

    Article Title: RBPJ binds to consensus and methylated cis elements within phased nucleosomes and controls gene expression in human aortic smooth muscle cells in cooperation with SRF

    doi: 10.1093/nar/gky562

    Figure Lengend Snippet: Differential chromatin structure and transcription factor binding between consensus and Alu RBPJ binding sites. ( A ) Heat map of clustered reads densities for the indicated genome-wide determination or DNA sequence feature centered around RBPJ peak summits for all 28 220 RBPJ binding sites. ( B ) The same analysis as in A for the 4921 RBPJ peak summits that intersected with an Alu element within 200 bp. ( C ) Quantification of DNA ends densities for MNAse digested input, RBPJ immunoprecipitated, and DNase hypersensitive DNA for cluster 1 (without Alu) and cluster 5 (with Alu) regions.

    Article Snippet: Cells were fixed with 0.7% formaldehyde and DNA was digested by 10 ul MNAse (New England Biolabs) to an average length of 120 bp.

    Techniques: Binding Assay, Genome Wide, Sequencing, Immunoprecipitation

    Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

    Journal: EMBO Reports

    Article Title: Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum

    doi: 10.15252/embr.201846331

    Figure Lengend Snippet: Ectopically expressed histone H3p localizes to the nucleus during parasite asexual development and incorporates into nucleosomes Indirect immunofluorescence assays were performed to determine the localization of ectopically expressed PfH3p‐HA in ring (R), trophozoite (T), and schizont (S) stages of Plasmodium falciparum asexual growth. PfH3p‐HA was detected using anti‐HA antibodies (green) and endogenous histone H3 with anti‐histone H3 N‐terminal antibodies (red). DAPI (blue) was used to stain the nucleus. Scale bar = 5 μm. Nuclei isolated from wild‐type (WT) or PfH3p‐HA‐expressing (WT + PfH3p‐HA) schizont‐stage parasites were treated with 4 U/ml of micrococcal nuclease (MNase) for the indicated amounts of time, the DNA purified and migrated on a 2% agarose gel, and stained with ethidium bromide. Mononucleosomes purified after 10 min of MNase treatment were separated using denaturing polyacrylamide gel electrophoresis and either stained with Coomassie Brilliant Blue (C.B.) or visualized by immunoblotting with anti‐HA (α‐HA) or anti‐C‐terminal histone H3 (α‐H3c) antibodies. Co‐immunoprecipitation (IP) experiments of purified mononucleosomes obtained from wild‐type (WT) or transfected (WT + PfH3p‐HA) schizont‐stage parasites were performed with either anti‐HA antibodies or mouse IgG. Immunoprecipitated products (right panel) were analyzed by immunoblotting using anti‐HA or anti‐histone H4 antibodies. Source data are available online for this figure.

    Article Snippet: Mononucleosomes were prepared from freshly isolated nuclei by digestion with 4 U MNase (NEB) for different amounts of time at 33°C; digestion was stopped by adding 10 mM EGTA pH 8.0.

    Techniques: Immunofluorescence, Staining, Isolation, Expressing, Purification, Agarose Gel Electrophoresis, Polyacrylamide Gel Electrophoresis, Immunoprecipitation, Transfection

    Characterization of the NRS sequences. ( A ) EMSA comparing binding of the DNA binding domain of GR to a GBS sequence flanked by either the control sequence (left) or by NRS2 (right). ( B ) DNase I accessibility assay was performed with populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR ( FKBP5 : control accessible region; IGFBP1 : control inaccessible region; integr. GBS: integrated reporter region). Results are shown as % of input remaining after DNAse I digestion ±SEM (n = 3). ( C ) Nucleosome occupancy was analyzed using micrococcal nuclease (MNase) assays for populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR. Integr. GBS: integrated reporter region. Results are shown as % of input remaining after MNase digestion ±SEM (n = 3).

    Journal: Nucleic Acids Research

    Article Title: Identification and characterization of DNA sequences that prevent glucocorticoid receptor binding to nearby response elements

    doi: 10.1093/nar/gkw203

    Figure Lengend Snippet: Characterization of the NRS sequences. ( A ) EMSA comparing binding of the DNA binding domain of GR to a GBS sequence flanked by either the control sequence (left) or by NRS2 (right). ( B ) DNase I accessibility assay was performed with populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR ( FKBP5 : control accessible region; IGFBP1 : control inaccessible region; integr. GBS: integrated reporter region). Results are shown as % of input remaining after DNAse I digestion ±SEM (n = 3). ( C ) Nucleosome occupancy was analyzed using micrococcal nuclease (MNase) assays for populations of transgenic cells with stably integrated reporters as indicated. Regions of interest were analyzed by qPCR. Integr. GBS: integrated reporter region. Results are shown as % of input remaining after MNase digestion ±SEM (n = 3).

    Article Snippet: For each sample containing 0.8 μg genomic DNA in 50 μl storage buffer, an equal volume of MNase reaction buffer (50 mM Tris pH 7.4; 25 mM KCl; 2.5 mM CaCl2 ; 5 mM MgCl2 ; 12.5 % glycerol) was added containing 1 μl MNase (NEB; ∼200 kunitz) for MNase-treated samples.

    Techniques: Binding Assay, Sequencing, Transgenic Assay, Stable Transfection, Real-time Polymerase Chain Reaction