mlui hf  (New England Biolabs)


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    Structured Review

    New England Biolabs mlui hf
    ZBTB38 binds the methylated M2 motif in vitro and it does not regulate its methylation in vivo . (a) Analysis of M2 motif methylation at two ZBTB38 binding sites and two control regions using the DNA methylation-sensitive <t>MluI</t> restriction enzyme. Genomic DNAs prepared from HeLa S3 cells transfected with siRNAs against ZBTB38 or control siRNAs were digested overnight by BamHI or BamHI+MluI and analysed by PCR amplification. (b) Analysis of M2 motif methylation at two ZBTB38 binding sites and two control regions using the DNA methylation-sensitive MluI restriction enzyme in HeLa-S3 cells expressing the HA-Flag-ZBTB38 protein and parental cells. (c) In vitro binding assays. GST-fusions of ZBTB38 central zinc fingers and mutated zinc fingers (H491R) or GST alone were incubated with equimolar mix of methylated, unmethylated, and mutated DNA probe containing the M2 motif. Left panel: relative quantification of methyl, unmethyl, and mutated level of DNAs recovered on the beads. Right panel: migration on agarose gel stained with ethidium bromide of total DNAs recovered on the beads prior enzymatic digestion.
    Mlui Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mlui hf/product/New England Biolabs
    Average 95 stars, based on 40 article reviews
    Price from $9.99 to $1999.99
    mlui hf - by Bioz Stars, 2022-12
    95/100 stars

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    1) Product Images from "Context-dependent CpG methylation directs cell-specific binding of transcription factor ZBTB38"

    Article Title: Context-dependent CpG methylation directs cell-specific binding of transcription factor ZBTB38

    Journal: Epigenetics

    doi: 10.1080/15592294.2022.2111135

    ZBTB38 binds the methylated M2 motif in vitro and it does not regulate its methylation in vivo . (a) Analysis of M2 motif methylation at two ZBTB38 binding sites and two control regions using the DNA methylation-sensitive MluI restriction enzyme. Genomic DNAs prepared from HeLa S3 cells transfected with siRNAs against ZBTB38 or control siRNAs were digested overnight by BamHI or BamHI+MluI and analysed by PCR amplification. (b) Analysis of M2 motif methylation at two ZBTB38 binding sites and two control regions using the DNA methylation-sensitive MluI restriction enzyme in HeLa-S3 cells expressing the HA-Flag-ZBTB38 protein and parental cells. (c) In vitro binding assays. GST-fusions of ZBTB38 central zinc fingers and mutated zinc fingers (H491R) or GST alone were incubated with equimolar mix of methylated, unmethylated, and mutated DNA probe containing the M2 motif. Left panel: relative quantification of methyl, unmethyl, and mutated level of DNAs recovered on the beads. Right panel: migration on agarose gel stained with ethidium bromide of total DNAs recovered on the beads prior enzymatic digestion.
    Figure Legend Snippet: ZBTB38 binds the methylated M2 motif in vitro and it does not regulate its methylation in vivo . (a) Analysis of M2 motif methylation at two ZBTB38 binding sites and two control regions using the DNA methylation-sensitive MluI restriction enzyme. Genomic DNAs prepared from HeLa S3 cells transfected with siRNAs against ZBTB38 or control siRNAs were digested overnight by BamHI or BamHI+MluI and analysed by PCR amplification. (b) Analysis of M2 motif methylation at two ZBTB38 binding sites and two control regions using the DNA methylation-sensitive MluI restriction enzyme in HeLa-S3 cells expressing the HA-Flag-ZBTB38 protein and parental cells. (c) In vitro binding assays. GST-fusions of ZBTB38 central zinc fingers and mutated zinc fingers (H491R) or GST alone were incubated with equimolar mix of methylated, unmethylated, and mutated DNA probe containing the M2 motif. Left panel: relative quantification of methyl, unmethyl, and mutated level of DNAs recovered on the beads. Right panel: migration on agarose gel stained with ethidium bromide of total DNAs recovered on the beads prior enzymatic digestion.

    Techniques Used: Methylation, In Vitro, In Vivo, Binding Assay, DNA Methylation Assay, Transfection, Polymerase Chain Reaction, Amplification, Expressing, Zinc-Fingers, Incubation, Migration, Agarose Gel Electrophoresis, Staining

    ZBTB38 binding sites contain a methylated CpG consensus . (a) Overlap between ZBTB38 binding regions and 63 chromatin binding factors in HeLa-S3 cells (ENCODE data). (b) De novo DNA motifs discovery in ZBTB38 binding regions using HOMER tools identify two sequence motifs: M1 ( P -value of 1.0 × 10 −453 ) and M2 ( P -value of 1.0 × 10 −198 ). The logo and enrichment statistics for M1 and M2 are presented. (c) Venn diagram indicating the proportion of ZBTB38 binding regions containing M1, M2, M1 + M2, or none of these motifs. (d) Average tag intensity of ZBTB38 ChIP-sequencing and Input samples in ZBTB38-bound regions containing M1, M2, M1 + M2, or none. (e) Genomic distribution of ZBTB38 binding regions containing M1, M2, M1 + M2, or none determined using HOMER tools. (f) Methylation level of the M1 motif at ZBTB38 binding regions (left panel) and at whole genome (right panel). (g) Methylation level of the M2 motif at ZBTB38 binding regions (left panel) and at whole genome (right panel). (h) Input DNAs, ZBTB38 ChIPed DNAs, and control IgG-bound DNAs were digested with BamHI and MluI prior to analysis on a 2% agarose gel stained with ethidium bromide. A control 72 base pair fragment without MluI and BamHI site was used as a control ChIP specificity.
    Figure Legend Snippet: ZBTB38 binding sites contain a methylated CpG consensus . (a) Overlap between ZBTB38 binding regions and 63 chromatin binding factors in HeLa-S3 cells (ENCODE data). (b) De novo DNA motifs discovery in ZBTB38 binding regions using HOMER tools identify two sequence motifs: M1 ( P -value of 1.0 × 10 −453 ) and M2 ( P -value of 1.0 × 10 −198 ). The logo and enrichment statistics for M1 and M2 are presented. (c) Venn diagram indicating the proportion of ZBTB38 binding regions containing M1, M2, M1 + M2, or none of these motifs. (d) Average tag intensity of ZBTB38 ChIP-sequencing and Input samples in ZBTB38-bound regions containing M1, M2, M1 + M2, or none. (e) Genomic distribution of ZBTB38 binding regions containing M1, M2, M1 + M2, or none determined using HOMER tools. (f) Methylation level of the M1 motif at ZBTB38 binding regions (left panel) and at whole genome (right panel). (g) Methylation level of the M2 motif at ZBTB38 binding regions (left panel) and at whole genome (right panel). (h) Input DNAs, ZBTB38 ChIPed DNAs, and control IgG-bound DNAs were digested with BamHI and MluI prior to analysis on a 2% agarose gel stained with ethidium bromide. A control 72 base pair fragment without MluI and BamHI site was used as a control ChIP specificity.

    Techniques Used: Binding Assay, Methylation, Sequencing, Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Staining

    2) Product Images from "Using a reverse genetics system to generate recombinant SARS-CoV-2 expressing robust levels of reporter genes"

    Article Title: Using a reverse genetics system to generate recombinant SARS-CoV-2 expressing robust levels of reporter genes

    Journal: bioRxiv

    doi: 10.1101/2022.05.21.492922

    Assembly of entire SARS-CoV-2 genome into a BAC. a. The released five fragments (F1-F5) from pUC57 plasmids. b. Colony PCR screening for positive pBeloBAC-F1. c. Restriction digestion analysis of pBeloBAC-F13 using PacI and MluI. d. Restriction digestion analysis of pBeloBAC-F134 using PacI and MluI (F3); and MluI and BstBI (F4). e. Restriction digestion analysis of pBeloBAC-F1342 using KasI and PacI (F2); PacI and MluI (F3); and MluI and BstBI (F4). f. Restrict digestion confirmation of the pBeloBAC-FL using KasI and PacI (F2); PacI and MluI (F3); MluI and BstBI (F4); and BstBI and BamHI (F5).
    Figure Legend Snippet: Assembly of entire SARS-CoV-2 genome into a BAC. a. The released five fragments (F1-F5) from pUC57 plasmids. b. Colony PCR screening for positive pBeloBAC-F1. c. Restriction digestion analysis of pBeloBAC-F13 using PacI and MluI. d. Restriction digestion analysis of pBeloBAC-F134 using PacI and MluI (F3); and MluI and BstBI (F4). e. Restriction digestion analysis of pBeloBAC-F1342 using KasI and PacI (F2); PacI and MluI (F3); and MluI and BstBI (F4). f. Restrict digestion confirmation of the pBeloBAC-FL using KasI and PacI (F2); PacI and MluI (F3); MluI and BstBI (F4); and BstBI and BamHI (F5).

    Techniques Used: BAC Assay, Polymerase Chain Reaction

    Recovery of rSARS-CoV-2 from the pBeloBAC-FL. a. Protocol used for recovery of rSARS-CoV-2 from the pBeloBAC-FL using Lipofectamine 2000 transfection in Vero E6 cells. b. CPE caused by rSARS-CoV-2/WT in Vero E6 cells. Scale bars, 100 µm. c. Confirmation of the rescued rSARS-CoV-2/WT by IFA using a mouse antibody against viral N protein and a TRITC labeled donkey anti-mouse IgG secondary antibody. The nucleus was stained by DAPI. d. Sanger sequencing of the M gene of the natural isolate SARS-CoV-2 (top) and the recombinant SARS-CoV-2(bottom) that the MluI was removed by silent mutation.
    Figure Legend Snippet: Recovery of rSARS-CoV-2 from the pBeloBAC-FL. a. Protocol used for recovery of rSARS-CoV-2 from the pBeloBAC-FL using Lipofectamine 2000 transfection in Vero E6 cells. b. CPE caused by rSARS-CoV-2/WT in Vero E6 cells. Scale bars, 100 µm. c. Confirmation of the rescued rSARS-CoV-2/WT by IFA using a mouse antibody against viral N protein and a TRITC labeled donkey anti-mouse IgG secondary antibody. The nucleus was stained by DAPI. d. Sanger sequencing of the M gene of the natural isolate SARS-CoV-2 (top) and the recombinant SARS-CoV-2(bottom) that the MluI was removed by silent mutation.

    Techniques Used: Transfection, Immunofluorescence, Labeling, Staining, Sequencing, Recombinant, Mutagenesis

    Overview of a BAC-based RG system for generation of rSARS-CoV-2. a. The schematic representation of the SARS-CoV-2 genome. Unique restriction sites used for vial genome assembly are indicated. b. Commercially synthesized five fragments (F1-F5) in pUC57 plasmids. Restriction sites used to release each of the viral fragments are indicated. The MluI and BstBI highlighted in red are the restriction sites that have been removed by silent mutation and used as genetics tags. c. Assembly of the entire SARS-CoV-2 genome into the empty pBeloBAC to generate the full-length rescue plasmid (pBeloBAC-FL).
    Figure Legend Snippet: Overview of a BAC-based RG system for generation of rSARS-CoV-2. a. The schematic representation of the SARS-CoV-2 genome. Unique restriction sites used for vial genome assembly are indicated. b. Commercially synthesized five fragments (F1-F5) in pUC57 plasmids. Restriction sites used to release each of the viral fragments are indicated. The MluI and BstBI highlighted in red are the restriction sites that have been removed by silent mutation and used as genetics tags. c. Assembly of the entire SARS-CoV-2 genome into the empty pBeloBAC to generate the full-length rescue plasmid (pBeloBAC-FL).

    Techniques Used: BAC Assay, Synthesized, Mutagenesis, Plasmid Preparation

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    New England Biolabs mlui hf
    Mlui Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mlui hf/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mlui hf - by Bioz Stars, 2022-12
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