u87mg dna  (New England Biolabs)


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  • 91
    Name:
    Msp I Methyltransferase
    Description:
    Msp I Methyltransferase 100 units
    Catalog Number:
    m0215s
    Price:
    76
    Size:
    100 units
    Category:
    DNA Methylases
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    Structured Review

    New England Biolabs u87mg dna
    Msp I Methyltransferase
    Msp I Methyltransferase 100 units
    https://www.bioz.com/result/u87mg dna/product/New England Biolabs
    Average 91 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    u87mg dna - by Bioz Stars, 2020-02
    91/100 stars

    Images

    1) Product Images from "In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays"

    Article Title: In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays

    Journal: Frontiers in Neuroscience

    doi: 10.3389/neuro.15.005.2009

    Bisulfite sequencing validation results superimposed on MA plots . Bisulfite sequencing results (Table 2 ) were mapped on the corresponding MA plots for normal brain, U87MG and GBM DNA samples. Red dots represent validated methylated fragments (all CCGG sites are methylated in tandem), black dots represent fully unmethylated fragments, and blue dots represent mixed methylation of CCGG sites which are expected to be called unmethylated by MSRE. Light blue dots represent insensitive fragments, and light green dotes represent sensitive fragments. Light red highlighted sensitive fragments have M -values with p ≤ 0.001 and HpaII channel intensity with p ≤ 0.95. Also, the position of MGMT Fragment 2 (265 bp) has been indicated with a blue triangle.
    Figure Legend Snippet: Bisulfite sequencing validation results superimposed on MA plots . Bisulfite sequencing results (Table 2 ) were mapped on the corresponding MA plots for normal brain, U87MG and GBM DNA samples. Red dots represent validated methylated fragments (all CCGG sites are methylated in tandem), black dots represent fully unmethylated fragments, and blue dots represent mixed methylation of CCGG sites which are expected to be called unmethylated by MSRE. Light blue dots represent insensitive fragments, and light green dotes represent sensitive fragments. Light red highlighted sensitive fragments have M -values with p ≤ 0.001 and HpaII channel intensity with p ≤ 0.95. Also, the position of MGMT Fragment 2 (265 bp) has been indicated with a blue triangle.

    Techniques Used: Methylation Sequencing, Methylation

    BfaI fragment coverage of the MGMT CpG island and bisulfite validation . (A) UCSC browser representation of MGMT CpG island showing two MSRE sensitive fragments in gray, predicted CpG island in blue, Agilent probes in green (note partial binding probes *), and methylation-specific PCR primers in red. Fragment 1 is 502 bp and contains 12 CCGG sites. Fragment 2 is 265 bp and contains 3 CCGG sites and binds MSP primers. Fragment 2 contains a portion of exon 1. (B) MSP results of human DNA samples confirm bisulfite sequencing with normal brain showing an unmethylated band, and GBM and U87MG showing a methylated band (as well as a unmethylated band). For controls, methylated SssI-treated DNA (pos.), and unmethylated whole genome amplified DNA (neg.) were analyzed in parallel. (C) Schematic diagram of bisulfite sequencing results on all CpG sites in Fragment 2 of uncloned GBM, U87MG, and normal DNA. Open circles indicate unmethylated CGs, and closed circles indicated methylated CGs. CGs in context of CCGG are enclosed in rectangles. Half-filled circles indicated sites where C peak (methylated) and T peak (unmethylated) from bisulfite sequencing were detected evenly.
    Figure Legend Snippet: BfaI fragment coverage of the MGMT CpG island and bisulfite validation . (A) UCSC browser representation of MGMT CpG island showing two MSRE sensitive fragments in gray, predicted CpG island in blue, Agilent probes in green (note partial binding probes *), and methylation-specific PCR primers in red. Fragment 1 is 502 bp and contains 12 CCGG sites. Fragment 2 is 265 bp and contains 3 CCGG sites and binds MSP primers. Fragment 2 contains a portion of exon 1. (B) MSP results of human DNA samples confirm bisulfite sequencing with normal brain showing an unmethylated band, and GBM and U87MG showing a methylated band (as well as a unmethylated band). For controls, methylated SssI-treated DNA (pos.), and unmethylated whole genome amplified DNA (neg.) were analyzed in parallel. (C) Schematic diagram of bisulfite sequencing results on all CpG sites in Fragment 2 of uncloned GBM, U87MG, and normal DNA. Open circles indicate unmethylated CGs, and closed circles indicated methylated CGs. CGs in context of CCGG are enclosed in rectangles. Half-filled circles indicated sites where C peak (methylated) and T peak (unmethylated) from bisulfite sequencing were detected evenly.

    Techniques Used: Binding Assay, Methylation, Polymerase Chain Reaction, Methylation Sequencing, Amplification

    Comparison of methylation between U87MG, and GBM DNA samples . (A) Estimation of methylated fragments in all samples determined by using p ≤ 0.001 for each sample identifies 3,125 fragments (covering 2,417 CpG Islands). 4,414 fragments (3,321 CpG Islands) were common to both GBM and U87MG, 4,471 fragments (3,452 CpG Islands) common to both GBM and normal, and 3,518 fragments (2,704 CpG Islands) common to both normal and U87MG. (B) Estimation of uniquely methylated fragments using methylation determination based on p ≤ 0.001 and unmethylated determination based on p > 0.01. Fragments within the intermediate region with 0.001 ≤ p ≤ 0.01 are not categorized.
    Figure Legend Snippet: Comparison of methylation between U87MG, and GBM DNA samples . (A) Estimation of methylated fragments in all samples determined by using p ≤ 0.001 for each sample identifies 3,125 fragments (covering 2,417 CpG Islands). 4,414 fragments (3,321 CpG Islands) were common to both GBM and U87MG, 4,471 fragments (3,452 CpG Islands) common to both GBM and normal, and 3,518 fragments (2,704 CpG Islands) common to both normal and U87MG. (B) Estimation of uniquely methylated fragments using methylation determination based on p ≤ 0.001 and unmethylated determination based on p > 0.01. Fragments within the intermediate region with 0.001 ≤ p ≤ 0.01 are not categorized.

    Techniques Used: Methylation

    2) Product Images from "In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays"

    Article Title: In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays

    Journal: Frontiers in Neuroscience

    doi: 10.3389/neuro.15.005.2009

    Bisulfite sequencing validation results superimposed on MA plots . Bisulfite sequencing results (Table 2 ) were mapped on the corresponding MA plots for normal brain, U87MG and GBM DNA samples. Red dots represent validated methylated fragments (all CCGG sites are methylated in tandem), black dots represent fully unmethylated fragments, and blue dots represent mixed methylation of CCGG sites which are expected to be called unmethylated by MSRE. Light blue dots represent insensitive fragments, and light green dotes represent sensitive fragments. Light red highlighted sensitive fragments have M -values with p ≤ 0.001 and HpaII channel intensity with p ≤ 0.95. Also, the position of MGMT Fragment 2 (265 bp) has been indicated with a blue triangle.
    Figure Legend Snippet: Bisulfite sequencing validation results superimposed on MA plots . Bisulfite sequencing results (Table 2 ) were mapped on the corresponding MA plots for normal brain, U87MG and GBM DNA samples. Red dots represent validated methylated fragments (all CCGG sites are methylated in tandem), black dots represent fully unmethylated fragments, and blue dots represent mixed methylation of CCGG sites which are expected to be called unmethylated by MSRE. Light blue dots represent insensitive fragments, and light green dotes represent sensitive fragments. Light red highlighted sensitive fragments have M -values with p ≤ 0.001 and HpaII channel intensity with p ≤ 0.95. Also, the position of MGMT Fragment 2 (265 bp) has been indicated with a blue triangle.

    Techniques Used: Methylation Sequencing, Methylation

    BfaI fragment coverage of the MGMT CpG island and bisulfite validation . (A) UCSC browser representation of MGMT CpG island showing two MSRE sensitive fragments in gray, predicted CpG island in blue, Agilent probes in green (note partial binding probes *), and methylation-specific PCR primers in red. Fragment 1 is 502 bp and contains 12 CCGG sites. Fragment 2 is 265 bp and contains 3 CCGG sites and binds MSP primers. Fragment 2 contains a portion of exon 1. (B) MSP results of human DNA samples confirm bisulfite sequencing with normal brain showing an unmethylated band, and GBM and U87MG showing a methylated band (as well as a unmethylated band). For controls, methylated SssI-treated DNA (pos.), and unmethylated whole genome amplified DNA (neg.) were analyzed in parallel. (C) Schematic diagram of bisulfite sequencing results on all CpG sites in Fragment 2 of uncloned GBM, U87MG, and normal DNA. Open circles indicate unmethylated CGs, and closed circles indicated methylated CGs. CGs in context of CCGG are enclosed in rectangles. Half-filled circles indicated sites where C peak (methylated) and T peak (unmethylated) from bisulfite sequencing were detected evenly.
    Figure Legend Snippet: BfaI fragment coverage of the MGMT CpG island and bisulfite validation . (A) UCSC browser representation of MGMT CpG island showing two MSRE sensitive fragments in gray, predicted CpG island in blue, Agilent probes in green (note partial binding probes *), and methylation-specific PCR primers in red. Fragment 1 is 502 bp and contains 12 CCGG sites. Fragment 2 is 265 bp and contains 3 CCGG sites and binds MSP primers. Fragment 2 contains a portion of exon 1. (B) MSP results of human DNA samples confirm bisulfite sequencing with normal brain showing an unmethylated band, and GBM and U87MG showing a methylated band (as well as a unmethylated band). For controls, methylated SssI-treated DNA (pos.), and unmethylated whole genome amplified DNA (neg.) were analyzed in parallel. (C) Schematic diagram of bisulfite sequencing results on all CpG sites in Fragment 2 of uncloned GBM, U87MG, and normal DNA. Open circles indicate unmethylated CGs, and closed circles indicated methylated CGs. CGs in context of CCGG are enclosed in rectangles. Half-filled circles indicated sites where C peak (methylated) and T peak (unmethylated) from bisulfite sequencing were detected evenly.

    Techniques Used: Binding Assay, Methylation, Polymerase Chain Reaction, Methylation Sequencing, Amplification

    Comparison of methylation between U87MG, and GBM DNA samples . (A) Estimation of methylated fragments in all samples determined by using p ≤ 0.001 for each sample identifies 3,125 fragments (covering 2,417 CpG Islands). 4,414 fragments (3,321 CpG Islands) were common to both GBM and U87MG, 4,471 fragments (3,452 CpG Islands) common to both GBM and normal, and 3,518 fragments (2,704 CpG Islands) common to both normal and U87MG. (B) Estimation of uniquely methylated fragments using methylation determination based on p ≤ 0.001 and unmethylated determination based on p > 0.01. Fragments within the intermediate region with 0.001 ≤ p ≤ 0.01 are not categorized.
    Figure Legend Snippet: Comparison of methylation between U87MG, and GBM DNA samples . (A) Estimation of methylated fragments in all samples determined by using p ≤ 0.001 for each sample identifies 3,125 fragments (covering 2,417 CpG Islands). 4,414 fragments (3,321 CpG Islands) were common to both GBM and U87MG, 4,471 fragments (3,452 CpG Islands) common to both GBM and normal, and 3,518 fragments (2,704 CpG Islands) common to both normal and U87MG. (B) Estimation of uniquely methylated fragments using methylation determination based on p ≤ 0.001 and unmethylated determination based on p > 0.01. Fragments within the intermediate region with 0.001 ≤ p ≤ 0.01 are not categorized.

    Techniques Used: Methylation

    Related Articles

    Diagnostic Assay:

    Article Title: Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose
    Article Snippet: DNA was methylated with MspI methyltransferase (NEB) for at least 4 h according to the manufacturer’s instructions. .. Successful transformation was verified by plasmid rescue followed by a diagnostic restriction digest.

    Centrifugation:

    Article Title: The structure of SgrAI bound to DNA; recognition of an 8 base pair target
    Article Snippet: Protein purification SgrAI restriction endonuclease was expressed in Escherichia coli strain ER2566, from plasmid pET21a_SgrA1R, in the presence of the MspI methyltransferase expressed from the plasmid pBAKMspIM (New England Biolabs). .. Cells were induced to overexpress at an OD600 of 0.5–0.6 with 1 mM isopropyl β-d -thiogalactopyranoside, and incubated for further 4 h, then harvested by centrifugation.

    Article Title: Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose ▿
    Article Snippet: The DNA was methylated overnight with 5 units of MspI methyltransferase (New England BioLabs, Ipswich, MA) and then purified with the DNA Clean and Concentrator kit (Zymo Research Inc., Orange, CA). .. The cells were recovered for 24 h at 34°C, then the cells were collected by centrifugation, and cell pellet was spread on modified VM cellobiose agar plates supplemented with 10 μg/ml erythromycin.

    Amplification:

    Article Title: Phase II Study of Bevacizumab Plus Temozolomide During and After Radiation Therapy for Patients With Newly Diagnosed Glioblastoma Multiforme
    Article Snippet: .. Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscataway, NJ), respectively. .. See accompanying editorial on page Supported in part by Genentech, American Brain Tumor Association/Mark Linder Small Project Grant, and Grant No. NIH/NCI K08CA124479 from the National Institutes of Health.

    Article Title: In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays
    Article Snippet: .. Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (NEB, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, NJ, USA), respectively. .. Bisulfite sequencing validation Primers were designed with the assistance of the online tool MethPrimer (Li and Dahiya, ).

    Article Title: Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage
    Article Snippet: .. Samples were subjected to a two-stage nested PCR strategy using: first-stage primers (5′GGATAT GTTGGGATAGTT-3′ and 5′-CCAAAAACCCCAA ACCC-3′) and second-stage primers (unmethylated reaction: 5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′ and 5′-AACTCCACACTCTTCCAAAAACAAAACA-3′; methylated reaction: 5′-TTTCGACGTTCGTAGGTTT TCGC-3′ and 5′-CACTCTTCCGAAAACGAAACG-3′).Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscat-away, NJ, USA). .. RT-PCR was performed using an Illustra Ready-to-Go RT-PCR kit (GE Healthcare, Little Chalfont, UK).

    Whole Genome Amplification:

    Article Title: Phase II Study of Bevacizumab Plus Temozolomide During and After Radiation Therapy for Patients With Newly Diagnosed Glioblastoma Multiforme
    Article Snippet: .. Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscataway, NJ), respectively. .. See accompanying editorial on page Supported in part by Genentech, American Brain Tumor Association/Mark Linder Small Project Grant, and Grant No. NIH/NCI K08CA124479 from the National Institutes of Health.

    Article Title: In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays
    Article Snippet: .. Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (NEB, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, NJ, USA), respectively. .. Bisulfite sequencing validation Primers were designed with the assistance of the online tool MethPrimer (Li and Dahiya, ).

    Article Title: Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage
    Article Snippet: .. Samples were subjected to a two-stage nested PCR strategy using: first-stage primers (5′GGATAT GTTGGGATAGTT-3′ and 5′-CCAAAAACCCCAA ACCC-3′) and second-stage primers (unmethylated reaction: 5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′ and 5′-AACTCCACACTCTTCCAAAAACAAAACA-3′; methylated reaction: 5′-TTTCGACGTTCGTAGGTTT TCGC-3′ and 5′-CACTCTTCCGAAAACGAAACG-3′).Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscat-away, NJ, USA). .. RT-PCR was performed using an Illustra Ready-to-Go RT-PCR kit (GE Healthcare, Little Chalfont, UK).

    Incubation:

    Article Title: The structure of SgrAI bound to DNA; recognition of an 8 base pair target
    Article Snippet: Protein purification SgrAI restriction endonuclease was expressed in Escherichia coli strain ER2566, from plasmid pET21a_SgrA1R, in the presence of the MspI methyltransferase expressed from the plasmid pBAKMspIM (New England Biolabs). .. Cells were induced to overexpress at an OD600 of 0.5–0.6 with 1 mM isopropyl β-d -thiogalactopyranoside, and incubated for further 4 h, then harvested by centrifugation.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Phase II Study of Bevacizumab Plus Temozolomide During and After Radiation Therapy for Patients With Newly Diagnosed Glioblastoma Multiforme
    Article Snippet: To generate bisulfite modified DNA, genomic DNA isolated from formalin-fixed, paraffin-embedded using Recoverall Total Nucleic Acid Isolation Kit (Ambion, Austin, TX) was modified using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA) following the manufacturer's protocol. .. Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscataway, NJ), respectively.

    Mass Spectrometry:

    Article Title: Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations
    Article Snippet: The plasmid DNA was treated with MspI methyltransferase (New England Biolabs, Ipswich, MA) for 3 h, followed by purification with the DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA). .. The voltage was 1.5 kV, and the time constant was 5 ms.

    Article Title: Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum
    Article Snippet: Plasmid DNA was methylated with MspI methyltransferase (NEB), followed by DNA purification and quantification. .. For each transformation, a 100 μl cell suspension was mixed with 2.0 μg of methylated plasmids and then electroporated in a 2-mm cuvette (1.25 kV, 5 ms, 1 square pulse) with a Gene Pulser Xcell (Bio-Rad) in the anaerobic chamber.

    Modification:

    Article Title: Phase II Study of Bevacizumab Plus Temozolomide During and After Radiation Therapy for Patients With Newly Diagnosed Glioblastoma Multiforme
    Article Snippet: To generate bisulfite modified DNA, genomic DNA isolated from formalin-fixed, paraffin-embedded using Recoverall Total Nucleic Acid Isolation Kit (Ambion, Austin, TX) was modified using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA) following the manufacturer's protocol. .. Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscataway, NJ), respectively.

    Article Title: Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose ▿
    Article Snippet: Cells were grown for 17 to 24 h in 10-ml cultures of modified VM medium to late exponential phase (optical density at 600 nm [OD600 ], 0.5 to 1.0; 5 × 106 CFU/ml). .. The DNA was methylated overnight with 5 units of MspI methyltransferase (New England BioLabs, Ipswich, MA) and then purified with the DNA Clean and Concentrator kit (Zymo Research Inc., Orange, CA).

    Article Title: Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations
    Article Snippet: Cells were grown in complex modified VM medium containing 5 g/l cellobiose and 2 g/l yeast extract for 17 to 24 hours to reach early to middle log phase (optical density at 600 nm of 0.3 to 0.5). .. The plasmid DNA was treated with MspI methyltransferase (New England Biolabs, Ipswich, MA) for 3 h, followed by purification with the DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA).

    Article Title: Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage
    Article Snippet: To generate bisulfite modified DNA, genomic DNA was modified with the EZ DNA Methylation-Gold Kit (ZymoResearch, Orange, CA, USA). .. Samples were subjected to a two-stage nested PCR strategy using: first-stage primers (5′GGATAT GTTGGGATAGTT-3′ and 5′-CCAAAAACCCCAA ACCC-3′) and second-stage primers (unmethylated reaction: 5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′ and 5′-AACTCCACACTCTTCCAAAAACAAAACA-3′; methylated reaction: 5′-TTTCGACGTTCGTAGGTTT TCGC-3′ and 5′-CACTCTTCCGAAAACGAAACG-3′).Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscat-away, NJ, USA).

    Transformation Assay:

    Article Title: Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose
    Article Snippet: Paragraph title: Transformation of C. cellulolyticum ... DNA was methylated with MspI methyltransferase (NEB) for at least 4 h according to the manufacturer’s instructions.

    Article Title: Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose ▿
    Article Snippet: Paragraph title: Transformation. ... The DNA was methylated overnight with 5 units of MspI methyltransferase (New England BioLabs, Ipswich, MA) and then purified with the DNA Clean and Concentrator kit (Zymo Research Inc., Orange, CA).

    Article Title: Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations
    Article Snippet: Paragraph title: Transformation ... The plasmid DNA was treated with MspI methyltransferase (New England Biolabs, Ipswich, MA) for 3 h, followed by purification with the DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA).

    Article Title: Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum
    Article Snippet: Paragraph title: C. cellulolyticum Transformation ... Plasmid DNA was methylated with MspI methyltransferase (NEB), followed by DNA purification and quantification.

    Electroporation:

    Article Title: Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose
    Article Snippet: The competent cells were mixed with 1–5 µg of in vitro methylated DNA and transferred to a 0.2-mm gap cuvette (BioRad, Hercules, CA, USA) before electroporation with a BioRad Micropulser set at 1.5 kV. .. DNA was methylated with MspI methyltransferase (NEB) for at least 4 h according to the manufacturer’s instructions.

    Article Title: Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose ▿
    Article Snippet: The cells were resuspended in 600 μl of electroporation buffer. .. The DNA was methylated overnight with 5 units of MspI methyltransferase (New England BioLabs, Ipswich, MA) and then purified with the DNA Clean and Concentrator kit (Zymo Research Inc., Orange, CA).

    Article Title: Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations
    Article Snippet: The washed cells were resuspended in electroporation buffer and stored on ice. .. The plasmid DNA was treated with MspI methyltransferase (New England Biolabs, Ipswich, MA) for 3 h, followed by purification with the DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA).

    Article Title: Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum
    Article Snippet: Lastly, competent cells made from every 10 ml of cell culture were resuspended in 100 μl chilled electroporation buffer for further use. .. Plasmid DNA was methylated with MspI methyltransferase (NEB), followed by DNA purification and quantification.

    Chromatography:

    Article Title: Activation of DNA Cleavage by Oligomerization of DNA Bound SgrAI
    Article Snippet: Briefly, SgrAI was expressed in E. coli strain ER2566 in the presence of the MspI methyltransferase (New England Biolabs). .. The enzyme was purified using FPLC (GE Healthcare Biosciences) chromatography and the following chromatographic resins: Heparin FF Sepharose (Pharmacia), SP FF Sepharose (GE Healthcare Biosciences), Q FF Sepharose (GE Healthcare Biosciences), and then a second Heparin FF Sepharose (GE Healthcare Biosciences) chromatographic step.

    Article Title: Domain Swapping in Allosteric Modulation of DNA Specificity
    Article Snippet: Briefly, the enzymes were expressed in E. coli strain ER2566 in the presence of the MspI methyltransferase (New England Biolabs). .. The enzymes were purified using FPLC (Pharmacia) chromatography and the following chromatographic resins: Heparin FF Sepharose (Pharmacia), SP FF Sepharose (Pharmacia), Q FF Sepharose (Pharmacia), and then a second Heparin FF Sepharose (Pharmacia) chromatographic step.

    Cell Culture:

    Article Title: Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum
    Article Snippet: Lastly, competent cells made from every 10 ml of cell culture were resuspended in 100 μl chilled electroporation buffer for further use. .. Plasmid DNA was methylated with MspI methyltransferase (NEB), followed by DNA purification and quantification.

    Polymerase Chain Reaction:

    Article Title: Phase II Study of Bevacizumab Plus Temozolomide During and After Radiation Therapy for Patients With Newly Diagnosed Glioblastoma Multiforme
    Article Snippet: PCR products were analyzed on 3% agarose gels. .. Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscataway, NJ), respectively.

    Article Title: Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage
    Article Snippet: MGMT methylation analysis was performed by methylation-specific PCR (MSP) according to a previously published protocol with slight modifications [ ]. .. Samples were subjected to a two-stage nested PCR strategy using: first-stage primers (5′GGATAT GTTGGGATAGTT-3′ and 5′-CCAAAAACCCCAA ACCC-3′) and second-stage primers (unmethylated reaction: 5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′ and 5′-AACTCCACACTCTTCCAAAAACAAAACA-3′; methylated reaction: 5′-TTTCGACGTTCGTAGGTTT TCGC-3′ and 5′-CACTCTTCCGAAAACGAAACG-3′).Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscat-away, NJ, USA).

    Sonication:

    Article Title: The structure of SgrAI bound to DNA; recognition of an 8 base pair target
    Article Snippet: Protein purification SgrAI restriction endonuclease was expressed in Escherichia coli strain ER2566, from plasmid pET21a_SgrA1R, in the presence of the MspI methyltransferase expressed from the plasmid pBAKMspIM (New England Biolabs). .. Cells were resuspended in 20 mM potassium phosphate buffer (pH 7.0), 50 mM NaCl, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 5% v/v glycerol, 50 μM PMSF and 100 μM benzamide, then lysed on ice by sonication.

    Methylation:

    Article Title: Phase II Study of Bevacizumab Plus Temozolomide During and After Radiation Therapy for Patients With Newly Diagnosed Glioblastoma Multiforme
    Article Snippet: Paragraph title: MGMT methylation analysis. ... Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscataway, NJ), respectively.

    Article Title: Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose
    Article Snippet: .. DNA was methylated with MspI methyltransferase (NEB) for at least 4 h according to the manufacturer’s instructions. ..

    Article Title: Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose ▿
    Article Snippet: .. The DNA was methylated overnight with 5 units of MspI methyltransferase (New England BioLabs, Ipswich, MA) and then purified with the DNA Clean and Concentrator kit (Zymo Research Inc., Orange, CA). .. In 2-mm-gap electroporation cuvettes (Molecular BioProducts, San Diego, CA), the cells and plasmid DNA were electroporated (1.5 kV, 25 μF, and 48 Ω) with a Bio-Rad gene pulser apparatus (Bio-Rad Laboratories, Richmond, CA).

    Article Title: In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays
    Article Snippet: Paragraph title: Methylation-specific (MSP) ... Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (NEB, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, NJ, USA), respectively.

    Article Title: Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations
    Article Snippet: The plasmid DNA was treated with MspI methyltransferase (New England Biolabs, Ipswich, MA) for 3 h, followed by purification with the DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA). .. For each transformation, a 50-μl cell suspension was mixed with 2.0 μg of methylated plasmid DNA.

    Article Title: Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum
    Article Snippet: .. Plasmid DNA was methylated with MspI methyltransferase (NEB), followed by DNA purification and quantification. .. For each transformation, a 100 μl cell suspension was mixed with 2.0 μg of methylated plasmids and then electroporated in a 2-mm cuvette (1.25 kV, 5 ms, 1 square pulse) with a Gene Pulser Xcell (Bio-Rad) in the anaerobic chamber.

    Article Title: Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage
    Article Snippet: .. Samples were subjected to a two-stage nested PCR strategy using: first-stage primers (5′GGATAT GTTGGGATAGTT-3′ and 5′-CCAAAAACCCCAA ACCC-3′) and second-stage primers (unmethylated reaction: 5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′ and 5′-AACTCCACACTCTTCCAAAAACAAAACA-3′; methylated reaction: 5′-TTTCGACGTTCGTAGGTTT TCGC-3′ and 5′-CACTCTTCCGAAAACGAAACG-3′).Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscat-away, NJ, USA). .. RT-PCR was performed using an Illustra Ready-to-Go RT-PCR kit (GE Healthcare, Little Chalfont, UK).

    Isolation:

    Article Title: Phase II Study of Bevacizumab Plus Temozolomide During and After Radiation Therapy for Patients With Newly Diagnosed Glioblastoma Multiforme
    Article Snippet: To generate bisulfite modified DNA, genomic DNA isolated from formalin-fixed, paraffin-embedded using Recoverall Total Nucleic Acid Isolation Kit (Ambion, Austin, TX) was modified using the EZ DNA Methylation-Gold Kit (Zymo Research, Orange, CA) following the manufacturer's protocol. .. Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscataway, NJ), respectively.

    Purification:

    Article Title: Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose ▿
    Article Snippet: .. The DNA was methylated overnight with 5 units of MspI methyltransferase (New England BioLabs, Ipswich, MA) and then purified with the DNA Clean and Concentrator kit (Zymo Research Inc., Orange, CA). .. In 2-mm-gap electroporation cuvettes (Molecular BioProducts, San Diego, CA), the cells and plasmid DNA were electroporated (1.5 kV, 25 μF, and 48 Ω) with a Bio-Rad gene pulser apparatus (Bio-Rad Laboratories, Richmond, CA).

    Article Title: Activation of DNA Cleavage by Oligomerization of DNA Bound SgrAI
    Article Snippet: Briefly, SgrAI was expressed in E. coli strain ER2566 in the presence of the MspI methyltransferase (New England Biolabs). .. The enzyme was purified using FPLC (GE Healthcare Biosciences) chromatography and the following chromatographic resins: Heparin FF Sepharose (Pharmacia), SP FF Sepharose (GE Healthcare Biosciences), Q FF Sepharose (GE Healthcare Biosciences), and then a second Heparin FF Sepharose (GE Healthcare Biosciences) chromatographic step.

    Article Title: Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations
    Article Snippet: .. The plasmid DNA was treated with MspI methyltransferase (New England Biolabs, Ipswich, MA) for 3 h, followed by purification with the DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA). .. For each transformation, a 50-μl cell suspension was mixed with 2.0 μg of methylated plasmid DNA.

    Article Title: Domain Swapping in Allosteric Modulation of DNA Specificity
    Article Snippet: Briefly, the enzymes were expressed in E. coli strain ER2566 in the presence of the MspI methyltransferase (New England Biolabs). .. The enzymes were purified using FPLC (Pharmacia) chromatography and the following chromatographic resins: Heparin FF Sepharose (Pharmacia), SP FF Sepharose (Pharmacia), Q FF Sepharose (Pharmacia), and then a second Heparin FF Sepharose (Pharmacia) chromatographic step.

    Protein Purification:

    Article Title: The structure of SgrAI bound to DNA; recognition of an 8 base pair target
    Article Snippet: .. Protein purification SgrAI restriction endonuclease was expressed in Escherichia coli strain ER2566, from plasmid pET21a_SgrA1R, in the presence of the MspI methyltransferase expressed from the plasmid pBAKMspIM (New England Biolabs). ..

    Article Title: Activation of DNA Cleavage by Oligomerization of DNA Bound SgrAI
    Article Snippet: Paragraph title: Protein Purification ... Briefly, SgrAI was expressed in E. coli strain ER2566 in the presence of the MspI methyltransferase (New England Biolabs).

    Article Title: Domain Swapping in Allosteric Modulation of DNA Specificity
    Article Snippet: Paragraph title: Protein Purification ... Briefly, the enzymes were expressed in E. coli strain ER2566 in the presence of the MspI methyltransferase (New England Biolabs).

    Fast Protein Liquid Chromatography:

    Article Title: Activation of DNA Cleavage by Oligomerization of DNA Bound SgrAI
    Article Snippet: Briefly, SgrAI was expressed in E. coli strain ER2566 in the presence of the MspI methyltransferase (New England Biolabs). .. The enzyme was purified using FPLC (GE Healthcare Biosciences) chromatography and the following chromatographic resins: Heparin FF Sepharose (Pharmacia), SP FF Sepharose (GE Healthcare Biosciences), Q FF Sepharose (GE Healthcare Biosciences), and then a second Heparin FF Sepharose (GE Healthcare Biosciences) chromatographic step.

    Article Title: Domain Swapping in Allosteric Modulation of DNA Specificity
    Article Snippet: Briefly, the enzymes were expressed in E. coli strain ER2566 in the presence of the MspI methyltransferase (New England Biolabs). .. The enzymes were purified using FPLC (Pharmacia) chromatography and the following chromatographic resins: Heparin FF Sepharose (Pharmacia), SP FF Sepharose (Pharmacia), Q FF Sepharose (Pharmacia), and then a second Heparin FF Sepharose (Pharmacia) chromatographic step.

    Nested PCR:

    Article Title: Phase II Study of Bevacizumab Plus Temozolomide During and After Radiation Therapy for Patients With Newly Diagnosed Glioblastoma Multiforme
    Article Snippet: Samples were subjected to a two-stage nested PCR strategy using: first-stage primers (5′-GGATATGTTGGGATAGTT-3′ and 5′-CCAAAAACCCCAAACCC-3′) and second-stage primers (unmethylated reaction: 5′-TTTGTGTTTTGATGTTTGTA-GGTTTTTGT-3′ and 5′-AACTCCACACTCTTCCAAAAACAAAACA-3′; methylated reaction: 5′-TTTCGACGTTCGTAGGTTTTCGC-3′ and 5′-GCACTCTTCCGAAAACGAA-ACG-3′). .. Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscataway, NJ), respectively.

    Article Title: Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage
    Article Snippet: .. Samples were subjected to a two-stage nested PCR strategy using: first-stage primers (5′GGATAT GTTGGGATAGTT-3′ and 5′-CCAAAAACCCCAA ACCC-3′) and second-stage primers (unmethylated reaction: 5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′ and 5′-AACTCCACACTCTTCCAAAAACAAAACA-3′; methylated reaction: 5′-TTTCGACGTTCGTAGGTTT TCGC-3′ and 5′-CACTCTTCCGAAAACGAAACG-3′).Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscat-away, NJ, USA). .. RT-PCR was performed using an Illustra Ready-to-Go RT-PCR kit (GE Healthcare, Little Chalfont, UK).

    Plasmid Preparation:

    Article Title: Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose
    Article Snippet: DNA was methylated with MspI methyltransferase (NEB) for at least 4 h according to the manufacturer’s instructions. .. Successful transformation was verified by plasmid rescue followed by a diagnostic restriction digest.

    Article Title: The structure of SgrAI bound to DNA; recognition of an 8 base pair target
    Article Snippet: .. Protein purification SgrAI restriction endonuclease was expressed in Escherichia coli strain ER2566, from plasmid pET21a_SgrA1R, in the presence of the MspI methyltransferase expressed from the plasmid pBAKMspIM (New England Biolabs). ..

    Article Title: Metabolic Engineering of Clostridium cellulolyticum for Production of Isobutanol from Cellulose ▿
    Article Snippet: For each transformation, 200 μl of the cells was mixed with 2 μg of MspI-methylated plasmid DNA. .. The DNA was methylated overnight with 5 units of MspI methyltransferase (New England BioLabs, Ipswich, MA) and then purified with the DNA Clean and Concentrator kit (Zymo Research Inc., Orange, CA).

    Article Title: Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations
    Article Snippet: .. The plasmid DNA was treated with MspI methyltransferase (New England Biolabs, Ipswich, MA) for 3 h, followed by purification with the DNA Clean and Concentrator Kit (Zymo Research, Irvine, CA). .. For each transformation, a 50-μl cell suspension was mixed with 2.0 μg of methylated plasmid DNA.

    Article Title: Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum
    Article Snippet: .. Plasmid DNA was methylated with MspI methyltransferase (NEB), followed by DNA purification and quantification. .. For each transformation, a 100 μl cell suspension was mixed with 2.0 μg of methylated plasmids and then electroporated in a 2-mm cuvette (1.25 kV, 5 ms, 1 square pulse) with a Gene Pulser Xcell (Bio-Rad) in the anaerobic chamber.

    Negative Control:

    Article Title: Phase II Study of Bevacizumab Plus Temozolomide During and After Radiation Therapy for Patients With Newly Diagnosed Glioblastoma Multiforme
    Article Snippet: .. Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscataway, NJ), respectively. .. See accompanying editorial on page Supported in part by Genentech, American Brain Tumor Association/Mark Linder Small Project Grant, and Grant No. NIH/NCI K08CA124479 from the National Institutes of Health.

    Article Title: In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays
    Article Snippet: .. Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (NEB, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, NJ, USA), respectively. .. Bisulfite sequencing validation Primers were designed with the assistance of the online tool MethPrimer (Li and Dahiya, ).

    Article Title: Lyophilized brain tumor specimens can be used for histologic, nucleic acid, and protein analyses after 1 year of room temperature storage
    Article Snippet: .. Samples were subjected to a two-stage nested PCR strategy using: first-stage primers (5′GGATAT GTTGGGATAGTT-3′ and 5′-CCAAAAACCCCAA ACCC-3′) and second-stage primers (unmethylated reaction: 5′-TTTGTGTTTTGATGTTTGTAGGTTTTTGT-3′ and 5′-AACTCCACACTCTTCCAAAAACAAAACA-3′; methylated reaction: 5′-TTTCGACGTTCGTAGGTTT TCGC-3′ and 5′-CACTCTTCCGAAAACGAAACG-3′).Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (New England Biolabs, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, Piscat-away, NJ, USA). .. RT-PCR was performed using an Illustra Ready-to-Go RT-PCR kit (GE Healthcare, Little Chalfont, UK).

    In Vitro:

    Article Title: Metabolic engineering of Clostridium cellulolyticum for the production of n-butanol from crystalline cellulose
    Article Snippet: The competent cells were mixed with 1–5 µg of in vitro methylated DNA and transferred to a 0.2-mm gap cuvette (BioRad, Hercules, CA, USA) before electroporation with a BioRad Micropulser set at 1.5 kV. .. DNA was methylated with MspI methyltransferase (NEB) for at least 4 h according to the manufacturer’s instructions.

    DNA Purification:

    Article Title: Cas9 Nickase-Assisted RNA Repression Enables Stable and Efficient Manipulation of Essential Metabolic Genes in Clostridium cellulolyticum
    Article Snippet: .. Plasmid DNA was methylated with MspI methyltransferase (NEB), followed by DNA purification and quantification. .. For each transformation, a 100 μl cell suspension was mixed with 2.0 μg of methylated plasmids and then electroporated in a 2-mm cuvette (1.25 kV, 5 ms, 1 square pulse) with a Gene Pulser Xcell (Bio-Rad) in the anaerobic chamber.

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    New England Biolabs u87mg dna
    Bisulfite sequencing validation results superimposed on MA plots . Bisulfite sequencing results (Table 2 ) were mapped on the corresponding MA plots for normal brain, <t>U87MG</t> and GBM <t>DNA</t> samples. Red dots represent validated methylated fragments (all CCGG sites are methylated in tandem), black dots represent fully unmethylated fragments, and blue dots represent mixed methylation of CCGG sites which are expected to be called unmethylated by MSRE. Light blue dots represent insensitive fragments, and light green dotes represent sensitive fragments. Light red highlighted sensitive fragments have M -values with p ≤ 0.001 and HpaII channel intensity with p ≤ 0.95. Also, the position of MGMT Fragment 2 (265 bp) has been indicated with a blue triangle.
    U87mg Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bisulfite sequencing validation results superimposed on MA plots . Bisulfite sequencing results (Table 2 ) were mapped on the corresponding MA plots for normal brain, U87MG and GBM DNA samples. Red dots represent validated methylated fragments (all CCGG sites are methylated in tandem), black dots represent fully unmethylated fragments, and blue dots represent mixed methylation of CCGG sites which are expected to be called unmethylated by MSRE. Light blue dots represent insensitive fragments, and light green dotes represent sensitive fragments. Light red highlighted sensitive fragments have M -values with p ≤ 0.001 and HpaII channel intensity with p ≤ 0.95. Also, the position of MGMT Fragment 2 (265 bp) has been indicated with a blue triangle.

    Journal: Frontiers in Neuroscience

    Article Title: In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays

    doi: 10.3389/neuro.15.005.2009

    Figure Lengend Snippet: Bisulfite sequencing validation results superimposed on MA plots . Bisulfite sequencing results (Table 2 ) were mapped on the corresponding MA plots for normal brain, U87MG and GBM DNA samples. Red dots represent validated methylated fragments (all CCGG sites are methylated in tandem), black dots represent fully unmethylated fragments, and blue dots represent mixed methylation of CCGG sites which are expected to be called unmethylated by MSRE. Light blue dots represent insensitive fragments, and light green dotes represent sensitive fragments. Light red highlighted sensitive fragments have M -values with p ≤ 0.001 and HpaII channel intensity with p ≤ 0.95. Also, the position of MGMT Fragment 2 (265 bp) has been indicated with a blue triangle.

    Article Snippet: Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (NEB, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, NJ, USA), respectively.

    Techniques: Methylation Sequencing, Methylation

    BfaI fragment coverage of the MGMT CpG island and bisulfite validation . (A) UCSC browser representation of MGMT CpG island showing two MSRE sensitive fragments in gray, predicted CpG island in blue, Agilent probes in green (note partial binding probes *), and methylation-specific PCR primers in red. Fragment 1 is 502 bp and contains 12 CCGG sites. Fragment 2 is 265 bp and contains 3 CCGG sites and binds MSP primers. Fragment 2 contains a portion of exon 1. (B) MSP results of human DNA samples confirm bisulfite sequencing with normal brain showing an unmethylated band, and GBM and U87MG showing a methylated band (as well as a unmethylated band). For controls, methylated SssI-treated DNA (pos.), and unmethylated whole genome amplified DNA (neg.) were analyzed in parallel. (C) Schematic diagram of bisulfite sequencing results on all CpG sites in Fragment 2 of uncloned GBM, U87MG, and normal DNA. Open circles indicate unmethylated CGs, and closed circles indicated methylated CGs. CGs in context of CCGG are enclosed in rectangles. Half-filled circles indicated sites where C peak (methylated) and T peak (unmethylated) from bisulfite sequencing were detected evenly.

    Journal: Frontiers in Neuroscience

    Article Title: In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays

    doi: 10.3389/neuro.15.005.2009

    Figure Lengend Snippet: BfaI fragment coverage of the MGMT CpG island and bisulfite validation . (A) UCSC browser representation of MGMT CpG island showing two MSRE sensitive fragments in gray, predicted CpG island in blue, Agilent probes in green (note partial binding probes *), and methylation-specific PCR primers in red. Fragment 1 is 502 bp and contains 12 CCGG sites. Fragment 2 is 265 bp and contains 3 CCGG sites and binds MSP primers. Fragment 2 contains a portion of exon 1. (B) MSP results of human DNA samples confirm bisulfite sequencing with normal brain showing an unmethylated band, and GBM and U87MG showing a methylated band (as well as a unmethylated band). For controls, methylated SssI-treated DNA (pos.), and unmethylated whole genome amplified DNA (neg.) were analyzed in parallel. (C) Schematic diagram of bisulfite sequencing results on all CpG sites in Fragment 2 of uncloned GBM, U87MG, and normal DNA. Open circles indicate unmethylated CGs, and closed circles indicated methylated CGs. CGs in context of CCGG are enclosed in rectangles. Half-filled circles indicated sites where C peak (methylated) and T peak (unmethylated) from bisulfite sequencing were detected evenly.

    Article Snippet: Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (NEB, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, NJ, USA), respectively.

    Techniques: Binding Assay, Methylation, Polymerase Chain Reaction, Methylation Sequencing, Amplification

    Comparison of methylation between U87MG, and GBM DNA samples . (A) Estimation of methylated fragments in all samples determined by using p ≤ 0.001 for each sample identifies 3,125 fragments (covering 2,417 CpG Islands). 4,414 fragments (3,321 CpG Islands) were common to both GBM and U87MG, 4,471 fragments (3,452 CpG Islands) common to both GBM and normal, and 3,518 fragments (2,704 CpG Islands) common to both normal and U87MG. (B) Estimation of uniquely methylated fragments using methylation determination based on p ≤ 0.001 and unmethylated determination based on p > 0.01. Fragments within the intermediate region with 0.001 ≤ p ≤ 0.01 are not categorized.

    Journal: Frontiers in Neuroscience

    Article Title: In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays

    doi: 10.3389/neuro.15.005.2009

    Figure Lengend Snippet: Comparison of methylation between U87MG, and GBM DNA samples . (A) Estimation of methylated fragments in all samples determined by using p ≤ 0.001 for each sample identifies 3,125 fragments (covering 2,417 CpG Islands). 4,414 fragments (3,321 CpG Islands) were common to both GBM and U87MG, 4,471 fragments (3,452 CpG Islands) common to both GBM and normal, and 3,518 fragments (2,704 CpG Islands) common to both normal and U87MG. (B) Estimation of uniquely methylated fragments using methylation determination based on p ≤ 0.001 and unmethylated determination based on p > 0.01. Fragments within the intermediate region with 0.001 ≤ p ≤ 0.01 are not categorized.

    Article Snippet: Positive and negative control samples for the MSP reaction were U87MG DNA treated with SssI methyltransferase (NEB, Ipswich, MA, USA) and whole-genome amplification of U87MG DNA using the GenomiPhi V2 Amplification kit (Amersham Biosciences, NJ, USA), respectively.

    Techniques: Methylation