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    New England Biolabs mcrbc
    The suvh1–1 mutation does not affect <t>DNA</t> methylation. ( A and B ) <t>McrBC-qPCR</t> analysis of DNA methylation levels at the d35S promoter and the LUC coding region in YJ (A) and LUCH (B). qPCR was performed using genomic DNA treated with or without McrBC. The relative levels of amplified DNA for UBQ5, LUC and d35S in samples treated with McrBC compared to untreated samples were shown. Error bars were from three technical replicates. Two biological replicates were performed and gave similar results. ( C and D ) The levels of CG, CHG and CHH DNA methylation at the d35S promoter (C) and LUC coding region (D) in YJ and YJ suvh1–1 as determined through MethylC sequencing. Results from two biological replicates (rep) are shown. (E) Total genomic CG, CHG and CHH DNA methylation in YJ and YJ suvh1–1 as determined through MethylC-seq. Results from two biological replicates (rep) are shown.
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    The suvh1–1 mutation does not affect DNA methylation. ( A and B ) McrBC-qPCR analysis of DNA methylation levels at the d35S promoter and the LUC coding region in YJ (A) and LUCH (B). qPCR was performed using genomic DNA treated with or without McrBC. The relative levels of amplified DNA for UBQ5, LUC and d35S in samples treated with McrBC compared to untreated samples were shown. Error bars were from three technical replicates. Two biological replicates were performed and gave similar results. ( C and D ) The levels of CG, CHG and CHH DNA methylation at the d35S promoter (C) and LUC coding region (D) in YJ and YJ suvh1–1 as determined through MethylC sequencing. Results from two biological replicates (rep) are shown. (E) Total genomic CG, CHG and CHH DNA methylation in YJ and YJ suvh1–1 as determined through MethylC-seq. Results from two biological replicates (rep) are shown.

    Journal: Nucleic Acids Research

    Article Title: SUVH1, a Su(var)3–9 family member, promotes the expression of genes targeted by DNA methylation

    doi: 10.1093/nar/gkv958

    Figure Lengend Snippet: The suvh1–1 mutation does not affect DNA methylation. ( A and B ) McrBC-qPCR analysis of DNA methylation levels at the d35S promoter and the LUC coding region in YJ (A) and LUCH (B). qPCR was performed using genomic DNA treated with or without McrBC. The relative levels of amplified DNA for UBQ5, LUC and d35S in samples treated with McrBC compared to untreated samples were shown. Error bars were from three technical replicates. Two biological replicates were performed and gave similar results. ( C and D ) The levels of CG, CHG and CHH DNA methylation at the d35S promoter (C) and LUC coding region (D) in YJ and YJ suvh1–1 as determined through MethylC sequencing. Results from two biological replicates (rep) are shown. (E) Total genomic CG, CHG and CHH DNA methylation in YJ and YJ suvh1–1 as determined through MethylC-seq. Results from two biological replicates (rep) are shown.

    Article Snippet: One hundred nanogram DNA was treated with 2 units of McrBC (New England Biolabs, M0272S) at 37°C for 30 min, and a mix without McrBC was performed in parallel as the control.

    Techniques: Mutagenesis, DNA Methylation Assay, Real-time Polymerase Chain Reaction, Amplification, Sequencing

    Transposons, but not genes, are methylated in maize. Genomic DNA was digested with McrBC during 0 min, 25 min, or 8 h, with or without Sss )

    Journal: Genome Research

    Article Title: Genes and Transposons Are Differentially Methylated in Plants, but Not in Mammals

    doi: 10.1101/gr.1784803

    Figure Lengend Snippet: Transposons, but not genes, are methylated in maize. Genomic DNA was digested with McrBC during 0 min, 25 min, or 8 h, with or without Sss )

    Article Snippet: Sss I pre-treatment and McrBC digestion was performed as recommended by the supplier (New England Biolabs).

    Techniques: Methylation

    McrPCR. Equal samples of genomic DNA are either pretreated with CpG methylase ( Sss I methylase), or not, and both samples are digested in a time course with McrBC (only time zero and complete digestion are shown). PCR is then performed using specific primers (arrows). If the target sequence (gray box) is methylated in the genome, there will be a decrease in the amount of PCR product following McrBC digestion with or without Sss I methylase pretreatment. If the target is not methylated, a decrease in PCR yield will only be evident following Sss I methylase pretreatment.

    Journal: Genome Research

    Article Title: Genes and Transposons Are Differentially Methylated in Plants, but Not in Mammals

    doi: 10.1101/gr.1784803

    Figure Lengend Snippet: McrPCR. Equal samples of genomic DNA are either pretreated with CpG methylase ( Sss I methylase), or not, and both samples are digested in a time course with McrBC (only time zero and complete digestion are shown). PCR is then performed using specific primers (arrows). If the target sequence (gray box) is methylated in the genome, there will be a decrease in the amount of PCR product following McrBC digestion with or without Sss I methylase pretreatment. If the target is not methylated, a decrease in PCR yield will only be evident following Sss I methylase pretreatment.

    Article Snippet: Sss I pre-treatment and McrBC digestion was performed as recommended by the supplier (New England Biolabs).

    Techniques: Polymerase Chain Reaction, Sequencing, Methylation

    qPCR validation of microarray results for remarkable genes showing significant changes in DNA methylation and transcript levels between d1 and d7 in neonatal murine hearts. The left panels show DNA methylation and gene expression microarray results represented by red bars and black markers, respectively; DNA methylation is expressed as MeDIP enrichment and gene expression levels as normalized microarray signals. The black markers preceding that indicating the neonatal d1 represent gene expression levels in embryonic hearts E16, E18, E19, E20. The middle panels present CpG methylation estimated with Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) where the DNA methylation levels correspond to the amounts of DNA undigested by CpG methylation dependent McrBC enzyme (1-(McrBC/Input). The right panels demonstrate transcript levels determined with qPCR as the ratios to the reference transcript of the Tbp gene (TATA binding protein gene). The microarray results were determined for pooled samples of 3 mice. The qPCR results represent average values obtained for three individuals for each developmental time-point. The statistical significance has been determined with two-tailed heteroscedastic Student’s t -test. The complete results of qPCR validation are collected in Additional file 2 : F2

    Journal: BMC Genomics

    Article Title: Changes in gene methylation patterns in neonatal murine hearts: Implications for the regenerative potential

    doi: 10.1186/s12864-016-2545-1

    Figure Lengend Snippet: qPCR validation of microarray results for remarkable genes showing significant changes in DNA methylation and transcript levels between d1 and d7 in neonatal murine hearts. The left panels show DNA methylation and gene expression microarray results represented by red bars and black markers, respectively; DNA methylation is expressed as MeDIP enrichment and gene expression levels as normalized microarray signals. The black markers preceding that indicating the neonatal d1 represent gene expression levels in embryonic hearts E16, E18, E19, E20. The middle panels present CpG methylation estimated with Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) where the DNA methylation levels correspond to the amounts of DNA undigested by CpG methylation dependent McrBC enzyme (1-(McrBC/Input). The right panels demonstrate transcript levels determined with qPCR as the ratios to the reference transcript of the Tbp gene (TATA binding protein gene). The microarray results were determined for pooled samples of 3 mice. The qPCR results represent average values obtained for three individuals for each developmental time-point. The statistical significance has been determined with two-tailed heteroscedastic Student’s t -test. The complete results of qPCR validation are collected in Additional file 2 : F2

    Article Snippet: Validation of CpG methylation CpG methylation levels were examined by using Methylation Dependent Restriction Digestion followed by quantitative PCR (MDRE-qPCR) with McrBC restriction endonuclease (NEB, cat. no. M0272).

    Techniques: Real-time Polymerase Chain Reaction, Microarray, DNA Methylation Assay, Expressing, Methylated DNA Immunoprecipitation, CpG Methylation Assay, Methylation, Binding Assay, Mouse Assay, Two Tailed Test