mspji  (New England Biolabs)


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    Name:
    MspJI
    Description:
    MspJI 1 000 units
    Catalog Number:
    r0661l
    Price:
    450
    Size:
    1 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs mspji
    MspJI
    MspJI 1 000 units
    https://www.bioz.com/result/mspji/product/New England Biolabs
    Average 95 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    mspji - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI"

    Article Title: Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI

    Journal: Genome Research

    doi: 10.1101/gr.222885.117

    MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).
    Figure Legend Snippet: MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).

    Techniques Used: Ligation, Fractionation, Amplification, Sequencing

    2) Product Images from "Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9"

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    Journal: mBio

    doi: 10.1128/mBio.00648-15

    Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.
    Figure Legend Snippet: Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Techniques Used: Modification, Mobility Shift, Sequencing, Methylation

    3) Product Images from "A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI"

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    Journal: BMC Biotechnology

    doi: 10.1186/s12896-015-0139-7

    BAC clone sequencing based on the MspJI-based DNA fragmentation approach. ( a ) Amplified BAC DNA directly from glycerol stocks, using the Φ29 enzyme. In the amplification solution, 30 μM 5-methyl-dCTP were included. ( b ) Resequencing results of the BAC clones. Read coverage depth for each clone was visualised on the Tablet software.
    Figure Legend Snippet: BAC clone sequencing based on the MspJI-based DNA fragmentation approach. ( a ) Amplified BAC DNA directly from glycerol stocks, using the Φ29 enzyme. In the amplification solution, 30 μM 5-methyl-dCTP were included. ( b ) Resequencing results of the BAC clones. Read coverage depth for each clone was visualised on the Tablet software.

    Techniques Used: BAC Assay, Sequencing, Amplification, Clone Assay, Software

    Illumina MiSeq short read-sequencing results of the libraries constructed from MspJI-digested and physically sheared DNA. The sorted alignment was visualised using the Tablet viewers. ( a ) Alignment results of Φ29 enzyme-amplified Agro gDNA-derived reads and physically sheared Agro gDNA-derived reads to the reference Agrobacterium genome sequences for the 1,000-1,250 and 2,000-2250 kb regions of the circular and linear chromosomes. ( b ) Alignment results of the Arabidopsis genome with the MspJI-enzymatic fragmentation and physical shearing methods. Read coverage depth for the 10,000-10,250, 20,000-20,250 and 30,000-30,250 kb regions of chromosome 1 was visualised.
    Figure Legend Snippet: Illumina MiSeq short read-sequencing results of the libraries constructed from MspJI-digested and physically sheared DNA. The sorted alignment was visualised using the Tablet viewers. ( a ) Alignment results of Φ29 enzyme-amplified Agro gDNA-derived reads and physically sheared Agro gDNA-derived reads to the reference Agrobacterium genome sequences for the 1,000-1,250 and 2,000-2250 kb regions of the circular and linear chromosomes. ( b ) Alignment results of the Arabidopsis genome with the MspJI-enzymatic fragmentation and physical shearing methods. Read coverage depth for the 10,000-10,250, 20,000-20,250 and 30,000-30,250 kb regions of chromosome 1 was visualised.

    Techniques Used: Sequencing, Construct, Amplification, Derivative Assay

    Short read-sequencing results of the libraries constructed from MspJI, FspEI and LpnPI-digested DNA. Read coverage depth was visualised using the Tablet viewer.
    Figure Legend Snippet: Short read-sequencing results of the libraries constructed from MspJI, FspEI and LpnPI-digested DNA. Read coverage depth was visualised using the Tablet viewer.

    Techniques Used: Sequencing, Construct

    Sequencing of the fungal endophyte genome with the MspJI-based DNA fragmentation method. Two independent experiments (rep1 and rep2) were performed using the single endophyte strain. ( a ) WGA of the endophyte genome with the Φ29 enzyme in the presence of 5-methyl-dCTP (X μM). ( b ) MspJI digestion of the endophyte genome-derived amplicons. ( c ) Flowchart of the MspJI- and physical shearing-based library prep procedures from WGA products.
    Figure Legend Snippet: Sequencing of the fungal endophyte genome with the MspJI-based DNA fragmentation method. Two independent experiments (rep1 and rep2) were performed using the single endophyte strain. ( a ) WGA of the endophyte genome with the Φ29 enzyme in the presence of 5-methyl-dCTP (X μM). ( b ) MspJI digestion of the endophyte genome-derived amplicons. ( c ) Flowchart of the MspJI- and physical shearing-based library prep procedures from WGA products.

    Techniques Used: Sequencing, Whole Genome Amplification, Derivative Assay

    MspJI-enzymatic digestion of 5 m C-containing PCR and Φ29 products. ( a ) DNA fragments amplified with the locus-specific PCR primers for the Agro _gc50 sequence under the presence of 5-methyl-dCTP (0, 2, 4 or 8 μM). ( b ) MspJI-digested DNA fragments derived from PCR products with each locus-specific primers and Agro gDNA as DNA template. Molar concentration denotes the 5-methyl-dCTP-concentration in PCR solution. ( c ) MspJI-enzymatic digestion of Φ29 enzyme-amplified DNA with randomly incorporated 5 m C from a range of DNA templates. 0, 10, 15 and 20 μM denote final concentrations of 5-methyl-dCTP in the REPLI-g WGA mixture.
    Figure Legend Snippet: MspJI-enzymatic digestion of 5 m C-containing PCR and Φ29 products. ( a ) DNA fragments amplified with the locus-specific PCR primers for the Agro _gc50 sequence under the presence of 5-methyl-dCTP (0, 2, 4 or 8 μM). ( b ) MspJI-digested DNA fragments derived from PCR products with each locus-specific primers and Agro gDNA as DNA template. Molar concentration denotes the 5-methyl-dCTP-concentration in PCR solution. ( c ) MspJI-enzymatic digestion of Φ29 enzyme-amplified DNA with randomly incorporated 5 m C from a range of DNA templates. 0, 10, 15 and 20 μM denote final concentrations of 5-methyl-dCTP in the REPLI-g WGA mixture.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Derivative Assay, Concentration Assay, Whole Genome Amplification

    Resequencing of PCR amplicons with the MspJI-based DNA fragmentation method. ( a ) Pooled PCR amplicons containing 5 m C. Locus-specific amplification was performed for each sequence independently. ( b ) MspJI digestion of the PCR amplicons. ( c ) Flowchart of the MspJI- and physical shearing-based library prep procedures from PCR amplicons.
    Figure Legend Snippet: Resequencing of PCR amplicons with the MspJI-based DNA fragmentation method. ( a ) Pooled PCR amplicons containing 5 m C. Locus-specific amplification was performed for each sequence independently. ( b ) MspJI digestion of the PCR amplicons. ( c ) Flowchart of the MspJI- and physical shearing-based library prep procedures from PCR amplicons.

    Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing

    4) Product Images from "Heritable Epigenetic Variation among Maize Inbreds"

    Article Title: Heritable Epigenetic Variation among Maize Inbreds

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1002372

    Variable DNA methylation patterns in near-isogenic lines and diverse inbreds. (A) The relative DNA methylation levels in selected near-isogenic lines was tested by digestion with the methylation dependent restriction enzyme MspjI followed by qPCR. Different subsets of NILs were selected and analyzed for each of 13 DMRs. The first two columns show the data from B73 and Mo17. Open circles reflect low methylation levels and black circles indicate high methylation levels. Intermediate methylation levels are indicated by gray color. The next group of 2–7 genotypes show the data from NILs that have B73 as the recurrent parent ( > 95% of the genome) and have introgression of the Mo17 haplotype in the region containing the DMR. The variable number of genotypes tested reflects the fact that some DMR loci are have more NILs with an introgression than others. The next group of 1–3 genotypes are NILs that are primarily Mo17 but have B73 introgressed at the DMR. The next two groups provide “control” genotypes of B73-like or Mo17-like NILs that do not have an introgression at the DMR. The expected patterns for cis (local) inheritance of DNA methylation or trans (unlinked) control of DNA methylation are shown. Note that the expected pattern for trans control would include a small number of genotypes with the methylation pattern from the introgressed genotype in cases where the trans-acting locus is introgressed. (B) The same type of assays were performed on a panel of 12 diverse inbred genotypes, including two inbred teosinte lines, to monitor the frequency for the hyper- and hypo-methylated states.
    Figure Legend Snippet: Variable DNA methylation patterns in near-isogenic lines and diverse inbreds. (A) The relative DNA methylation levels in selected near-isogenic lines was tested by digestion with the methylation dependent restriction enzyme MspjI followed by qPCR. Different subsets of NILs were selected and analyzed for each of 13 DMRs. The first two columns show the data from B73 and Mo17. Open circles reflect low methylation levels and black circles indicate high methylation levels. Intermediate methylation levels are indicated by gray color. The next group of 2–7 genotypes show the data from NILs that have B73 as the recurrent parent ( > 95% of the genome) and have introgression of the Mo17 haplotype in the region containing the DMR. The variable number of genotypes tested reflects the fact that some DMR loci are have more NILs with an introgression than others. The next group of 1–3 genotypes are NILs that are primarily Mo17 but have B73 introgressed at the DMR. The next two groups provide “control” genotypes of B73-like or Mo17-like NILs that do not have an introgression at the DMR. The expected patterns for cis (local) inheritance of DNA methylation or trans (unlinked) control of DNA methylation are shown. Note that the expected pattern for trans control would include a small number of genotypes with the methylation pattern from the introgressed genotype in cases where the trans-acting locus is introgressed. (B) The same type of assays were performed on a panel of 12 diverse inbred genotypes, including two inbred teosinte lines, to monitor the frequency for the hyper- and hypo-methylated states.

    Techniques Used: DNA Methylation Assay, Methylation, Real-time Polymerase Chain Reaction

    Related Articles

    Amplification:

    Article Title: The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
    Article Snippet: Paragraph title: Generating PCR amplified target DNA fragments ... MspJI digestion ( ) was carried out at 37°C in 30-μl reaction mixture consisting of 0.2–1 μg of PCR products, 3 μl of 10× NEBuffer 4, 1 μl of 100× bovine serum albumin, 1 μl of 10 μM MspJI activator solution, 2–4 U of MspJI (NEB) and nuclease-free water to 30 μl.

    Article Title: Identification of a DNA methylation marker that detects the presence of lymph node metastases of gastric cancers
    Article Snippet: DNA (1 μg)was treated with Msp JI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR sequence ( , ), in a 30 μl reaction [4 U of Msp JI, 1× NEB buffer 4 (New England Biolabs) and 0.1 mg/ml BSA] at 37°C for 20 h. Following purification, the DNA was treated with Msp JI again and dissolved in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) at a concentration of 5 ng/μl without purification. .. To normalize the quantity of input DNA, the number of copies of a standard sequence, which may be amplified with a primer pair (5′-TTGCTTGAAGTTTTGTTGCTGTAGT-3′ and 5′-AATAAACTCAGTTGTGACATGGACA-3′) and contains no Msp JI site, was measured by qPCR.

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence . .. Subsequently, amplification of the enzyme-treated samples and paired untreated samples was carried out using specific primers that encompassed the target MspJI/FspEI site.

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI
    Article Snippet: .. Restriction enzyme digestion The amplified DNA (5 μl) was digested with 3 U of the MspJI, FspEI or LpnPI restriction enzyme (NEB) following manufacturer’s protocol. ..

    Polymerase Chain Reaction:

    Article Title: The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
    Article Snippet: .. MspJI digestion ( ) was carried out at 37°C in 30-μl reaction mixture consisting of 0.2–1 μg of PCR products, 3 μl of 10× NEBuffer 4, 1 μl of 100× bovine serum albumin, 1 μl of 10 μM MspJI activator solution, 2–4 U of MspJI (NEB) and nuclease-free water to 30 μl. .. The MspJI activator was prepared through self-annealing of the oligo kjz.ml1, which was first incubated at 65°C for 1 h in 1× saline-sodium citrate (SSC) buffer using a PCR machine and then slowly cool down in room temperature.

    Article Title: C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD
    Article Snippet: DNA (1 μg) from post-mortem cerebellar tissue was restriction enzyme digested for 4 h with Hpa II (10 U), Msp I (10 U), or MspJ I (4 U) (New England Biolabs) at 37°C followed by phenol:chloroform:isoamyl alcohol extraction. .. 100 ng of digested DNA was used for repeat primed PCR as described previously [ ].

    Article Title: Florigen-Encoding Genes of Day-Neutral and Photoperiod-Sensitive Maize Are Regulated by Different Chromatin Modifications at the Floral Transition 1Florigen-Encoding Genes of Day-Neutral and Photoperiod-Sensitive Maize Are Regulated by Different Chromatin Modifications at the Floral Transition 1 [OPEN]
    Article Snippet: Digestion with Msp JI (New England Biolabs) was performed following the manufacturer’s instructions. .. One microliter of bisulfite-treated DNA was used for PCR under the following conditions: 1.5 m m MgCl2 , 0.2 m m of each deoxyribonucleoside triphosphate, 0.4 μ m of each primer, and 1.25 units of Taq Platinum (Life Technologies).

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: Paragraph title: PCR following treatment with a methylation-dependent restriction enzyme (PTMR) ... In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence .

    Construct:

    Article Title: Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease
    Article Snippet: The authors thank Jeffrey Lary and James Cole at the Biotechnology and Bioservices Center in the University of Connecticut for performing analytical ultracentrifugation analysis of MspJI, Brenda Baker, Nancy Considine and John Buswell at the organic synthesis unit of New England Biolabs for synthesizing the oligonucleotides, Geoffrey Wilson, Hua Wang, Elisabeth Raleigh and William Jack for discussion, Keith Lunen and Daniel Heiter for technical advice. .. J.R.H. performed crystallographic work, M.Y.M constructed and assessed all the mutants, M.Y.M and D.C.-K. performed nicking and gel shift experiments, D.C.-K. and M.S. performed large-scale purification, X.Z and R.M.G performed purification and early crystallization trials, R.J.R, Y.Z. and X.C. organized and designed the scope of the study, and all were involved in analyzing data and preparing the manuscript.

    Real-time Polymerase Chain Reaction:

    Article Title: An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation
    Article Snippet: Paragraph title: DNA methylation-dependent qPCR assay (MD-qPCR) ... Purified samples were subjected to digestion by MspJI (NEB, R0166L) overnight at 37°C.

    Article Title: Identification of a DNA methylation marker that detects the presence of lymph node metastases of gastric cancers
    Article Snippet: Paragraph title: Quantitative PCR following treatment with a methylation-dependent restriction enzyme (qPTMR) ... DNA (1 μg)was treated with Msp JI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR sequence ( , ), in a 30 μl reaction [4 U of Msp JI, 1× NEB buffer 4 (New England Biolabs) and 0.1 mg/ml BSA] at 37°C for 20 h. Following purification, the DNA was treated with Msp JI again and dissolved in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) at a concentration of 5 ng/μl without purification.

    Incubation:

    Article Title: The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
    Article Snippet: MspJI digestion ( ) was carried out at 37°C in 30-μl reaction mixture consisting of 0.2–1 μg of PCR products, 3 μl of 10× NEBuffer 4, 1 μl of 100× bovine serum albumin, 1 μl of 10 μM MspJI activator solution, 2–4 U of MspJI (NEB) and nuclease-free water to 30 μl. .. The MspJI activator was prepared through self-annealing of the oligo kjz.ml1, which was first incubated at 65°C for 1 h in 1× saline-sodium citrate (SSC) buffer using a PCR machine and then slowly cool down in room temperature.

    Article Title: DNA Methylation Influences Chlorogenic Acid Biosynthesis in Lonicera japonica by Mediating LjbZIP8 to Regulate Phenylalanine Ammonia-Lyase 2 Expression
    Article Snippet: The Msp JI digestion solution included 100 ng of genomic DNA, 10 units of Msp JI (New England Biolabs, Ipswich, MA, United States), 1 × NEBuffer 4 (New England Biolabs, Ipswich, MA, United States), 0.5 μM reaction activator, and deionized water to a final volume of 20 μL. .. Reactions were incubated at 37°C for 4 h, and followed by heat inactivation at 65°C for 15 min.

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence . .. One microgram of the extracted DNA was incubated with 4 units of the appropriate two enzymes at 37 °C for 8 h in 30 μl of readymade reaction buffer (New England Biolabs) .

    Article Title: Crystal structure of human nucleosome core particle containing enzymatically introduced CpG methylation
    Article Snippet: Large‐scale methylation of CpG146 DNA and its verification For the preparation of CpG‐methylated nucleosomal DNA, 50 μg·mL−1 of the CpG146 DNA was incubated at 37 °C for 16 h with M.Sss I (6 units·μg−1 DNA), in 10 mm Tris‐HCl buffer (pH 8.0), containing 50 mm NaCl, 2.5 mm EDTA, and 640 μm SAM. .. A small portion of the reacted DNA was digested with Eco72 I (Thermo Fisher Scientific; cat. ER0361) or Msp JI (NEB, cat. R0661S), at 1.8 units·pmol−1 DNA and 0.45 units·pmol−1 DNA, respectively.

    Crystallization Assay:

    Article Title: Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease
    Article Snippet: The authors thank Jeffrey Lary and James Cole at the Biotechnology and Bioservices Center in the University of Connecticut for performing analytical ultracentrifugation analysis of MspJI, Brenda Baker, Nancy Considine and John Buswell at the organic synthesis unit of New England Biolabs for synthesizing the oligonucleotides, Geoffrey Wilson, Hua Wang, Elisabeth Raleigh and William Jack for discussion, Keith Lunen and Daniel Heiter for technical advice. .. J.R.H. performed crystallographic work, M.Y.M constructed and assessed all the mutants, M.Y.M and D.C.-K. performed nicking and gel shift experiments, D.C.-K. and M.S. performed large-scale purification, X.Z and R.M.G performed purification and early crystallization trials, R.J.R, Y.Z. and X.C. organized and designed the scope of the study, and all were involved in analyzing data and preparing the manuscript.

    Derivative Assay:

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples
    Article Snippet: One hundred twenty-five to 500 ng of gDNA extracted from P. vivax -infected whole-blood samples was digested with 5 units of FspEI (New England Biolabs) and 5 units of MspJI (New England Biolabs) enzymes in a 30-µl reaction mixture. .. Samples were digested for 2 h at 37°C and then heat inactivated at 80°C for 15 min. For coverage analysis, rarefaction analysis was performed on Illumina MiSeq sequencing reads derived from samples digested with MspJI or FspEI (digest and SWGA) or mock digested with no enzyme prior to SWGA with primer set pvset1920.

    Ligation:

    Article Title: The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
    Article Snippet: MspJI digestion ( ) was carried out at 37°C in 30-μl reaction mixture consisting of 0.2–1 μg of PCR products, 3 μl of 10× NEBuffer 4, 1 μl of 100× bovine serum albumin, 1 μl of 10 μM MspJI activator solution, 2–4 U of MspJI (NEB) and nuclease-free water to 30 μl. .. After MspJI digestion, fragments with complementary cohesive ends were purified by agarose gel electrophoresis and were then ligated with T4 DNA ligase at 16°C for 2 h. Ligation reaction was terminated by adding 5 μl of 6× DNA loading buffer, denatured at 65°C for 15 min and then analysed by agarose gel electrophoresis.

    Infection:

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples
    Article Snippet: .. One hundred twenty-five to 500 ng of gDNA extracted from P. vivax -infected whole-blood samples was digested with 5 units of FspEI (New England Biolabs) and 5 units of MspJI (New England Biolabs) enzymes in a 30-µl reaction mixture. ..

    other:

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9
    Article Snippet: One microgram of T4(C), T4(HMC), or T4(glc-HMC) was digested with AluI (R0137s; NEB), MspJI (R0661S; NEB), or T4 phage β-glucosyltransferase (M0357S; NEB) in accordance with NEB-specified protocols.

    Article Title: Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI
    Article Snippet: LpnPI, FspEI, and MspJI (New England Biolabs) digestions were carried out according to the manufacturer's protocol.

    Sequencing:

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples
    Article Snippet: One hundred twenty-five to 500 ng of gDNA extracted from P. vivax -infected whole-blood samples was digested with 5 units of FspEI (New England Biolabs) and 5 units of MspJI (New England Biolabs) enzymes in a 30-µl reaction mixture. .. Samples were digested for 2 h at 37°C and then heat inactivated at 80°C for 15 min. For coverage analysis, rarefaction analysis was performed on Illumina MiSeq sequencing reads derived from samples digested with MspJI or FspEI (digest and SWGA) or mock digested with no enzyme prior to SWGA with primer set pvset1920.

    Article Title: Identification of a DNA methylation marker that detects the presence of lymph node metastases of gastric cancers
    Article Snippet: .. DNA (1 μg)was treated with Msp JI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR sequence ( , ), in a 30 μl reaction [4 U of Msp JI, 1× NEB buffer 4 (New England Biolabs) and 0.1 mg/ml BSA] at 37°C for 20 h. Following purification, the DNA was treated with Msp JI again and dissolved in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) at a concentration of 5 ng/μl without purification. .. Using 1 μl of the solution, quantitative PCR (qPCR) was performed by real-time PCR with primers that encompassed a target Msp JI site ( ).

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: .. In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence . .. One microgram of the extracted DNA was incubated with 4 units of the appropriate two enzymes at 37 °C for 8 h in 30 μl of readymade reaction buffer (New England Biolabs) .

    Binding Assay:

    Article Title: Crystal structure of human nucleosome core particle containing enzymatically introduced CpG methylation
    Article Snippet: A small portion of the reacted DNA was digested with Eco72 I (Thermo Fisher Scientific; cat. ER0361) or Msp JI (NEB, cat. R0661S), at 1.8 units·pmol−1 DNA and 0.45 units·pmol−1 DNA, respectively. .. The CpG‐methylated CpG146 DNA was also used in the binding assay with MBD2, using an EpiXplore methylated DNA enrichment kit (Takara Bio, Shiga, Japan; cat. 631963).

    Methylation:

    Article Title: An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation
    Article Snippet: Paragraph title: DNA methylation-dependent qPCR assay (MD-qPCR) ... Purified samples were subjected to digestion by MspJI (NEB, R0166L) overnight at 37°C.

    Article Title: C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD
    Article Snippet: Paragraph title: Hexanucleotide repeat methylation assay ... DNA (1 μg) from post-mortem cerebellar tissue was restriction enzyme digested for 4 h with Hpa II (10 U), Msp I (10 U), or MspJ I (4 U) (New England Biolabs) at 37°C followed by phenol:chloroform:isoamyl alcohol extraction.

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples
    Article Snippet: Paragraph title: Methylation digest. ... One hundred twenty-five to 500 ng of gDNA extracted from P. vivax -infected whole-blood samples was digested with 5 units of FspEI (New England Biolabs) and 5 units of MspJI (New England Biolabs) enzymes in a 30-µl reaction mixture.

    Article Title: Identification of a DNA methylation marker that detects the presence of lymph node metastases of gastric cancers
    Article Snippet: Paragraph title: Quantitative PCR following treatment with a methylation-dependent restriction enzyme (qPTMR) ... DNA (1 μg)was treated with Msp JI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR sequence ( , ), in a 30 μl reaction [4 U of Msp JI, 1× NEB buffer 4 (New England Biolabs) and 0.1 mg/ml BSA] at 37°C for 20 h. Following purification, the DNA was treated with Msp JI again and dissolved in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) at a concentration of 5 ng/μl without purification.

    Article Title: DNA methylation analysis in malignant pheochromocytoma and paraganglioma
    Article Snippet: .. In this study, we employed two types of methylation-dependent restriction enzymes: MspJI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR (N = A, T, G or C; R = G or C) sequence , , and FspEI (New England Biolabs), which cleaves DNA 12 bp downstream from the Cm C sequence . .. One microgram of the extracted DNA was incubated with 4 units of the appropriate two enzymes at 37 °C for 8 h in 30 μl of readymade reaction buffer (New England Biolabs) .

    Article Title: Crystal structure of human nucleosome core particle containing enzymatically introduced CpG methylation
    Article Snippet: Paragraph title: Large‐scale methylation of CpG146 DNA and its verification ... A small portion of the reacted DNA was digested with Eco72 I (Thermo Fisher Scientific; cat. ER0361) or Msp JI (NEB, cat. R0661S), at 1.8 units·pmol−1 DNA and 0.45 units·pmol−1 DNA, respectively.

    Isolation:

    Article Title: An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation
    Article Snippet: DNA methylation-dependent qPCR assay (MD-qPCR) Genomic DNA was harvested using a standard phenol:chloroform isolation, followed by ethanol precipitation. .. Purified samples were subjected to digestion by MspJI (NEB, R0166L) overnight at 37°C.

    Electrophoretic Mobility Shift Assay:

    Article Title: Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease
    Article Snippet: The authors thank Jeffrey Lary and James Cole at the Biotechnology and Bioservices Center in the University of Connecticut for performing analytical ultracentrifugation analysis of MspJI, Brenda Baker, Nancy Considine and John Buswell at the organic synthesis unit of New England Biolabs for synthesizing the oligonucleotides, Geoffrey Wilson, Hua Wang, Elisabeth Raleigh and William Jack for discussion, Keith Lunen and Daniel Heiter for technical advice. .. J.R.H. performed crystallographic work, M.Y.M constructed and assessed all the mutants, M.Y.M and D.C.-K. performed nicking and gel shift experiments, D.C.-K. and M.S. performed large-scale purification, X.Z and R.M.G performed purification and early crystallization trials, R.J.R, Y.Z. and X.C. organized and designed the scope of the study, and all were involved in analyzing data and preparing the manuscript.

    Purification:

    Article Title: The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
    Article Snippet: MspJI digestion ( ) was carried out at 37°C in 30-μl reaction mixture consisting of 0.2–1 μg of PCR products, 3 μl of 10× NEBuffer 4, 1 μl of 100× bovine serum albumin, 1 μl of 10 μM MspJI activator solution, 2–4 U of MspJI (NEB) and nuclease-free water to 30 μl. .. After MspJI digestion, fragments with complementary cohesive ends were purified by agarose gel electrophoresis and were then ligated with T4 DNA ligase at 16°C for 2 h. Ligation reaction was terminated by adding 5 μl of 6× DNA loading buffer, denatured at 65°C for 15 min and then analysed by agarose gel electrophoresis.

    Article Title: Structure and cleavage activity of the tetrameric MspJI DNA modification-dependent restriction endonuclease
    Article Snippet: The authors thank Jeffrey Lary and James Cole at the Biotechnology and Bioservices Center in the University of Connecticut for performing analytical ultracentrifugation analysis of MspJI, Brenda Baker, Nancy Considine and John Buswell at the organic synthesis unit of New England Biolabs for synthesizing the oligonucleotides, Geoffrey Wilson, Hua Wang, Elisabeth Raleigh and William Jack for discussion, Keith Lunen and Daniel Heiter for technical advice. .. J.R.H. performed crystallographic work, M.Y.M constructed and assessed all the mutants, M.Y.M and D.C.-K. performed nicking and gel shift experiments, D.C.-K. and M.S. performed large-scale purification, X.Z and R.M.G performed purification and early crystallization trials, R.J.R, Y.Z. and X.C. organized and designed the scope of the study, and all were involved in analyzing data and preparing the manuscript.

    Article Title: An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation
    Article Snippet: .. Purified samples were subjected to digestion by MspJI (NEB, R0166L) overnight at 37°C. .. Digested DNA was purified and quantified by PicoGreen according to manufacturer's protocol (Life Technologies, P11495) using the NanoDrop 3300 fluorospectrometer.

    Article Title: Identification of a DNA methylation marker that detects the presence of lymph node metastases of gastric cancers
    Article Snippet: .. DNA (1 μg)was treated with Msp JI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR sequence ( , ), in a 30 μl reaction [4 U of Msp JI, 1× NEB buffer 4 (New England Biolabs) and 0.1 mg/ml BSA] at 37°C for 20 h. Following purification, the DNA was treated with Msp JI again and dissolved in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) at a concentration of 5 ng/μl without purification. .. Using 1 μl of the solution, quantitative PCR (qPCR) was performed by real-time PCR with primers that encompassed a target Msp JI site ( ).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: DNA Methylation Influences Chlorogenic Acid Biosynthesis in Lonicera japonica by Mediating LjbZIP8 to Regulate Phenylalanine Ammonia-Lyase 2 Expression
    Article Snippet: The Msp JI digestion solution included 100 ng of genomic DNA, 10 units of Msp JI (New England Biolabs, Ipswich, MA, United States), 1 × NEBuffer 4 (New England Biolabs, Ipswich, MA, United States), 0.5 μM reaction activator, and deionized water to a final volume of 20 μL. .. The RT-PCR solution included 10 μL 2 × I-5TM High-Fidelity Master Mix (MLAB, Los Angeles, CA, United States), 0.4 μL 50 × ROX (Applied Biosystems, Foster City, CA, United States), 0.4 μL forward and reverse primers (10 μM), 1 μL 20 × EvaGreen (Biotium, Hayward, CA, United States), and 2 μL reaction temple (endonuclease-treated samples diluted 1:10 with double-deionized water).

    Software:

    Article Title: C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD
    Article Snippet: DNA (1 μg) from post-mortem cerebellar tissue was restriction enzyme digested for 4 h with Hpa II (10 U), Msp I (10 U), or MspJ I (4 U) (New England Biolabs) at 37°C followed by phenol:chloroform:isoamyl alcohol extraction. .. Fragment length analysis was done using the Genetic Analyzer 3130x (Life Technologies) and Peak Scanner software (Life Technologies).

    Agarose Gel Electrophoresis:

    Article Title: The MASTER (methylation-assisted tailorable ends rational) ligation method for seamless DNA assembly
    Article Snippet: MspJI digestion ( ) was carried out at 37°C in 30-μl reaction mixture consisting of 0.2–1 μg of PCR products, 3 μl of 10× NEBuffer 4, 1 μl of 100× bovine serum albumin, 1 μl of 10 μM MspJI activator solution, 2–4 U of MspJI (NEB) and nuclease-free water to 30 μl. .. After MspJI digestion, fragments with complementary cohesive ends were purified by agarose gel electrophoresis and were then ligated with T4 DNA ligase at 16°C for 2 h. Ligation reaction was terminated by adding 5 μl of 6× DNA loading buffer, denatured at 65°C for 15 min and then analysed by agarose gel electrophoresis.

    In Vitro:

    Article Title: C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD
    Article Snippet: DNA (1 μg) from post-mortem cerebellar tissue was restriction enzyme digested for 4 h with Hpa II (10 U), Msp I (10 U), or MspJ I (4 U) (New England Biolabs) at 37°C followed by phenol:chloroform:isoamyl alcohol extraction. .. To ensure that methylated DNA could be digested by MspJ I under these conditions, 1 μg of DNA from repeat expanded or control cerebellum was in vitro methylated using M.SssI (New England Biolabs) for 4 h at 37 °C followed by phenol:chloroform:isoamyl alcohol extraction.

    Ethanol Precipitation:

    Article Title: An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation
    Article Snippet: DNA methylation-dependent qPCR assay (MD-qPCR) Genomic DNA was harvested using a standard phenol:chloroform isolation, followed by ethanol precipitation. .. Purified samples were subjected to digestion by MspJI (NEB, R0166L) overnight at 37°C.

    DNA Methylation Assay:

    Article Title: An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation
    Article Snippet: Purified samples were subjected to digestion by MspJI (NEB, R0166L) overnight at 37°C. .. Change in DNA methylation is represented by relative fold change in the Cq values as follows: 2∧ [Cq(U)-Cq(I)], where Cq(U) is the Cq for the undifferentiated (ESC) sample, and Cq(I) represents a sample from a given time point post differentiation.

    Article Title: Florigen-Encoding Genes of Day-Neutral and Photoperiod-Sensitive Maize Are Regulated by Different Chromatin Modifications at the Floral Transition 1Florigen-Encoding Genes of Day-Neutral and Photoperiod-Sensitive Maize Are Regulated by Different Chromatin Modifications at the Floral Transition 1 [OPEN]
    Article Snippet: Paragraph title: DNA Methylation Analysis ... Digestion with Msp JI (New England Biolabs) was performed following the manufacturer’s instructions.

    Article Title: DNA Methylation Influences Chlorogenic Acid Biosynthesis in Lonicera japonica by Mediating LjbZIP8 to Regulate Phenylalanine Ammonia-Lyase 2 Expression
    Article Snippet: Paragraph title: Quantitative DNA Methylation Analysis ... The Msp JI digestion solution included 100 ng of genomic DNA, 10 units of Msp JI (New England Biolabs, Ipswich, MA, United States), 1 × NEBuffer 4 (New England Biolabs, Ipswich, MA, United States), 0.5 μM reaction activator, and deionized water to a final volume of 20 μL.

    Concentration Assay:

    Article Title: Identification of a DNA methylation marker that detects the presence of lymph node metastases of gastric cancers
    Article Snippet: .. DNA (1 μg)was treated with Msp JI (New England Biolabs, Beverly, MA, USA), which cleaves DNA 9 bp downstream from the m CNNR sequence ( , ), in a 30 μl reaction [4 U of Msp JI, 1× NEB buffer 4 (New England Biolabs) and 0.1 mg/ml BSA] at 37°C for 20 h. Following purification, the DNA was treated with Msp JI again and dissolved in TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA) at a concentration of 5 ng/μl without purification. .. Using 1 μl of the solution, quantitative PCR (qPCR) was performed by real-time PCR with primers that encompassed a target Msp JI site ( ).

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    New England Biolabs mspji
    MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with <t>LpnPI,</t> followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, <t>MspJI,</t> and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).
    Mspji, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).

    Journal: Genome Research

    Article Title: Genome-wide DNA methylation profiling using the methylation-dependent restriction enzyme LpnPI

    doi: 10.1101/gr.222885.117

    Figure Lengend Snippet: MeD-seq wet laboratory and bioinformatics platform. ( A ) Genomic DNA is digested with LpnPI, followed by DNA repair, adaptor ligation to 32-bp fragments, size fractionation, amplification, and sequencing. ( B ) Sequencing reads are trimmed, filtered based on the CpG sequence, and aligned to the genome. ( C ) Nucleotide frequency plotted against the position in the sequencing reads, showing enrichment of CG nucleotides around 16–17 bp from the start. ( D ) Alignment of LpnPI, MspJI, and FspEI CpG-filtered MeD-seq reads obtained using fibroblast DNA. ( E ) Sequencing read profiles of the HOXA and KCNQ1 loci obtained with fibroblast DNA using LpnPI, MspJI, and FspEI. ( F ) Pearson correlation analysis of MeD-seq read counts of TSSs for LpnPI-MspJI ( left ), LpnPI-FspEI ( middle ), and FspEI-MspJI ( right ) comparisons. ( G ) Pearson correlation analysis of read counts of TSSs for technical replicates digested with LpnPI ( left ) and MspJI ( right ).

    Article Snippet: LpnPI, FspEI, and MspJI (New England Biolabs) digestions were carried out according to the manufacturer's protocol.

    Techniques: Ligation, Fractionation, Amplification, Sequencing

    Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Journal: mBio

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    doi: 10.1128/mBio.00648-15

    Figure Lengend Snippet: Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Article Snippet: One microgram of T4(C), T4(HMC), or T4(glc-HMC) was digested with AluI (R0137s; NEB), MspJI (R0661S; NEB), or T4 phage β-glucosyltransferase (M0357S; NEB) in accordance with NEB-specified protocols.

    Techniques: Modification, Mobility Shift, Sequencing, Methylation

    Hypermethylation of the C9orf72 promoter. a Cerebellar DNA from control ( n = 8, left ) and repeat-expanded cases ( n = 8, right ) were mock digested (no enzyme) or digested with Msp I, Hpa II or MspJ I. DNA was subject to repeat primed PCR and representative

    Journal: Acta neuropathologica

    Article Title: C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD

    doi: 10.1007/s00401-014-1286-y

    Figure Lengend Snippet: Hypermethylation of the C9orf72 promoter. a Cerebellar DNA from control ( n = 8, left ) and repeat-expanded cases ( n = 8, right ) were mock digested (no enzyme) or digested with Msp I, Hpa II or MspJ I. DNA was subject to repeat primed PCR and representative

    Article Snippet: DNA (1 μg) from post-mortem cerebellar tissue was restriction enzyme digested for 4 h with Hpa II (10 U), Msp I (10 U), or MspJ I (4 U) (New England Biolabs) at 37°C followed by phenol:chloroform:isoamyl alcohol extraction.

    Techniques: Polymerase Chain Reaction

    BAC clone sequencing based on the MspJI-based DNA fragmentation approach. ( a ) Amplified BAC DNA directly from glycerol stocks, using the Φ29 enzyme. In the amplification solution, 30 μM 5-methyl-dCTP were included. ( b ) Resequencing results of the BAC clones. Read coverage depth for each clone was visualised on the Tablet software.

    Journal: BMC Biotechnology

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    doi: 10.1186/s12896-015-0139-7

    Figure Lengend Snippet: BAC clone sequencing based on the MspJI-based DNA fragmentation approach. ( a ) Amplified BAC DNA directly from glycerol stocks, using the Φ29 enzyme. In the amplification solution, 30 μM 5-methyl-dCTP were included. ( b ) Resequencing results of the BAC clones. Read coverage depth for each clone was visualised on the Tablet software.

    Article Snippet: Restriction enzyme digestion The amplified DNA (5 μl) was digested with 3 U of the MspJI, FspEI or LpnPI restriction enzyme (NEB) following manufacturer’s protocol.

    Techniques: BAC Assay, Sequencing, Amplification, Clone Assay, Software

    Illumina MiSeq short read-sequencing results of the libraries constructed from MspJI-digested and physically sheared DNA. The sorted alignment was visualised using the Tablet viewers. ( a ) Alignment results of Φ29 enzyme-amplified Agro gDNA-derived reads and physically sheared Agro gDNA-derived reads to the reference Agrobacterium genome sequences for the 1,000-1,250 and 2,000-2250 kb regions of the circular and linear chromosomes. ( b ) Alignment results of the Arabidopsis genome with the MspJI-enzymatic fragmentation and physical shearing methods. Read coverage depth for the 10,000-10,250, 20,000-20,250 and 30,000-30,250 kb regions of chromosome 1 was visualised.

    Journal: BMC Biotechnology

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    doi: 10.1186/s12896-015-0139-7

    Figure Lengend Snippet: Illumina MiSeq short read-sequencing results of the libraries constructed from MspJI-digested and physically sheared DNA. The sorted alignment was visualised using the Tablet viewers. ( a ) Alignment results of Φ29 enzyme-amplified Agro gDNA-derived reads and physically sheared Agro gDNA-derived reads to the reference Agrobacterium genome sequences for the 1,000-1,250 and 2,000-2250 kb regions of the circular and linear chromosomes. ( b ) Alignment results of the Arabidopsis genome with the MspJI-enzymatic fragmentation and physical shearing methods. Read coverage depth for the 10,000-10,250, 20,000-20,250 and 30,000-30,250 kb regions of chromosome 1 was visualised.

    Article Snippet: Restriction enzyme digestion The amplified DNA (5 μl) was digested with 3 U of the MspJI, FspEI or LpnPI restriction enzyme (NEB) following manufacturer’s protocol.

    Techniques: Sequencing, Construct, Amplification, Derivative Assay

    Short read-sequencing results of the libraries constructed from MspJI, FspEI and LpnPI-digested DNA. Read coverage depth was visualised using the Tablet viewer.

    Journal: BMC Biotechnology

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    doi: 10.1186/s12896-015-0139-7

    Figure Lengend Snippet: Short read-sequencing results of the libraries constructed from MspJI, FspEI and LpnPI-digested DNA. Read coverage depth was visualised using the Tablet viewer.

    Article Snippet: Restriction enzyme digestion The amplified DNA (5 μl) was digested with 3 U of the MspJI, FspEI or LpnPI restriction enzyme (NEB) following manufacturer’s protocol.

    Techniques: Sequencing, Construct

    Sequencing of the fungal endophyte genome with the MspJI-based DNA fragmentation method. Two independent experiments (rep1 and rep2) were performed using the single endophyte strain. ( a ) WGA of the endophyte genome with the Φ29 enzyme in the presence of 5-methyl-dCTP (X μM). ( b ) MspJI digestion of the endophyte genome-derived amplicons. ( c ) Flowchart of the MspJI- and physical shearing-based library prep procedures from WGA products.

    Journal: BMC Biotechnology

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    doi: 10.1186/s12896-015-0139-7

    Figure Lengend Snippet: Sequencing of the fungal endophyte genome with the MspJI-based DNA fragmentation method. Two independent experiments (rep1 and rep2) were performed using the single endophyte strain. ( a ) WGA of the endophyte genome with the Φ29 enzyme in the presence of 5-methyl-dCTP (X μM). ( b ) MspJI digestion of the endophyte genome-derived amplicons. ( c ) Flowchart of the MspJI- and physical shearing-based library prep procedures from WGA products.

    Article Snippet: Restriction enzyme digestion The amplified DNA (5 μl) was digested with 3 U of the MspJI, FspEI or LpnPI restriction enzyme (NEB) following manufacturer’s protocol.

    Techniques: Sequencing, Whole Genome Amplification, Derivative Assay

    MspJI-enzymatic digestion of 5 m C-containing PCR and Φ29 products. ( a ) DNA fragments amplified with the locus-specific PCR primers for the Agro _gc50 sequence under the presence of 5-methyl-dCTP (0, 2, 4 or 8 μM). ( b ) MspJI-digested DNA fragments derived from PCR products with each locus-specific primers and Agro gDNA as DNA template. Molar concentration denotes the 5-methyl-dCTP-concentration in PCR solution. ( c ) MspJI-enzymatic digestion of Φ29 enzyme-amplified DNA with randomly incorporated 5 m C from a range of DNA templates. 0, 10, 15 and 20 μM denote final concentrations of 5-methyl-dCTP in the REPLI-g WGA mixture.

    Journal: BMC Biotechnology

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    doi: 10.1186/s12896-015-0139-7

    Figure Lengend Snippet: MspJI-enzymatic digestion of 5 m C-containing PCR and Φ29 products. ( a ) DNA fragments amplified with the locus-specific PCR primers for the Agro _gc50 sequence under the presence of 5-methyl-dCTP (0, 2, 4 or 8 μM). ( b ) MspJI-digested DNA fragments derived from PCR products with each locus-specific primers and Agro gDNA as DNA template. Molar concentration denotes the 5-methyl-dCTP-concentration in PCR solution. ( c ) MspJI-enzymatic digestion of Φ29 enzyme-amplified DNA with randomly incorporated 5 m C from a range of DNA templates. 0, 10, 15 and 20 μM denote final concentrations of 5-methyl-dCTP in the REPLI-g WGA mixture.

    Article Snippet: Restriction enzyme digestion The amplified DNA (5 μl) was digested with 3 U of the MspJI, FspEI or LpnPI restriction enzyme (NEB) following manufacturer’s protocol.

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Derivative Assay, Concentration Assay, Whole Genome Amplification

    Resequencing of PCR amplicons with the MspJI-based DNA fragmentation method. ( a ) Pooled PCR amplicons containing 5 m C. Locus-specific amplification was performed for each sequence independently. ( b ) MspJI digestion of the PCR amplicons. ( c ) Flowchart of the MspJI- and physical shearing-based library prep procedures from PCR amplicons.

    Journal: BMC Biotechnology

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    doi: 10.1186/s12896-015-0139-7

    Figure Lengend Snippet: Resequencing of PCR amplicons with the MspJI-based DNA fragmentation method. ( a ) Pooled PCR amplicons containing 5 m C. Locus-specific amplification was performed for each sequence independently. ( b ) MspJI digestion of the PCR amplicons. ( c ) Flowchart of the MspJI- and physical shearing-based library prep procedures from PCR amplicons.

    Article Snippet: Restriction enzyme digestion The amplified DNA (5 μl) was digested with 3 U of the MspJI, FspEI or LpnPI restriction enzyme (NEB) following manufacturer’s protocol.

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing