mspi  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    MspI
    Description:
    MspI 25 000 units
    Catalog Number:
    R0106L
    Price:
    269
    Category:
    Restriction Enzymes
    Size:
    25 000 units
    Buy from Supplier


    Structured Review

    New England Biolabs mspi
    MspI
    MspI 25 000 units
    https://www.bioz.com/result/mspi/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    mspi - by Bioz Stars, 2021-06
    99/100 stars

    Images

    1) Product Images from "PADI4 Haplotypes in Association with RA Mexican Patients, a New Prospect for Antigen Modulation"

    Article Title: PADI4 Haplotypes in Association with RA Mexican Patients, a New Prospect for Antigen Modulation

    Journal: Clinical and Developmental Immunology

    doi: 10.1155/2013/383681

    PADI4 SNPs enzyme digestion. The figure shows digestion of three SNPs in the PADI4 gene. (a) Shows digestion of PADI4_89, with HaeIII enzyme; lane 1 represents the A/A genotype, lane 2 A/G and 3 G/G. (b) Demonstrates PADI4_90 amplification (221 bp) in lane 1 and digested products with MscI enzyme in lanes 2 (C/C genotype), 3 (C/T genotype), and 4 (T/T genotype). (c) Shows amplification product of PADI4_92 in lane 1 (363 bp) and restriction products obtained with the enzyme MspI; lane 2 corresponds to the G/G genotype, lane 3 G/C, and lane 4 C/C. Visualized in 8% (29 : 1) polyacrylamide gel with silver staining. M: molecular weight marker (50 bp).
    Figure Legend Snippet: PADI4 SNPs enzyme digestion. The figure shows digestion of three SNPs in the PADI4 gene. (a) Shows digestion of PADI4_89, with HaeIII enzyme; lane 1 represents the A/A genotype, lane 2 A/G and 3 G/G. (b) Demonstrates PADI4_90 amplification (221 bp) in lane 1 and digested products with MscI enzyme in lanes 2 (C/C genotype), 3 (C/T genotype), and 4 (T/T genotype). (c) Shows amplification product of PADI4_92 in lane 1 (363 bp) and restriction products obtained with the enzyme MspI; lane 2 corresponds to the G/G genotype, lane 3 G/C, and lane 4 C/C. Visualized in 8% (29 : 1) polyacrylamide gel with silver staining. M: molecular weight marker (50 bp).

    Techniques Used: Amplification, Silver Staining, Molecular Weight, Marker

    2) Product Images from "Loci specific epigenetic drug sensitivity"

    Article Title: Loci specific epigenetic drug sensitivity

    Journal: bioRxiv

    doi: 10.1101/686139

    An overview of MAPMEDS. (a) Schematic structure of barcoded lentiviral constructs. The library vector contains short barcode, CMV promoter and mClover fluorescent protein as a reporter. The barcode is random 16-bp-long DNA with repeats of A,G and T. MspI restriction site is integrated upstream CMV promoter for genomic location mapping. (b) Barcoded lentivirus was packed and transduced into K562 cells at low MOI to create founder cells with singly integrated reporter. (c-d) Barcoded founder cells, selected by flow cytometry, were expanded for two weeks and split into two pools. Cells in the first pool were collected for locating reporter integration site. Founder cells in the second pool were sorted into 96-well plates to establish clonal cell lines. Barcode of each clone was simultaneously identified by split-pool encoding and deep sequencing. Library of characterized reporter clones is a useful resource to examine loci specific epigenetic drug sensitivity. (e) Loci specific effects were decoupled from global effects through mixing individual barcoded clones of interests with control cells expressing mClover and IRFP from multiple integration sites. Co-cultured cells were treated with TSA, JQ1 and 5’AZA for 24 hours. Expression of reporter proteins were measured by flow cytometers. Distribution of mClover expression in control cells was used to remove global effects of drugs. (f) A cartoon illustrating known mechanisms of actions of Trichostatin A, JQ1 and 5-Azacytidine.
    Figure Legend Snippet: An overview of MAPMEDS. (a) Schematic structure of barcoded lentiviral constructs. The library vector contains short barcode, CMV promoter and mClover fluorescent protein as a reporter. The barcode is random 16-bp-long DNA with repeats of A,G and T. MspI restriction site is integrated upstream CMV promoter for genomic location mapping. (b) Barcoded lentivirus was packed and transduced into K562 cells at low MOI to create founder cells with singly integrated reporter. (c-d) Barcoded founder cells, selected by flow cytometry, were expanded for two weeks and split into two pools. Cells in the first pool were collected for locating reporter integration site. Founder cells in the second pool were sorted into 96-well plates to establish clonal cell lines. Barcode of each clone was simultaneously identified by split-pool encoding and deep sequencing. Library of characterized reporter clones is a useful resource to examine loci specific epigenetic drug sensitivity. (e) Loci specific effects were decoupled from global effects through mixing individual barcoded clones of interests with control cells expressing mClover and IRFP from multiple integration sites. Co-cultured cells were treated with TSA, JQ1 and 5’AZA for 24 hours. Expression of reporter proteins were measured by flow cytometers. Distribution of mClover expression in control cells was used to remove global effects of drugs. (f) A cartoon illustrating known mechanisms of actions of Trichostatin A, JQ1 and 5-Azacytidine.

    Techniques Used: Construct, Plasmid Preparation, Flow Cytometry, Sequencing, Clone Assay, Expressing, Cell Culture

    Diverse insertion landscapes of barcoded reporter. (a) Reporter mapping by inverse PCR. Genomic DNA of founder cells was extracted, digested with restriction enzyme MspI and self-ligated to stitch barcode with its neighboring genome. Ligated product was amplified and followed by next generation sequencing. (b) Ideogram plot displaying reporter integration sites of individual clones in the library. Centromere position is indicated in red and stalk is marked in light blue. Heterochromatic region, which tend to be rich with adenine and thymine and relatively gene-poor, is represented by black and variation of grey. R-band in white on the ideogram is less condensed chromatin that is transcriptionally more active.
    Figure Legend Snippet: Diverse insertion landscapes of barcoded reporter. (a) Reporter mapping by inverse PCR. Genomic DNA of founder cells was extracted, digested with restriction enzyme MspI and self-ligated to stitch barcode with its neighboring genome. Ligated product was amplified and followed by next generation sequencing. (b) Ideogram plot displaying reporter integration sites of individual clones in the library. Centromere position is indicated in red and stalk is marked in light blue. Heterochromatic region, which tend to be rich with adenine and thymine and relatively gene-poor, is represented by black and variation of grey. R-band in white on the ideogram is less condensed chromatin that is transcriptionally more active.

    Techniques Used: Inverse PCR, Amplification, Next-Generation Sequencing, Clone Assay

    3) Product Images from "R213W mutation in the retinoschisis 1 gene causes X-linked juvenile retinoschisis in a large Chinese family"

    Article Title: R213W mutation in the retinoschisis 1 gene causes X-linked juvenile retinoschisis in a large Chinese family

    Journal: Molecular Vision

    doi:

    Representative 2% agarose gel of the restriction enzyme digestion analysis using MspI. The enzyme cut the PCR products into two fragments in unaffected males (I-4, II-20) and the normal female (II-4) but could not cut the products in affected males (II-10, III-22, III-23). Female carriers (I-3, II-6, II-19) exhibit a heterozygous status.
    Figure Legend Snippet: Representative 2% agarose gel of the restriction enzyme digestion analysis using MspI. The enzyme cut the PCR products into two fragments in unaffected males (I-4, II-20) and the normal female (II-4) but could not cut the products in affected males (II-10, III-22, III-23). Female carriers (I-3, II-6, II-19) exhibit a heterozygous status.

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction

    4) Product Images from "Differential DNA Methylation Analysis without a Reference Genome"

    Article Title: Differential DNA Methylation Analysis without a Reference Genome

    Journal: Cell Reports

    doi: 10.1016/j.celrep.2015.11.024

    DNA Methylation Analysis without a Reference Genome Workflow for reference-genome-independent analysis of differential DNA methylation using an optimized RRBS protocol and the RefFreeDMA software. Colored bars represent RRBS sequencing reads, and identical colors indicate high sequence similarity. Bisulfite-converted MspI restriction sites are shown at the beginning of each read (CGG for methylated sites and TGG for unmethylated sites). To derive a deduced genome, reads from all samples are clustered by sequence similarity, and a consensus sequence is determined. These deduced genome fragments (black-edged bars) are concatenated into one deduced genome, to which the RRBS reads for each sample are mapped. DNA methylation levels are obtained by counting the number of Cs versus Ts for individual cytosines in the deduced genome (this step typically focuses on CpG sites, but the method also supports the analysis of non-CpG methylation). Differential methylation analysis is performed by comparing site-specific and fragment-specific DNA methylation levels between sample groups. Finally, the identified differentially methylated fragments are analyzed by cross-mapping to well-annotated genomes of other species (e.g., mouse or human) and by motif enrichment analysis (e.g., for identifying enriched transcription factor binding sites).
    Figure Legend Snippet: DNA Methylation Analysis without a Reference Genome Workflow for reference-genome-independent analysis of differential DNA methylation using an optimized RRBS protocol and the RefFreeDMA software. Colored bars represent RRBS sequencing reads, and identical colors indicate high sequence similarity. Bisulfite-converted MspI restriction sites are shown at the beginning of each read (CGG for methylated sites and TGG for unmethylated sites). To derive a deduced genome, reads from all samples are clustered by sequence similarity, and a consensus sequence is determined. These deduced genome fragments (black-edged bars) are concatenated into one deduced genome, to which the RRBS reads for each sample are mapped. DNA methylation levels are obtained by counting the number of Cs versus Ts for individual cytosines in the deduced genome (this step typically focuses on CpG sites, but the method also supports the analysis of non-CpG methylation). Differential methylation analysis is performed by comparing site-specific and fragment-specific DNA methylation levels between sample groups. Finally, the identified differentially methylated fragments are analyzed by cross-mapping to well-annotated genomes of other species (e.g., mouse or human) and by motif enrichment analysis (e.g., for identifying enriched transcription factor binding sites).

    Techniques Used: DNA Methylation Assay, Software, Sequencing, Methylation, CpG Methylation Assay, Binding Assay

    An Optimized RRBS Protocol Validated in Nine Species (A) Schematic outline of RRBS library preparation and the corresponding sequencing reads. (B) Computationally predicted (blue) and experimentally measured (red) fragment length distribution of RRBS libraries in nine vertebrate species. Predictions were based on in silico MspI restriction digests of the reference genomes using the BSgenome R package. Experimental results were obtained by electrophoresis (Experion DNA 1k chip). In species with a reference genome, concordance between predicted and experimentally measured peaks can be used to confirm successful RRBS library preparation.
    Figure Legend Snippet: An Optimized RRBS Protocol Validated in Nine Species (A) Schematic outline of RRBS library preparation and the corresponding sequencing reads. (B) Computationally predicted (blue) and experimentally measured (red) fragment length distribution of RRBS libraries in nine vertebrate species. Predictions were based on in silico MspI restriction digests of the reference genomes using the BSgenome R package. Experimental results were obtained by electrophoresis (Experion DNA 1k chip). In species with a reference genome, concordance between predicted and experimentally measured peaks can be used to confirm successful RRBS library preparation.

    Techniques Used: Sequencing, In Silico, Electrophoresis, Chromatin Immunoprecipitation

    5) Product Images from "The high mobility group A2 protein epigenetically silences the Cdh1 gene during epithelial-to-mesenchymal transition"

    Article Title: The high mobility group A2 protein epigenetically silences the Cdh1 gene during epithelial-to-mesenchymal transition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1293

    The Cdh1 promoter is epigenetically silenced in HMGA2-overexpressing NMuMG cells. ( A ) An illustration of the mouse Cdh1 promoter, position −230 to +200 base pairs relative to the transcription start site (+1), containing E-boxes (yellow) and CpG dinucleotides (black lines). The blue underline indicates the proximal region (position −108 to +3) examined in ChIP-qPCR assays in this study. ( B ) HMGA2 binding to proximal region of the mouse Cdh1 promoter was analysed by ChIP assays with non-specific IgG or HA antibody in NM-Mock and NM-Hmga2 cells. Precipitated DNA was analysed by qPCR and data are graphed as explained in the methods. ( C ) ChIP-qPCR assays were performed to examine levels of histone H3 and its lysine modifications (active marks, K4me3 and K9ac; repressive marks, K9me3 and K27me3) on the proximal region of the mouse Cdh1 promoter in NM-Mock and NM-Hmga2 cells. ( D ) HpaII–MspI digestion–methylation assay using primers which span the proximal region of the Cdh1 promoter in NM-Mock and NM-Hmga2 cells. The PCR product was subjected to agarose gel electrophoresis and a band observed after HpaII-digestion indicates that the amplified DNA is methylated (asterisk). ( E ) The DNA methylation status of the Cdh1 promoter in NM-Mock and NM-Hmga2 cells was analysed by bisulphite sequencing of the promoter region shown in panel A, where CpG sites are denoted by circles, and five independent clones of each cell line are shown here. White and black circles represent unmethylated and methylated CpG sites respectively. ( F ) Expression of HMGA2 and CDH1 in human breast cancer cell lines classified as basal A (red), basal B (grey) and luminal (blue) subtypes. Expression values derived from microarray analysis of gene expression are shown in logarithmic (log 2 ) scale.
    Figure Legend Snippet: The Cdh1 promoter is epigenetically silenced in HMGA2-overexpressing NMuMG cells. ( A ) An illustration of the mouse Cdh1 promoter, position −230 to +200 base pairs relative to the transcription start site (+1), containing E-boxes (yellow) and CpG dinucleotides (black lines). The blue underline indicates the proximal region (position −108 to +3) examined in ChIP-qPCR assays in this study. ( B ) HMGA2 binding to proximal region of the mouse Cdh1 promoter was analysed by ChIP assays with non-specific IgG or HA antibody in NM-Mock and NM-Hmga2 cells. Precipitated DNA was analysed by qPCR and data are graphed as explained in the methods. ( C ) ChIP-qPCR assays were performed to examine levels of histone H3 and its lysine modifications (active marks, K4me3 and K9ac; repressive marks, K9me3 and K27me3) on the proximal region of the mouse Cdh1 promoter in NM-Mock and NM-Hmga2 cells. ( D ) HpaII–MspI digestion–methylation assay using primers which span the proximal region of the Cdh1 promoter in NM-Mock and NM-Hmga2 cells. The PCR product was subjected to agarose gel electrophoresis and a band observed after HpaII-digestion indicates that the amplified DNA is methylated (asterisk). ( E ) The DNA methylation status of the Cdh1 promoter in NM-Mock and NM-Hmga2 cells was analysed by bisulphite sequencing of the promoter region shown in panel A, where CpG sites are denoted by circles, and five independent clones of each cell line are shown here. White and black circles represent unmethylated and methylated CpG sites respectively. ( F ) Expression of HMGA2 and CDH1 in human breast cancer cell lines classified as basal A (red), basal B (grey) and luminal (blue) subtypes. Expression values derived from microarray analysis of gene expression are shown in logarithmic (log 2 ) scale.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Methylation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, DNA Methylation Assay, Bisulfite Sequencing, Clone Assay, Expressing, Derivative Assay, Microarray

    DNMT3A upregulation by TGFβ in NMuMG cells. ( A ) Immunoblots for E-cadherin and GAPDH in NMuMG cells unstimulated or stimulated with TGFβ1 (5 ng/ml) for 24 or 48 h, in the presence of vehicle or 10 μM 5-aza. ( B ) Phase contrast microscopy of cells as described in (A). Scale bar: 50 μm. ( C ) Luciferase reporter assay of human Cdh1 promoter in NMuMG cells stimulated with TGFβ1 for the indicated time period. ( D ) HpaII–MspI digestion–methylation assay of Cdh1 promoter in NMuMG cells stimulated with TGFβ1 for the indicated time period. ( E ) Dnmt mRNA levels after TGFβ1 treatment of NMuMG cells for the indicated time periods, were normalized to Gapdh mRNA expression. Expression values of the 0-h time point are normalized to 1. ( F ) Dnmt3a mRNA expression in NMuMG cells pre-treated for 30 min with or without TGFβ type I receptor kinase inhibitor SB505124 (10 μM), in the absence or presence of TGFβ1 for 24 h. DMSO was used as a vehicle. qPCR values were normalized to that of Gapdh and the expression values of DMSO-treated, unstimulated cells were normalized to 1. ( G ) Phase contrast images (left panel) and immunoblot analysis (right panel) of DNMT3A and GAPDH protein levels in NMuMG cells transfected with si Control or si Dnmt3a , untreated or treated with TGFβ1 for 24 h. Scale bar : 50 μm. ( H ) E-cadherin, DNMT1, DNMT3A and α-tubulin protein levels in NMuMG cells transfected with si Control , si Dnmt1 or si Dnmt3a , in the absence or presence of TGFβ1 for 48 h.
    Figure Legend Snippet: DNMT3A upregulation by TGFβ in NMuMG cells. ( A ) Immunoblots for E-cadherin and GAPDH in NMuMG cells unstimulated or stimulated with TGFβ1 (5 ng/ml) for 24 or 48 h, in the presence of vehicle or 10 μM 5-aza. ( B ) Phase contrast microscopy of cells as described in (A). Scale bar: 50 μm. ( C ) Luciferase reporter assay of human Cdh1 promoter in NMuMG cells stimulated with TGFβ1 for the indicated time period. ( D ) HpaII–MspI digestion–methylation assay of Cdh1 promoter in NMuMG cells stimulated with TGFβ1 for the indicated time period. ( E ) Dnmt mRNA levels after TGFβ1 treatment of NMuMG cells for the indicated time periods, were normalized to Gapdh mRNA expression. Expression values of the 0-h time point are normalized to 1. ( F ) Dnmt3a mRNA expression in NMuMG cells pre-treated for 30 min with or without TGFβ type I receptor kinase inhibitor SB505124 (10 μM), in the absence or presence of TGFβ1 for 24 h. DMSO was used as a vehicle. qPCR values were normalized to that of Gapdh and the expression values of DMSO-treated, unstimulated cells were normalized to 1. ( G ) Phase contrast images (left panel) and immunoblot analysis (right panel) of DNMT3A and GAPDH protein levels in NMuMG cells transfected with si Control or si Dnmt3a , untreated or treated with TGFβ1 for 24 h. Scale bar : 50 μm. ( H ) E-cadherin, DNMT1, DNMT3A and α-tubulin protein levels in NMuMG cells transfected with si Control , si Dnmt1 or si Dnmt3a , in the absence or presence of TGFβ1 for 48 h.

    Techniques Used: Western Blot, Microscopy, Luciferase, Reporter Assay, Methylation, Expressing, Real-time Polymerase Chain Reaction, Transfection

    6) Product Images from "Poly(ADP-ribosyl)ation is involved in the epigenetic control of TET1 gene transcription"

    Article Title: Poly(ADP-ribosyl)ation is involved in the epigenetic control of TET1 gene transcription

    Journal: Oncotarget

    doi:

    DNA methylation negatively regulates TET1 transcription (A) Western blot analysis of TET1 protein in MOLT-3 and SKW-3 cell lines. LAMIN B1 (LMNB1) was used as loading control. (B) Schematic representation of TET1 gene where the expanded region is the fragment investigated by EpiTYPER assay for DNA methylation analysis. Each stick is a CpG and each circle represents CpGs detectable by EpiTYPER assay. (C) DNA methylation analysis of TET1 CGI performed by using EpiTYPER assay. (D) Analysis of TET1 DNA methylation using MspI/HpaII restriction and PCR amplification performed on SKW-3 cells treated for 48 hrs with different doses of 5-azacytidine (5-aza). U, uncut; H, HpaII; M, MspI. (E) qRT-PCR analysis of TET1 gene expression on SKW-3 cells treated for 48 hrs with different doses of 5-aza. The results are shown as means ± S.E.M. (n=3). P-value was determined by paired Student's t-test (**P
    Figure Legend Snippet: DNA methylation negatively regulates TET1 transcription (A) Western blot analysis of TET1 protein in MOLT-3 and SKW-3 cell lines. LAMIN B1 (LMNB1) was used as loading control. (B) Schematic representation of TET1 gene where the expanded region is the fragment investigated by EpiTYPER assay for DNA methylation analysis. Each stick is a CpG and each circle represents CpGs detectable by EpiTYPER assay. (C) DNA methylation analysis of TET1 CGI performed by using EpiTYPER assay. (D) Analysis of TET1 DNA methylation using MspI/HpaII restriction and PCR amplification performed on SKW-3 cells treated for 48 hrs with different doses of 5-azacytidine (5-aza). U, uncut; H, HpaII; M, MspI. (E) qRT-PCR analysis of TET1 gene expression on SKW-3 cells treated for 48 hrs with different doses of 5-aza. The results are shown as means ± S.E.M. (n=3). P-value was determined by paired Student's t-test (**P

    Techniques Used: DNA Methylation Assay, Western Blot, EpiTYPER Assay, Polymerase Chain Reaction, Amplification, Quantitative RT-PCR, Expressing

    PARylation regulates DNA methylation patterns of TET1 CpG island (A) Schematic representation of TET1 gene where fragments BC (arrows) and A (arrowheads), amplified by PCR after endonuclease restriction for DNA methylation analysis, are indicated. Fragment BC contains recognition sites for MspI/HpaII which are not present in fragment A. The expanded region corresponds to the fragment analyzed by bisulfite sequencing where each stick is a CpG dinucleotide. (B) Analysis of TET1 DNA methylation using MspI/HpaII restriction and PCR amplification performed on MOLT-3 and SKW-3 treated for 72 hrs with 1μM PJ-34 or 1μM ABT-888. Fragment A was used as loading control. (C) Analysis of TET1 DNA methylation by bisulfite sequencing performed on MOLT-3 treated for 72 hrs with 1μM PJ-34 or 1μM ABT-888. Each clone is represented by a row, and the CpG dinucleotide being investigated is arranged in columns. White and black circles represent unmethylated and methylated/modified cytosines, respectively. (D) Analysis of DNA hydroxymethylation of TET1 CGI performed using glucosyltransferase reaction and MspI restriction followed by PCR amplification. Fragment A was used as loading control. Analysis was performed on MOLT-3 treated for 72 hrs with 1μM PJ-34 or 1μM ABT-888.
    Figure Legend Snippet: PARylation regulates DNA methylation patterns of TET1 CpG island (A) Schematic representation of TET1 gene where fragments BC (arrows) and A (arrowheads), amplified by PCR after endonuclease restriction for DNA methylation analysis, are indicated. Fragment BC contains recognition sites for MspI/HpaII which are not present in fragment A. The expanded region corresponds to the fragment analyzed by bisulfite sequencing where each stick is a CpG dinucleotide. (B) Analysis of TET1 DNA methylation using MspI/HpaII restriction and PCR amplification performed on MOLT-3 and SKW-3 treated for 72 hrs with 1μM PJ-34 or 1μM ABT-888. Fragment A was used as loading control. (C) Analysis of TET1 DNA methylation by bisulfite sequencing performed on MOLT-3 treated for 72 hrs with 1μM PJ-34 or 1μM ABT-888. Each clone is represented by a row, and the CpG dinucleotide being investigated is arranged in columns. White and black circles represent unmethylated and methylated/modified cytosines, respectively. (D) Analysis of DNA hydroxymethylation of TET1 CGI performed using glucosyltransferase reaction and MspI restriction followed by PCR amplification. Fragment A was used as loading control. Analysis was performed on MOLT-3 treated for 72 hrs with 1μM PJ-34 or 1μM ABT-888.

    Techniques Used: DNA Methylation Assay, Amplification, Polymerase Chain Reaction, Methylation Sequencing, Methylation, Modification

    7) Product Images from "AFSM sequencing approach: a simple and rapid method for genome-wide SNP and methylation site discovery and genetic mapping"

    Article Title: AFSM sequencing approach: a simple and rapid method for genome-wide SNP and methylation site discovery and genetic mapping

    Journal: Scientific Reports

    doi: 10.1038/srep07300

    AFSM adapters and primers. (a, c) Sequences of three different types of double-stranded AFSM adapters. The barcode adapter terminates with a 5-bp barcode at the 3′ end of its top stand and a 5-bp overhang at the 5′ end of its bottom strand that is complementary to the sticky end generated by EcoRI. The HpaII-methylation adapter has a HpaII-compatible sticky end, and the MspI-methylation adapter has a MspI-compatible sticky end. (b) PCR primer sequences for EcoRI-HpaII. (d) PCR primer sequences for EcoRI-MspI.
    Figure Legend Snippet: AFSM adapters and primers. (a, c) Sequences of three different types of double-stranded AFSM adapters. The barcode adapter terminates with a 5-bp barcode at the 3′ end of its top stand and a 5-bp overhang at the 5′ end of its bottom strand that is complementary to the sticky end generated by EcoRI. The HpaII-methylation adapter has a HpaII-compatible sticky end, and the MspI-methylation adapter has a MspI-compatible sticky end. (b) PCR primer sequences for EcoRI-HpaII. (d) PCR primer sequences for EcoRI-MspI.

    Techniques Used: Generated, Methylation, Polymerase Chain Reaction

    Preparation and sequencing of AFSM tags. Sample preparation for AFSM genotyping is accomplished by combining two restriction enzyme pairs (EcoRI-MspI and EcoRI-HpaII) to digest genomic DNA and incorporating barcodes for multiplex sequencing. EcoRI is used as a rare cutter, and the methylation-retraction enzymes HpaII and MspI are employed as frequent cutters. HpaII and MspI have different sensitivities to methylation of the inner or outer cytosines and can produce different products, reflecting the different methylation states of the cytosines.
    Figure Legend Snippet: Preparation and sequencing of AFSM tags. Sample preparation for AFSM genotyping is accomplished by combining two restriction enzyme pairs (EcoRI-MspI and EcoRI-HpaII) to digest genomic DNA and incorporating barcodes for multiplex sequencing. EcoRI is used as a rare cutter, and the methylation-retraction enzymes HpaII and MspI are employed as frequent cutters. HpaII and MspI have different sensitivities to methylation of the inner or outer cytosines and can produce different products, reflecting the different methylation states of the cytosines.

    Techniques Used: Sequencing, Sample Prep, Multiplex Assay, Methylation

    8) Product Images from "Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation"

    Article Title: Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation

    Journal: Epigenetics

    doi: 10.4161/epi.24280

    Figure 1. Schematic presentation of the HMST-Seq method. For (A) “C + mC” library, the genomic DNA was first glucosylated, and then digested with MspI. For (B) “C” library and (C) “C + mC + hmC”
    Figure Legend Snippet: Figure 1. Schematic presentation of the HMST-Seq method. For (A) “C + mC” library, the genomic DNA was first glucosylated, and then digested with MspI. For (B) “C” library and (C) “C + mC + hmC”

    Techniques Used:

    9) Product Images from "Genetic and Epigenetic Changes during the Upward Expansion of Deyeuxia angustifolia Kom. in the Alpine Tundra of the Changbai Mountains, China"

    Article Title: Genetic and Epigenetic Changes during the Upward Expansion of Deyeuxia angustifolia Kom. in the Alpine Tundra of the Changbai Mountains, China

    Journal: Plants

    doi: 10.3390/plants10020291

    Relative DNA methylation levels in all populations of D. angustifolia. I—fragments present in both profiles (1,1), unmethylated state; II—fragments were present only with EcoRI/HpaII (1,0), hemi-methylation of external cytosine; III—fragments were present only with EcoRI/MspI (0,1), full-methylation of internal cytosine; IV—fragments were absent with both profile (0,0), uninformative site.
    Figure Legend Snippet: Relative DNA methylation levels in all populations of D. angustifolia. I—fragments present in both profiles (1,1), unmethylated state; II—fragments were present only with EcoRI/HpaII (1,0), hemi-methylation of external cytosine; III—fragments were present only with EcoRI/MspI (0,1), full-methylation of internal cytosine; IV—fragments were absent with both profile (0,0), uninformative site.

    Techniques Used: DNA Methylation Assay, Methylation

    10) Product Images from "Microsphere-Based Multiplex Analysis of DNA Methylation in Acute Myeloid Leukemia"

    Article Title: Microsphere-Based Multiplex Analysis of DNA Methylation in Acute Myeloid Leukemia

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2013.10.010

    The MELP assay accurately reflects HELP-derived data. A: Comparison of HELP-derived HpaII/MspI ratios ( x axis) to MELP-derived ratios ( y axis) at the 18 loci used in the methylation classifier for AML ( r = 0.63 to 0.92, P
    Figure Legend Snippet: The MELP assay accurately reflects HELP-derived data. A: Comparison of HELP-derived HpaII/MspI ratios ( x axis) to MELP-derived ratios ( y axis) at the 18 loci used in the methylation classifier for AML ( r = 0.63 to 0.92, P

    Techniques Used: Derivative Assay, Methylation

    A: Schematic of the HELP assay. Genomic DNA is digested with either MspI (methylation insensitive) or HpaII (methylation sensitive). The resulting fragments are ligated to linkers and PCR amplified with linker-specific primers. Amplicons are fluorescently
    Figure Legend Snippet: A: Schematic of the HELP assay. Genomic DNA is digested with either MspI (methylation insensitive) or HpaII (methylation sensitive). The resulting fragments are ligated to linkers and PCR amplified with linker-specific primers. Amplicons are fluorescently

    Techniques Used: HELP Assay, Methylation, Polymerase Chain Reaction, Amplification

    11) Product Images from "Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource"

    Article Title: Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx900

    Detection of DNA methylation in Tnp1 and Pfn3 minigenes with DNMT3 overexpression. ( A ) Location of HpaII/MspI isochizomer target sequences in the Tnp1 and Pfn3 minigenes (red arrowheads). HpaII cleavage is inhibited by overlapping methylation, whereas MspI is insensitive. Primer binding sites flanking the restriction site (methyl-sensitive) and undigested control region are shown. ( B ) Methylation-sensitive restriction enzyme (MSRE)-PCR in basal and induced Tnp1 and Pfn3 minigenes. Minimal signal intensity in the HpaII-digested samples is indicative of minor to absent overlapping DNA methylation at the interrogated sites. ( C ) Immunoblotting for DNMT3 overexpression in Tnp1 and Pfn3 minigene host cells with transient transfection of mammalian expression constructs encoding DNMT3A or DNMT3B, as compared to vector control. Blotting for β-tubulin served as loading control. ( D ) MSRE-PCR in Tnp1 and Pfn3 host cells with overexpression of DNMT3A and DNMT3B. Gains in DNA methylation are evident through amplification with the methyl-sensitive primer sets following HpaII digest in DNMT3-transfected cells versus vector control (red boxes). (–) Dox samples shown; (+) Dox samples and control primers are presented in Supplementary Figure S7 . ( E ) MSRE-qPCR for quantification of resistance to HpaII digest (left), and concurrent susceptibility to MspI (right), in the DNMT3-transfected minigene host cells +/– Dox-induction. Values represent amplification with methyl-sensitive primers relative to the downstream control primer sets that do not overlap a HpaII/MspI site. Mean ± SD of two independent, biological replicates are shown. P -values = two-tailed Student's t -test comparing DNMT3A (left italic) and DNMT3B (right italics) to control. In addition, for Pfn3 + 5′3′ DNMT3A versus DNMT3B values were statistically significant ( P = 4.5e–3 and 5.0e–4 for minus and plus Dox, respectively).
    Figure Legend Snippet: Detection of DNA methylation in Tnp1 and Pfn3 minigenes with DNMT3 overexpression. ( A ) Location of HpaII/MspI isochizomer target sequences in the Tnp1 and Pfn3 minigenes (red arrowheads). HpaII cleavage is inhibited by overlapping methylation, whereas MspI is insensitive. Primer binding sites flanking the restriction site (methyl-sensitive) and undigested control region are shown. ( B ) Methylation-sensitive restriction enzyme (MSRE)-PCR in basal and induced Tnp1 and Pfn3 minigenes. Minimal signal intensity in the HpaII-digested samples is indicative of minor to absent overlapping DNA methylation at the interrogated sites. ( C ) Immunoblotting for DNMT3 overexpression in Tnp1 and Pfn3 minigene host cells with transient transfection of mammalian expression constructs encoding DNMT3A or DNMT3B, as compared to vector control. Blotting for β-tubulin served as loading control. ( D ) MSRE-PCR in Tnp1 and Pfn3 host cells with overexpression of DNMT3A and DNMT3B. Gains in DNA methylation are evident through amplification with the methyl-sensitive primer sets following HpaII digest in DNMT3-transfected cells versus vector control (red boxes). (–) Dox samples shown; (+) Dox samples and control primers are presented in Supplementary Figure S7 . ( E ) MSRE-qPCR for quantification of resistance to HpaII digest (left), and concurrent susceptibility to MspI (right), in the DNMT3-transfected minigene host cells +/– Dox-induction. Values represent amplification with methyl-sensitive primers relative to the downstream control primer sets that do not overlap a HpaII/MspI site. Mean ± SD of two independent, biological replicates are shown. P -values = two-tailed Student's t -test comparing DNMT3A (left italic) and DNMT3B (right italics) to control. In addition, for Pfn3 + 5′3′ DNMT3A versus DNMT3B values were statistically significant ( P = 4.5e–3 and 5.0e–4 for minus and plus Dox, respectively).

    Techniques Used: DNA Methylation Assay, Over Expression, Methylation, Binding Assay, Polymerase Chain Reaction, Transfection, Expressing, Construct, Plasmid Preparation, Amplification, Real-time Polymerase Chain Reaction, Two Tailed Test

    12) Product Images from "Cell-type-specific brain methylomes profiled via ultralow-input microfluidics"

    Article Title: Cell-type-specific brain methylomes profiled via ultralow-input microfluidics

    Journal: Nature biomedical engineering

    doi:

    MID-RRBS generated high quality data using sub-1 ng DNA (a) The percentage of the theoretical maximum of CpGs covered at 1× and 10× coverage with starting DNA samples of various amounts. MspI in silico . (e) Saturation analysis of MID-RRBS data in comparison with other works. The analysis was conducted by random selection of a number of raw reads followed by using the same pipeline to identify unique CpGs. Each data point was generated with 4 subsamplings per dataset. The error bars represent s.d. The centre represents mean. n = 3 for LCM-RRBS, n = 8 for mRRBS, n = 2 for the rest.
    Figure Legend Snippet: MID-RRBS generated high quality data using sub-1 ng DNA (a) The percentage of the theoretical maximum of CpGs covered at 1× and 10× coverage with starting DNA samples of various amounts. MspI in silico . (e) Saturation analysis of MID-RRBS data in comparison with other works. The analysis was conducted by random selection of a number of raw reads followed by using the same pipeline to identify unique CpGs. Each data point was generated with 4 subsamplings per dataset. The error bars represent s.d. The centre represents mean. n = 3 for LCM-RRBS, n = 8 for mRRBS, n = 2 for the rest.

    Techniques Used: Generated, In Silico, Selection, Laser Capture Microdissection

    13) Product Images from "Laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse"

    Article Title: Laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt230

    LCM-RRBS workflow. A complex tissue is dissected using LCM. Extracted DNA is digested by the methylation-insensitive enzyme MspI, end repaired and ligated with methylated Illumina adapters. After bisulfite conversion, each sample is ‘barcoded’ by introducing a sample-specific index (shown as green, blue or violet boxes) through low-cycle PCR. Samples are pooled and loaded onto a high-percentage gel for fragment separation and size selection. Using universal primers, the final library is amplified and sequenced on the Illumina platform.
    Figure Legend Snippet: LCM-RRBS workflow. A complex tissue is dissected using LCM. Extracted DNA is digested by the methylation-insensitive enzyme MspI, end repaired and ligated with methylated Illumina adapters. After bisulfite conversion, each sample is ‘barcoded’ by introducing a sample-specific index (shown as green, blue or violet boxes) through low-cycle PCR. Samples are pooled and loaded onto a high-percentage gel for fragment separation and size selection. Using universal primers, the final library is amplified and sequenced on the Illumina platform.

    Techniques Used: Laser Capture Microdissection, Methylation, Polymerase Chain Reaction, Selection, Amplification

    14) Product Images from "Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation"

    Article Title: Methylation detection oligonucleotide microarray analysis: a high-resolution method for detection of CpG island methylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp413

    Schematic of the procedure. Shown at the top is genomic DNA with a CpG island that is methylated. The DNA is cleaved with the restriction endonuclease MspI and adaptors ligated. The ligated material is divided evenly, one half being digested with McrBC and the other half being mock digested. This material is used as template for PCR amplification and the resulting product is used for microarray comparison.
    Figure Legend Snippet: Schematic of the procedure. Shown at the top is genomic DNA with a CpG island that is methylated. The DNA is cleaved with the restriction endonuclease MspI and adaptors ligated. The ligated material is divided evenly, one half being digested with McrBC and the other half being mock digested. This material is used as template for PCR amplification and the resulting product is used for microarray comparison.

    Techniques Used: Methylation, Polymerase Chain Reaction, Amplification, Microarray

    McrBC PCR of two different fragments of the MTSS1 CpG island for tumors identified of having methylation of this island compared to matched normal samples. Fragment 1 encompasses the MspI fragment we have identified as being methylated and overlaps the gene TSS. Fragment 5, we do not detect methylation. Both Normal and Tumor were digested with McrBC or mock digested for both matched pairs.
    Figure Legend Snippet: McrBC PCR of two different fragments of the MTSS1 CpG island for tumors identified of having methylation of this island compared to matched normal samples. Fragment 1 encompasses the MspI fragment we have identified as being methylated and overlaps the gene TSS. Fragment 5, we do not detect methylation. Both Normal and Tumor were digested with McrBC or mock digested for both matched pairs.

    Techniques Used: Polymerase Chain Reaction, Methylation

    15) Product Images from "Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides"

    Article Title: Biotinylation of Deoxyguanosine at the Abasic Site in Double-Stranded Oligodeoxynucleotides

    Journal: Journal of Analytical Methods in Chemistry

    doi: 10.1155/2016/4681421

    Restriction digestion and gel electrophoresis of the PCR amplified products using SS-G (a), SS-abasic site (b), or the biotinylated DS-G/abasic site (c) as the template. The restriction enzyme MspI which recognizes the CCGG sequence was used to cleave the PCR amplified product.
    Figure Legend Snippet: Restriction digestion and gel electrophoresis of the PCR amplified products using SS-G (a), SS-abasic site (b), or the biotinylated DS-G/abasic site (c) as the template. The restriction enzyme MspI which recognizes the CCGG sequence was used to cleave the PCR amplified product.

    Techniques Used: Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Amplification, Sequencing

    16) Product Images from "Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation"

    Article Title: Integrated detection of both 5-mC and 5-hmC by high-throughput tag sequencing technology highlights methylation reprogramming of bivalent genes during cellular differentiation

    Journal: Epigenetics

    doi: 10.4161/epi.24280

    Figure 1. Schematic presentation of the HMST-Seq method. For (A) “C + mC” library, the genomic DNA was first glucosylated, and then digested with MspI. For (B) “C” library and (C) “C + mC + hmC”
    Figure Legend Snippet: Figure 1. Schematic presentation of the HMST-Seq method. For (A) “C + mC” library, the genomic DNA was first glucosylated, and then digested with MspI. For (B) “C” library and (C) “C + mC + hmC”

    Techniques Used:

    17) Product Images from "Ascorbic acid–induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma"

    Article Title: Ascorbic acid–induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI98747

    AA leads to increased TET activity and 5hmC levels in ccRCC cells. ( A ) Schematic showing the role of AA as an essential cofactor for TET enzymatic activity. ( B ) Intracellular L2HG levels measured by MS in ccRCC cell line 786-O are much higher than in the immortalized normal kidney cell line HKC8 ( n = 2). ( C and D ) TET activity was measured in vitro with AA-treated RCC cells (769-P and 786-O) and was increased after treatment. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). Exposure time was 4 hours, mimicking bioavailability curves with i.v. AA, followed by 24-hour incubation with fresh media prior to harvesting the cells for nuclear extraction and TET activity analysis. We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.025 (0.05/2 to account for multiple comparisons). ( E ) 5hmC was measured by LC-ESI-MS/MS and was significantly increased after AA treatment of RCC cells 769-P. Addition of catalase did not change the percentage of 5hmC. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.0125 (0.05/4 to account for multiple variations). ( F ) Unsupervised clustering based on genome-wide methylation analysis conducted by HELP assay. Ward clustering shows global methylation changes are induced by AA treatment. ( G ) Histograms based on methylation (log [HpaII/MspI]) show increased hypomethylation after AA treatment. ( H ) Smad6 promoter becomes demethylated after AA treatment in both 786-O and 769-P ccRCC cells.
    Figure Legend Snippet: AA leads to increased TET activity and 5hmC levels in ccRCC cells. ( A ) Schematic showing the role of AA as an essential cofactor for TET enzymatic activity. ( B ) Intracellular L2HG levels measured by MS in ccRCC cell line 786-O are much higher than in the immortalized normal kidney cell line HKC8 ( n = 2). ( C and D ) TET activity was measured in vitro with AA-treated RCC cells (769-P and 786-O) and was increased after treatment. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). Exposure time was 4 hours, mimicking bioavailability curves with i.v. AA, followed by 24-hour incubation with fresh media prior to harvesting the cells for nuclear extraction and TET activity analysis. We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.025 (0.05/2 to account for multiple comparisons). ( E ) 5hmC was measured by LC-ESI-MS/MS and was significantly increased after AA treatment of RCC cells 769-P. Addition of catalase did not change the percentage of 5hmC. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.0125 (0.05/4 to account for multiple variations). ( F ) Unsupervised clustering based on genome-wide methylation analysis conducted by HELP assay. Ward clustering shows global methylation changes are induced by AA treatment. ( G ) Histograms based on methylation (log [HpaII/MspI]) show increased hypomethylation after AA treatment. ( H ) Smad6 promoter becomes demethylated after AA treatment in both 786-O and 769-P ccRCC cells.

    Techniques Used: Activity Assay, Mass Spectrometry, In Vitro, Incubation, IF-P, Genome Wide, Methylation, HELP Assay

    18) Product Images from "Colonization of the Cecal Mucosa by Helicobacter hepaticus Impacts the Diversity of the Indigenous Microbiota "

    Article Title: Colonization of the Cecal Mucosa by Helicobacter hepaticus Impacts the Diversity of the Indigenous Microbiota

    Journal: Infection and Immunity

    doi: 10.1128/IAI.73.10.6852-6961.2005

    Changes in diversity measures of the mucosa-associated microbiota of the murine cecum 30 days after experimental infection with H. hepaticus . The Shannon diversity index ( H ) and Shannon evenness ( H / H max ) were calculated for each mouse based on normalized T-RFLP profiles obtained with HhaI and MspI digestion. Compared to uninfected animals (“Uninfected” category), animals experimentally infected with H. hepaticus (“Infected” category) had significant decreases in both diversity and evenness. When the analysis was repeated, this time suppressing the H. hepaticus terminal restriction fragment before normalization of the profiles (“Infected [suppressed]” category), the change in diversity and evenness in infected animals was no longer apparent. Comparisons for all pairs of time points was performed by analysis of variance using Tukey-Kramer honestly significant difference. Categories within each plot not connected by the same roman numeral are significantly different with an alpha level set to 0.05.
    Figure Legend Snippet: Changes in diversity measures of the mucosa-associated microbiota of the murine cecum 30 days after experimental infection with H. hepaticus . The Shannon diversity index ( H ) and Shannon evenness ( H / H max ) were calculated for each mouse based on normalized T-RFLP profiles obtained with HhaI and MspI digestion. Compared to uninfected animals (“Uninfected” category), animals experimentally infected with H. hepaticus (“Infected” category) had significant decreases in both diversity and evenness. When the analysis was repeated, this time suppressing the H. hepaticus terminal restriction fragment before normalization of the profiles (“Infected [suppressed]” category), the change in diversity and evenness in infected animals was no longer apparent. Comparisons for all pairs of time points was performed by analysis of variance using Tukey-Kramer honestly significant difference. Categories within each plot not connected by the same roman numeral are significantly different with an alpha level set to 0.05.

    Techniques Used: Infection

    19) Product Images from "Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function"

    Article Title: Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function

    Journal: Developmental Cell

    doi: 10.1016/j.devcel.2017.05.021

    Mouse TDRD9 Is an ATPase, and Its Activity Is Essential for Transposon Silencing, but Not for piRNA Biogenesis (A) Domain architecture of mouse TDRD9 with putative consensus amino acid residues responsible for ATP binding and ATP hydrolysis is shown. The point mutation E257Q that abolishes ATPase activity is indicated. (B) Quality of recombinant mouse TDRD9 protein used for ATPase assays. Wild-type and E257Q point mutant versions were produced. (C) Thin-layer chromatography of ATPase reactions revealing the faster-migrating free phosphate in the presence of the wild-type TDRD9 protein. (D) Creation of the catalytic-dead Tdrd9 knockin (KI) mouse carrying the E257Q mutation in the ATPase motif (DEVH → DQVH). The same mouse line also allows creation of the knockout (−/−) mutant, by using loxP sites flanking exons 3–5. See also Figure S6 . (E) Representative image of adult testes from indicated genotypes, showing atrophied testes in homozygous Tdrd9 knockout and knockin mutants. (F) H E staining of adult testes from homozygous Tdrd9 knockin mutant showing arrested germ cell development. Scale bar, 40 μm. See also Figure S7 A. (G) Northern analysis of transposons in total testicular RNA, showing derepression of LINE1 retrotransposons in homozygous Tdrd9 knockout and knockin mutants. Age of donor animals is indicated. (H) Western analysis of total testicular lysates for L1ORF1p expression. MILI (germ cell marker) and TUBULIN (loading control) expression was also examined. (I) Methylation-sensitive Southern blotting for LINE1 genomic loci. The red arrows point to fragments appearing under conditions of reduced DNA methylation in the homozygous Tdrd9 mutants. H, HpaII-digested DNA; M, MspI-digested DNA. (J) Immunoprecipitation of PIWI proteins and 5′ end labeling of associated small RNAs from neonatal (P0) testes. (K) Comparison of MILI-associated piRNAs mapping to individual repeats. There is a striking enrichment of the piRNAs produced from LINE and LTR repeats in Tdrd9 mutants ( Tdrd9 KI / KI and Tdrd9 − / − ). See also Figure S8 . (L) Graphs show the distribution of MIWI2-associated piRNAs mapped along B1Mus1.SINE consensus sequence, revealing a depletion of piRNAs in the Tdrd9 KI / KI and Tdrd9 − / − mutants. (M) Immunofluorescence analysis of indicated proteins in embryonic testes (embryonic day 16.5) of the different genotypes. Note the nucleo-cytoplasmic distribution of TDRD9 in wild-type germ cells, while it is restricted to the cytoplasm in the Tdrd9 KI / KI mutant.
    Figure Legend Snippet: Mouse TDRD9 Is an ATPase, and Its Activity Is Essential for Transposon Silencing, but Not for piRNA Biogenesis (A) Domain architecture of mouse TDRD9 with putative consensus amino acid residues responsible for ATP binding and ATP hydrolysis is shown. The point mutation E257Q that abolishes ATPase activity is indicated. (B) Quality of recombinant mouse TDRD9 protein used for ATPase assays. Wild-type and E257Q point mutant versions were produced. (C) Thin-layer chromatography of ATPase reactions revealing the faster-migrating free phosphate in the presence of the wild-type TDRD9 protein. (D) Creation of the catalytic-dead Tdrd9 knockin (KI) mouse carrying the E257Q mutation in the ATPase motif (DEVH → DQVH). The same mouse line also allows creation of the knockout (−/−) mutant, by using loxP sites flanking exons 3–5. See also Figure S6 . (E) Representative image of adult testes from indicated genotypes, showing atrophied testes in homozygous Tdrd9 knockout and knockin mutants. (F) H E staining of adult testes from homozygous Tdrd9 knockin mutant showing arrested germ cell development. Scale bar, 40 μm. See also Figure S7 A. (G) Northern analysis of transposons in total testicular RNA, showing derepression of LINE1 retrotransposons in homozygous Tdrd9 knockout and knockin mutants. Age of donor animals is indicated. (H) Western analysis of total testicular lysates for L1ORF1p expression. MILI (germ cell marker) and TUBULIN (loading control) expression was also examined. (I) Methylation-sensitive Southern blotting for LINE1 genomic loci. The red arrows point to fragments appearing under conditions of reduced DNA methylation in the homozygous Tdrd9 mutants. H, HpaII-digested DNA; M, MspI-digested DNA. (J) Immunoprecipitation of PIWI proteins and 5′ end labeling of associated small RNAs from neonatal (P0) testes. (K) Comparison of MILI-associated piRNAs mapping to individual repeats. There is a striking enrichment of the piRNAs produced from LINE and LTR repeats in Tdrd9 mutants ( Tdrd9 KI / KI and Tdrd9 − / − ). See also Figure S8 . (L) Graphs show the distribution of MIWI2-associated piRNAs mapped along B1Mus1.SINE consensus sequence, revealing a depletion of piRNAs in the Tdrd9 KI / KI and Tdrd9 − / − mutants. (M) Immunofluorescence analysis of indicated proteins in embryonic testes (embryonic day 16.5) of the different genotypes. Note the nucleo-cytoplasmic distribution of TDRD9 in wild-type germ cells, while it is restricted to the cytoplasm in the Tdrd9 KI / KI mutant.

    Techniques Used: Activity Assay, Binding Assay, Mutagenesis, Recombinant, Produced, Thin Layer Chromatography, Knock-In, Knock-Out, Staining, Northern Blot, Western Blot, Expressing, Marker, Methylation, Southern Blot, DNA Methylation Assay, Immunoprecipitation, End Labeling, Sequencing, Immunofluorescence

    Catalytic Activity of MVH Is Essential for Transposon Silencing and Biogenesis of MIWI2 piRNAs (A) Creation of the catalytic-dead Mvh mouse carrying a point mutation E446Q in the ATPase motif (DEAD → DQAD). See also Figure S2 . (B) Representative testes from adult animals (P80; 80 days old) of indicated Mvh genotypes. (C) Testes weight in different genotypes. (D and E) H E staining of adult mouse testes showing arrested germ cell development in the Mvh − / KI mutant (D), and (E) presence of sperm in the lumen of the wild-type epididymis, but not from that of the mutant. sp, spermatocytes; rs, round spermatids; es, elongated spermatids. Scale bars, 50 μm. (F) Staining for γ-H2AX in adult testes sections. Arrows point to the XY body. Scale bar, 10 μm. (G) Northern analysis for indicated transposon transcripts in total testicular RNA. The donor animals are numbered and their ages indicated. Total testicular DNA from the same animals were used for Southern blotting in (I). (H) Staining for L1ORF1p in mouse testes from animals of indicated ages. Scale bars, 38 μm (upper) and 48 μm (lower). (I) Methylation-sensitive Southern blotting examining L1 genomic loci. The donor animals are the same as those used for northern analysis (indicated by animal numbers). The red arrows point to the cleavage fragment seen under conditions of reduced DNA methylation, and only in the Mvh − / KI mutant. H, HpaII-digested DNA; M, MspI-digested DNA. (J and K) Immunoprecipitation of PIWI proteins from neonatal (P0) testes and 5′ end labeling of associated piRNAs. RNA size markers are 5′ end labeled (length in nucleotides). (L) Immunofluorescence detection of indicated proteins in neonatal testes Scale bar, 10 μm.
    Figure Legend Snippet: Catalytic Activity of MVH Is Essential for Transposon Silencing and Biogenesis of MIWI2 piRNAs (A) Creation of the catalytic-dead Mvh mouse carrying a point mutation E446Q in the ATPase motif (DEAD → DQAD). See also Figure S2 . (B) Representative testes from adult animals (P80; 80 days old) of indicated Mvh genotypes. (C) Testes weight in different genotypes. (D and E) H E staining of adult mouse testes showing arrested germ cell development in the Mvh − / KI mutant (D), and (E) presence of sperm in the lumen of the wild-type epididymis, but not from that of the mutant. sp, spermatocytes; rs, round spermatids; es, elongated spermatids. Scale bars, 50 μm. (F) Staining for γ-H2AX in adult testes sections. Arrows point to the XY body. Scale bar, 10 μm. (G) Northern analysis for indicated transposon transcripts in total testicular RNA. The donor animals are numbered and their ages indicated. Total testicular DNA from the same animals were used for Southern blotting in (I). (H) Staining for L1ORF1p in mouse testes from animals of indicated ages. Scale bars, 38 μm (upper) and 48 μm (lower). (I) Methylation-sensitive Southern blotting examining L1 genomic loci. The donor animals are the same as those used for northern analysis (indicated by animal numbers). The red arrows point to the cleavage fragment seen under conditions of reduced DNA methylation, and only in the Mvh − / KI mutant. H, HpaII-digested DNA; M, MspI-digested DNA. (J and K) Immunoprecipitation of PIWI proteins from neonatal (P0) testes and 5′ end labeling of associated piRNAs. RNA size markers are 5′ end labeled (length in nucleotides). (L) Immunofluorescence detection of indicated proteins in neonatal testes Scale bar, 10 μm.

    Techniques Used: Activity Assay, Mutagenesis, Staining, Northern Blot, Southern Blot, Methylation, DNA Methylation Assay, Immunoprecipitation, End Labeling, Labeling, Immunofluorescence

    20) Product Images from "Characterization of the Genome of the Dairy Lactobacillus helveticus Bacteriophage ?AQ113"

    Article Title: Characterization of the Genome of the Dairy Lactobacillus helveticus Bacteriophage ?AQ113

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00620-13

    Restriction analysis of ΦAQ113 DNA. The phage DNA was digested with BglII, HhaI, PstI, HaeIII, MspI, EcoRI, and EcoRV. Lanes M1 and M2, 1-kb Plus and λ-HindIII DNA ladders; A and B indicate that the digests were unheated and heated prior
    Figure Legend Snippet: Restriction analysis of ΦAQ113 DNA. The phage DNA was digested with BglII, HhaI, PstI, HaeIII, MspI, EcoRI, and EcoRV. Lanes M1 and M2, 1-kb Plus and λ-HindIII DNA ladders; A and B indicate that the digests were unheated and heated prior

    Techniques Used:

    21) Product Images from "DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells"

    Article Title: DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz717

    Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.
    Figure Legend Snippet: Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.

    Techniques Used: Lysis, Methylation, Ligation, Sequencing

    22) Product Images from "SlyA Protein Activates fimB Gene Expression and Type 1 Fimbriation in Escherichia coli K-12 *"

    Article Title: SlyA Protein Activates fimB Gene Expression and Type 1 Fimbriation in Escherichia coli K-12 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.266619

    Competition between H-NS and SlyA binding upstream of the fimB promoter. A , binding of H-NS to the fim03 fragment labeled at the fim03f end. Decreasing concentrations of H-NS were mixed with the fim03 DNA with and without SlyA in the “HNS” buffer. Lane 1 , no proteins; lanes 2 and 7 , 400 n m H-NS; lanes 3 and 8 , 200 n m H-NS; lanes 4 and 9 , 100 n m H-NS; lanes 5 and 10 , 50 n m H-NS; lanes 6 and 12 , 25 n m H-NS; lanes 7–12 also contained 2 μ m SlyA. B , H-NS binding with and without SlyA in the “SlyA” buffer. Lane 1 , no proteins; lane 2 , 50 n m SlyA; lane 3 , 1 μ m SlyA; lane 4 , 50 n m H-NS; lane 5 , 100 n m H-NS; lane 6 , 1 μ m SlyA, and 50 n m H-NS; lane 7, 1 μ m SlyA and 100 n m H-NS. Proteins were incubated for 15 min at 25 °C before treatment with DNase I. Products were analyzed on 6% denaturing polyacrylamide gels. Regions protected by H-NS and SlyA are indicated. The arrows indicate hypersensitive DNaseI cleavages on the DNA in the presence of H-NS, which are observed in the “SlyA” buffer ( B ) but not in the “H-NS” buffer (H-NS). The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).
    Figure Legend Snippet: Competition between H-NS and SlyA binding upstream of the fimB promoter. A , binding of H-NS to the fim03 fragment labeled at the fim03f end. Decreasing concentrations of H-NS were mixed with the fim03 DNA with and without SlyA in the “HNS” buffer. Lane 1 , no proteins; lanes 2 and 7 , 400 n m H-NS; lanes 3 and 8 , 200 n m H-NS; lanes 4 and 9 , 100 n m H-NS; lanes 5 and 10 , 50 n m H-NS; lanes 6 and 12 , 25 n m H-NS; lanes 7–12 also contained 2 μ m SlyA. B , H-NS binding with and without SlyA in the “SlyA” buffer. Lane 1 , no proteins; lane 2 , 50 n m SlyA; lane 3 , 1 μ m SlyA; lane 4 , 50 n m H-NS; lane 5 , 100 n m H-NS; lane 6 , 1 μ m SlyA, and 50 n m H-NS; lane 7, 1 μ m SlyA and 100 n m H-NS. Proteins were incubated for 15 min at 25 °C before treatment with DNase I. Products were analyzed on 6% denaturing polyacrylamide gels. Regions protected by H-NS and SlyA are indicated. The arrows indicate hypersensitive DNaseI cleavages on the DNA in the presence of H-NS, which are observed in the “SlyA” buffer ( B ) but not in the “H-NS” buffer (H-NS). The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).

    Techniques Used: Binding Assay, Labeling, Incubation, Marker

    The interaction of SlyA with the fimB promoter region in vitro . A , effect of 0, 15, 30, 60, and 120 n m SlyA dimer on the electophoretic mobility of DNA amplicons fim03 and pBS (each 11 n m ). Amplicon fim03 is 282 bp of the region upstream of fimB and includes O SA1 and O SA2 ( Fig. 1 ). Amplicon pBS is a negative control as previously described ( 27 ). The SlyA-DNA complex is indicated with an arrow . Samples were separated on a 5% polyacrylamide gel. Electrophoresis was carried out at 160 V for 35 min as described under “Experimental Procedures.” B , DNase I footprinting. The fim03 fragment, labeled at the fim03r end, was mixed with decreasing concentrations of SlyA for 15 min at 25 °C before digestion with DNaseI. Lane 1 , no SlyA; lane 2 , 1 μ m SlyA; lane 3 , 500 n m ; lane 4 , 250 n m ; lane 5 , 125 n m ; lane 6 , 62.5 n m ; lane 7 , 31 n m ; lane 8 , 15.6 n m . The products were analyzed on a 6% denaturing polyacrylamide gel. Regions protected by SlyA are indicated. The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).
    Figure Legend Snippet: The interaction of SlyA with the fimB promoter region in vitro . A , effect of 0, 15, 30, 60, and 120 n m SlyA dimer on the electophoretic mobility of DNA amplicons fim03 and pBS (each 11 n m ). Amplicon fim03 is 282 bp of the region upstream of fimB and includes O SA1 and O SA2 ( Fig. 1 ). Amplicon pBS is a negative control as previously described ( 27 ). The SlyA-DNA complex is indicated with an arrow . Samples were separated on a 5% polyacrylamide gel. Electrophoresis was carried out at 160 V for 35 min as described under “Experimental Procedures.” B , DNase I footprinting. The fim03 fragment, labeled at the fim03r end, was mixed with decreasing concentrations of SlyA for 15 min at 25 °C before digestion with DNaseI. Lane 1 , no SlyA; lane 2 , 1 μ m SlyA; lane 3 , 500 n m ; lane 4 , 250 n m ; lane 5 , 125 n m ; lane 6 , 62.5 n m ; lane 7 , 31 n m ; lane 8 , 15.6 n m . The products were analyzed on a 6% denaturing polyacrylamide gel. Regions protected by SlyA are indicated. The marker is pBR322 digested with MspI, and the sizes of the fragments were used to calculate their positions relative to the transcriptional start site of fimB (+1).

    Techniques Used: In Vitro, Amplification, Negative Control, Polyacrylamide Gel Electrophoresis, Footprinting, Labeling, Marker

    23) Product Images from "First Case of Segniliparus rotundus Pneumonia in a Patient with Bronchiectasis ▿"

    Article Title: First Case of Segniliparus rotundus Pneumonia in a Patient with Bronchiectasis ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.01023-11

    PCR restriction-enzyme polymorphism analysis (PRA). (A) Simulation of PRA of the hsp65 and rpoB genes. (B) PRA electrophoresis results. M, size marker; lanes 1 to 3, hsp65 amplicons digested by MspI from S. rugosus CIP 108378 T , S. rotundus CIP 108380
    Figure Legend Snippet: PCR restriction-enzyme polymorphism analysis (PRA). (A) Simulation of PRA of the hsp65 and rpoB genes. (B) PRA electrophoresis results. M, size marker; lanes 1 to 3, hsp65 amplicons digested by MspI from S. rugosus CIP 108378 T , S. rotundus CIP 108380

    Techniques Used: Polymerase Chain Reaction, Electrophoresis, Marker

    24) Product Images from "Telomere damage induces internal loops that generate telomeric circles"

    Article Title: Telomere damage induces internal loops that generate telomeric circles

    Journal: bioRxiv

    doi: 10.1101/2020.01.29.924951

    A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.
    Figure Legend Snippet: A two-step procedure for the purification of mammalian telomeres A. Top: agarose gel showing the separation of the large telomeric repeat fragments from the bulk DNA in a sucrose gradient. Genomic DNA (~2.5 mg) from SV40-MEFs was digested with HinfI and MspI. The digested DNA was separated by centrifugation on a sucrose gradient. Seven fractions were collected and an aliquot (~1/500) of each fraction was loaded on an agarose gel. Bottom: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the high molecular weight (HMW) fractions. B. Left: agarose gel showing the separation of the large telomeric repeat fragments from the remaining non-telomeric DNA, in the second purification round. The HMW DNA, contained in the last four fractions of the sucrose gradient described in (A), was recovered and digested with RsaI, AluI, MboI, HinfI, MspI, HphI and MnlI. The digested DNA was separated on a preparative agarose gel and the DNA migrating in the area above 5 kb was extracted from the gel. The image shows an aliquot (~1/100) of the digested DNA, separated on an agarose gel. Right: the gel was blotted onto a membrane and hybridized with a TTAGGG repeats probe to verify that telomeric repeats remained in the HMW area. C. Dot blot analysis showing the enrichment of telomeric repeats. The indicated amounts of DNA from each enrichment step were spotted on a membrane and hybridized either with a probe recognizing the long interspersed BamHI repeats or TTAGGG repeats. The amount of TTAGGG repeat signal/ng was quantified and reported relative to the signal/ng value in the initial, non-enriched DNA. D. Single molecule analysis showing the enrichment of the telomeric repeats. The DNA was combed onto silanized coverslips, denatured in situ and labeled sequentially with an antibody against single-stranded DNA and a Cy3-labeled (TTAGGG) 3 PNA probe.

    Techniques Used: Purification, Agarose Gel Electrophoresis, Centrifugation, Molecular Weight, Dot Blot, In Situ, Labeling

    25) 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

    Schematic diagram of in silico annotated genomic DNA and MSRE method . (A) Using in silico simulation, genomic DNA can be separated into MSRE sensitive and insensitive BfaI fragments (flanked by gray boxes) based on the presence or absence of internal HpaII/MspI sites (CCGG) (blue squares). Microarray probes binding to sensitive fragments are represented with green rectangles. Probes binding to insensitive fragments are represented by blue rectangles. Sensitive fragments can have multiple CCGG sites that can be fully methylated, partially methylated, or completely unmethylated in tandem. Methylated CCGG sites (CC m GG) are indicated with a red dot placed above the blue square. (B) Genomic DNA is fragmented with BfaI, ligated to H12/H24 linkers, digested with HpaII and MspI in parallel, amplified, differentially labeled and co-hybridized to Agilent CpG island arrays. Uncleaved fragments will have high intensities compared to cleaved fragments since only uncleaved fragments are amplified. Fully methylated fragments are cut by MspI but not HpaII, resulting in amplification of HpaII digested fragments only with resultant M -value [log 2 (HpaII/MspI)] > 0. Completely unmethylated or partially methylated (not all CCGGs methylated in tandem) fragments are cut by both MspI and HpaII. With the absence of amplification, these fragments resulting in low signal intensities in both channels (HpaII and MspI ∼ 0) with M = 0. Insensitive fragments should not be cut by either enzyme, resulting in M = 0.
    Figure Legend Snippet: Schematic diagram of in silico annotated genomic DNA and MSRE method . (A) Using in silico simulation, genomic DNA can be separated into MSRE sensitive and insensitive BfaI fragments (flanked by gray boxes) based on the presence or absence of internal HpaII/MspI sites (CCGG) (blue squares). Microarray probes binding to sensitive fragments are represented with green rectangles. Probes binding to insensitive fragments are represented by blue rectangles. Sensitive fragments can have multiple CCGG sites that can be fully methylated, partially methylated, or completely unmethylated in tandem. Methylated CCGG sites (CC m GG) are indicated with a red dot placed above the blue square. (B) Genomic DNA is fragmented with BfaI, ligated to H12/H24 linkers, digested with HpaII and MspI in parallel, amplified, differentially labeled and co-hybridized to Agilent CpG island arrays. Uncleaved fragments will have high intensities compared to cleaved fragments since only uncleaved fragments are amplified. Fully methylated fragments are cut by MspI but not HpaII, resulting in amplification of HpaII digested fragments only with resultant M -value [log 2 (HpaII/MspI)] > 0. Completely unmethylated or partially methylated (not all CCGGs methylated in tandem) fragments are cut by both MspI and HpaII. With the absence of amplification, these fragments resulting in low signal intensities in both channels (HpaII and MspI ∼ 0) with M = 0. Insensitive fragments should not be cut by either enzyme, resulting in M = 0.

    Techniques Used: In Silico, Microarray, Binding Assay, Methylation, Amplification, Labeling

    Related Articles

    Polymerase Chain Reaction:

    Article Title: R213W mutation in the retinoschisis 1 gene causes X-linked juvenile retinoschisis in a large Chinese family
    Article Snippet: Once the candidate mutation was identified, restriction enzyme MspI (5′…C^CGG…3′) recognizing the potential mutation site was used for all 28 family members with genomic DNA available. .. The PCR amplified products were digested with MspI at 37 °C for 5 h, according to the manufacturer’s protocol (New England Biolabs, Ipswich, MA). .. Samples were then electrophoresed on a 2% (W/V) agarose gel with 0.5 μg/ml ethidium bromide.

    Article Title: Regional Mucosa-Associated Microbiota Determine Physiological Expression of TLR2 and TLR4 in Murine Colon
    Article Snippet: PCR products were verified by electrophoresis of aliquots of PCR mixtures (8 ul) in 1.0% agarose and purified by precipitation. .. Aliquots of purified PCR products were digested by 20 U Msp I (New England Biolabs Inc.) and subsequently subjected to capillary electrophoresis using the Applied Biosystems DNA sequencer 3130. .. Restriction-digest fragment abundance was determined using GeneMapper software (Applied Biosystems).

    Amplification:

    Article Title: R213W mutation in the retinoschisis 1 gene causes X-linked juvenile retinoschisis in a large Chinese family
    Article Snippet: Once the candidate mutation was identified, restriction enzyme MspI (5′…C^CGG…3′) recognizing the potential mutation site was used for all 28 family members with genomic DNA available. .. The PCR amplified products were digested with MspI at 37 °C for 5 h, according to the manufacturer’s protocol (New England Biolabs, Ipswich, MA). .. Samples were then electrophoresed on a 2% (W/V) agarose gel with 0.5 μg/ml ethidium bromide.

    Mass Spectrometry:

    Article Title: PADI4 Haplotypes in Association with RA Mexican Patients, a New Prospect for Antigen Modulation
    Article Snippet: The PCR products were visualized by electrophoresis in 8% (29 : 1) polyacrylamide gels at 150 V for 1 h, followed by silver staining. .. The PADI4_89, PADI4_90, and PADI4_92 genotypes were identified after restriction enzyme digestion with HaeIII , MscI, and MspI, respectively (New England Biolabs, MS, USA), shown in . .. Haplotypes Haplotype analysis was done using the software PHASE v 1.0 for haplotype reconstruction, and recombination rate estimation was done using the genotypic data [ ].

    Purification:

    Article Title: Regional Mucosa-Associated Microbiota Determine Physiological Expression of TLR2 and TLR4 in Murine Colon
    Article Snippet: PCR products were verified by electrophoresis of aliquots of PCR mixtures (8 ul) in 1.0% agarose and purified by precipitation. .. Aliquots of purified PCR products were digested by 20 U Msp I (New England Biolabs Inc.) and subsequently subjected to capillary electrophoresis using the Applied Biosystems DNA sequencer 3130. .. Restriction-digest fragment abundance was determined using GeneMapper software (Applied Biosystems).

    Electrophoresis:

    Article Title: Regional Mucosa-Associated Microbiota Determine Physiological Expression of TLR2 and TLR4 in Murine Colon
    Article Snippet: PCR products were verified by electrophoresis of aliquots of PCR mixtures (8 ul) in 1.0% agarose and purified by precipitation. .. Aliquots of purified PCR products were digested by 20 U Msp I (New England Biolabs Inc.) and subsequently subjected to capillary electrophoresis using the Applied Biosystems DNA sequencer 3130. .. Restriction-digest fragment abundance was determined using GeneMapper software (Applied Biosystems).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs msp i
    T-RFLP analysis of microbial communities in mouse colonic tissue and stool samples. (A) Representative T-RFLP patterns of bacterial populations in mouse stool sample (S), proximal (PC) and distal colon (DC). 16S rRNA genes were obtained from amplification of DNA template (50 ng DNA), digested by restriction enzyme <t>Msp</t> I and analyzed by capillary electrophoresis. Fragment size in base pairs is shown at the top and peak height is shown as relative fluorescence. (B) A representative phylogenetic tree was built up from 4 littermate mice based on T-RFLP analysis. Similarities of bacterial populations between stool sample (S), proximal colon (PC) and distal colon (DC) were compared by Bray-Curtis distance calculations. The scale bar shows the distance of similarity.
    Msp I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/msp i/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    msp i - by Bioz Stars, 2021-06
    99/100 stars
      Buy from Supplier

    Image Search Results


    T-RFLP analysis of microbial communities in mouse colonic tissue and stool samples. (A) Representative T-RFLP patterns of bacterial populations in mouse stool sample (S), proximal (PC) and distal colon (DC). 16S rRNA genes were obtained from amplification of DNA template (50 ng DNA), digested by restriction enzyme Msp I and analyzed by capillary electrophoresis. Fragment size in base pairs is shown at the top and peak height is shown as relative fluorescence. (B) A representative phylogenetic tree was built up from 4 littermate mice based on T-RFLP analysis. Similarities of bacterial populations between stool sample (S), proximal colon (PC) and distal colon (DC) were compared by Bray-Curtis distance calculations. The scale bar shows the distance of similarity.

    Journal: PLoS ONE

    Article Title: Regional Mucosa-Associated Microbiota Determine Physiological Expression of TLR2 and TLR4 in Murine Colon

    doi: 10.1371/journal.pone.0013607

    Figure Lengend Snippet: T-RFLP analysis of microbial communities in mouse colonic tissue and stool samples. (A) Representative T-RFLP patterns of bacterial populations in mouse stool sample (S), proximal (PC) and distal colon (DC). 16S rRNA genes were obtained from amplification of DNA template (50 ng DNA), digested by restriction enzyme Msp I and analyzed by capillary electrophoresis. Fragment size in base pairs is shown at the top and peak height is shown as relative fluorescence. (B) A representative phylogenetic tree was built up from 4 littermate mice based on T-RFLP analysis. Similarities of bacterial populations between stool sample (S), proximal colon (PC) and distal colon (DC) were compared by Bray-Curtis distance calculations. The scale bar shows the distance of similarity.

    Article Snippet: Aliquots of purified PCR products were digested by 20 U Msp I (New England Biolabs Inc.) and subsequently subjected to capillary electrophoresis using the Applied Biosystems DNA sequencer 3130.

    Techniques: Amplification, Electrophoresis, Fluorescence, Mouse Assay

    Representative 2% agarose gel of the restriction enzyme digestion analysis using MspI. The enzyme cut the PCR products into two fragments in unaffected males (I-4, II-20) and the normal female (II-4) but could not cut the products in affected males (II-10, III-22, III-23). Female carriers (I-3, II-6, II-19) exhibit a heterozygous status.

    Journal: Molecular Vision

    Article Title: R213W mutation in the retinoschisis 1 gene causes X-linked juvenile retinoschisis in a large Chinese family

    doi:

    Figure Lengend Snippet: Representative 2% agarose gel of the restriction enzyme digestion analysis using MspI. The enzyme cut the PCR products into two fragments in unaffected males (I-4, II-20) and the normal female (II-4) but could not cut the products in affected males (II-10, III-22, III-23). Female carriers (I-3, II-6, II-19) exhibit a heterozygous status.

    Article Snippet: The PCR amplified products were digested with MspI at 37 °C for 5 h, according to the manufacturer’s protocol (New England Biolabs, Ipswich, MA).

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction

    PADI4 SNPs enzyme digestion. The figure shows digestion of three SNPs in the PADI4 gene. (a) Shows digestion of PADI4_89, with HaeIII enzyme; lane 1 represents the A/A genotype, lane 2 A/G and 3 G/G. (b) Demonstrates PADI4_90 amplification (221 bp) in lane 1 and digested products with MscI enzyme in lanes 2 (C/C genotype), 3 (C/T genotype), and 4 (T/T genotype). (c) Shows amplification product of PADI4_92 in lane 1 (363 bp) and restriction products obtained with the enzyme MspI; lane 2 corresponds to the G/G genotype, lane 3 G/C, and lane 4 C/C. Visualized in 8% (29 : 1) polyacrylamide gel with silver staining. M: molecular weight marker (50 bp).

    Journal: Clinical and Developmental Immunology

    Article Title: PADI4 Haplotypes in Association with RA Mexican Patients, a New Prospect for Antigen Modulation

    doi: 10.1155/2013/383681

    Figure Lengend Snippet: PADI4 SNPs enzyme digestion. The figure shows digestion of three SNPs in the PADI4 gene. (a) Shows digestion of PADI4_89, with HaeIII enzyme; lane 1 represents the A/A genotype, lane 2 A/G and 3 G/G. (b) Demonstrates PADI4_90 amplification (221 bp) in lane 1 and digested products with MscI enzyme in lanes 2 (C/C genotype), 3 (C/T genotype), and 4 (T/T genotype). (c) Shows amplification product of PADI4_92 in lane 1 (363 bp) and restriction products obtained with the enzyme MspI; lane 2 corresponds to the G/G genotype, lane 3 G/C, and lane 4 C/C. Visualized in 8% (29 : 1) polyacrylamide gel with silver staining. M: molecular weight marker (50 bp).

    Article Snippet: The PADI4_89, PADI4_90, and PADI4_92 genotypes were identified after restriction enzyme digestion with HaeIII , MscI, and MspI, respectively (New England Biolabs, MS, USA), shown in .

    Techniques: Amplification, Silver Staining, Molecular Weight, Marker

    An overview of MAPMEDS. (a) Schematic structure of barcoded lentiviral constructs. The library vector contains short barcode, CMV promoter and mClover fluorescent protein as a reporter. The barcode is random 16-bp-long DNA with repeats of A,G and T. MspI restriction site is integrated upstream CMV promoter for genomic location mapping. (b) Barcoded lentivirus was packed and transduced into K562 cells at low MOI to create founder cells with singly integrated reporter. (c-d) Barcoded founder cells, selected by flow cytometry, were expanded for two weeks and split into two pools. Cells in the first pool were collected for locating reporter integration site. Founder cells in the second pool were sorted into 96-well plates to establish clonal cell lines. Barcode of each clone was simultaneously identified by split-pool encoding and deep sequencing. Library of characterized reporter clones is a useful resource to examine loci specific epigenetic drug sensitivity. (e) Loci specific effects were decoupled from global effects through mixing individual barcoded clones of interests with control cells expressing mClover and IRFP from multiple integration sites. Co-cultured cells were treated with TSA, JQ1 and 5’AZA for 24 hours. Expression of reporter proteins were measured by flow cytometers. Distribution of mClover expression in control cells was used to remove global effects of drugs. (f) A cartoon illustrating known mechanisms of actions of Trichostatin A, JQ1 and 5-Azacytidine.

    Journal: bioRxiv

    Article Title: Loci specific epigenetic drug sensitivity

    doi: 10.1101/686139

    Figure Lengend Snippet: An overview of MAPMEDS. (a) Schematic structure of barcoded lentiviral constructs. The library vector contains short barcode, CMV promoter and mClover fluorescent protein as a reporter. The barcode is random 16-bp-long DNA with repeats of A,G and T. MspI restriction site is integrated upstream CMV promoter for genomic location mapping. (b) Barcoded lentivirus was packed and transduced into K562 cells at low MOI to create founder cells with singly integrated reporter. (c-d) Barcoded founder cells, selected by flow cytometry, were expanded for two weeks and split into two pools. Cells in the first pool were collected for locating reporter integration site. Founder cells in the second pool were sorted into 96-well plates to establish clonal cell lines. Barcode of each clone was simultaneously identified by split-pool encoding and deep sequencing. Library of characterized reporter clones is a useful resource to examine loci specific epigenetic drug sensitivity. (e) Loci specific effects were decoupled from global effects through mixing individual barcoded clones of interests with control cells expressing mClover and IRFP from multiple integration sites. Co-cultured cells were treated with TSA, JQ1 and 5’AZA for 24 hours. Expression of reporter proteins were measured by flow cytometers. Distribution of mClover expression in control cells was used to remove global effects of drugs. (f) A cartoon illustrating known mechanisms of actions of Trichostatin A, JQ1 and 5-Azacytidine.

    Article Snippet: For each replica, 2 µg of genomic DNA was digested with 20 units of MspI (NEB) overnight at 37C in a volume of 100 µl.

    Techniques: Construct, Plasmid Preparation, Flow Cytometry, Sequencing, Clone Assay, Expressing, Cell Culture

    Diverse insertion landscapes of barcoded reporter. (a) Reporter mapping by inverse PCR. Genomic DNA of founder cells was extracted, digested with restriction enzyme MspI and self-ligated to stitch barcode with its neighboring genome. Ligated product was amplified and followed by next generation sequencing. (b) Ideogram plot displaying reporter integration sites of individual clones in the library. Centromere position is indicated in red and stalk is marked in light blue. Heterochromatic region, which tend to be rich with adenine and thymine and relatively gene-poor, is represented by black and variation of grey. R-band in white on the ideogram is less condensed chromatin that is transcriptionally more active.

    Journal: bioRxiv

    Article Title: Loci specific epigenetic drug sensitivity

    doi: 10.1101/686139

    Figure Lengend Snippet: Diverse insertion landscapes of barcoded reporter. (a) Reporter mapping by inverse PCR. Genomic DNA of founder cells was extracted, digested with restriction enzyme MspI and self-ligated to stitch barcode with its neighboring genome. Ligated product was amplified and followed by next generation sequencing. (b) Ideogram plot displaying reporter integration sites of individual clones in the library. Centromere position is indicated in red and stalk is marked in light blue. Heterochromatic region, which tend to be rich with adenine and thymine and relatively gene-poor, is represented by black and variation of grey. R-band in white on the ideogram is less condensed chromatin that is transcriptionally more active.

    Article Snippet: For each replica, 2 µg of genomic DNA was digested with 20 units of MspI (NEB) overnight at 37C in a volume of 100 µl.

    Techniques: Inverse PCR, Amplification, Next-Generation Sequencing, Clone Assay