eagi  (New England Biolabs)


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    EagI
    Description:
    EagI 2 500 units
    Catalog Number:
    r0505l
    Price:
    269
    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs eagi
    EagI
    EagI 2 500 units
    https://www.bioz.com/result/eagi/product/New England Biolabs
    Average 98 stars, based on 55 article reviews
    Price from $9.99 to $1999.99
    eagi - by Bioz Stars, 2020-07
    98/100 stars

    Images

    1) Product Images from "One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome"

    Article Title: One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

    Journal:

    doi: 10.1073/pnas.0811011106

    Validation of an intact M. genitalium genome by restriction analysis. ( A ) Diagram of the EagI (red), BssHII (blue), and AatII (green) restriction fragments expected for a complete and proper assembly of the synthetic JCVI-1.1 M. genitalium genome. The
    Figure Legend Snippet: Validation of an intact M. genitalium genome by restriction analysis. ( A ) Diagram of the EagI (red), BssHII (blue), and AatII (green) restriction fragments expected for a complete and proper assembly of the synthetic JCVI-1.1 M. genitalium genome. The

    Techniques Used:

    2) Product Images from "Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA"

    Article Title: Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA

    Journal: Biotechnology and bioengineering

    doi: 10.1002/bit.23224

    S1 nuclease converts supercoiled plasmid DNA to nicked circular and linear forms through digestion at a variety of locations in the plasmid. (A) Two μg of supercoiled pRH04.152-rbsB was incubated at 22°C (top) or 37°C (bottom) with different amounts of S1 nuclease for different lengths of time and then analyzed by agarose gel electrophoresis. The sixth and seventh lanes contain undigested supercoiled control and SpeI-digested linear control, respectively. The band that runs slower than the linear band was presumed to correspond to nicked circular plasmid. In the thirteenth lane, after the DNA was digested with 10 units for 24 hours an additional 10 units of S1 nuclease was added and the DNA was incubated for an additional hour. The first and last lanes contain the λDNA/HindIII molecular weight standards. (B) Plasmids with or without the f1 origin (pDIMC8-pfMBP and pRH04-pfMBP, respectively) were incubated with or without S1 nuclease, followed by incubation with or without NcoI, EagI, and SacII and then analyzed by agarose gel electrophoresis. (C) Plasmids with or without the f1 origin (pDIMC8-rbsb and pRH04-rbsb, respectively) were incubated with S1 nuclease or DNaseI, followed by incubation with XmnI and analyzed by agarose gel electrophoresis.
    Figure Legend Snippet: S1 nuclease converts supercoiled plasmid DNA to nicked circular and linear forms through digestion at a variety of locations in the plasmid. (A) Two μg of supercoiled pRH04.152-rbsB was incubated at 22°C (top) or 37°C (bottom) with different amounts of S1 nuclease for different lengths of time and then analyzed by agarose gel electrophoresis. The sixth and seventh lanes contain undigested supercoiled control and SpeI-digested linear control, respectively. The band that runs slower than the linear band was presumed to correspond to nicked circular plasmid. In the thirteenth lane, after the DNA was digested with 10 units for 24 hours an additional 10 units of S1 nuclease was added and the DNA was incubated for an additional hour. The first and last lanes contain the λDNA/HindIII molecular weight standards. (B) Plasmids with or without the f1 origin (pDIMC8-pfMBP and pRH04-pfMBP, respectively) were incubated with or without S1 nuclease, followed by incubation with or without NcoI, EagI, and SacII and then analyzed by agarose gel electrophoresis. (C) Plasmids with or without the f1 origin (pDIMC8-rbsb and pRH04-rbsb, respectively) were incubated with S1 nuclease or DNaseI, followed by incubation with XmnI and analyzed by agarose gel electrophoresis.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Molecular Weight

    3) Product Images from "A Genome-Wide Screen for Normally Methylated Human CpG Islands That Can Identify Novel Imprinted Genes"

    Article Title: A Genome-Wide Screen for Normally Methylated Human CpG Islands That Can Identify Novel Imprinted Genes

    Journal: Genome Research

    doi: 10.1101/gr.224102

    Overall strategy for cloning methylated CpG islands. In step 1, genomic DNA was digested with Mse I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.
    Figure Legend Snippet: Overall strategy for cloning methylated CpG islands. In step 1, genomic DNA was digested with Mse I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.

    Techniques Used: Clone Assay, Methylation, Transformation Assay

    4) Product Images from "Intronic Parent-of-Origin Dependent Differential Methylation at the Actn1 Gene Is Conserved in Rodents but Is Not Associated with Imprinted Expression"

    Article Title: Intronic Parent-of-Origin Dependent Differential Methylation at the Actn1 Gene Is Conserved in Rodents but Is Not Associated with Imprinted Expression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0048936

    Maternal methylation of a novel DMR at the Actn1 gene in diverse mouse tissues. A) A detailed map of the novel maternal Actn 1 DMR is shown in the lower part. The diagram directly above shows the design for the MS-RFLP and bisulfite sequencing validation assays. Also included in this diagram are the locations of the methylation-sensitive enzyme restriction sites tested with MS-RFLP ( BsaAI, EagI and HpaII) , the strain-specific cut sites ( AhdI (present in 129S1 but not in PWK, due to SNP rs32640406) and StyI (present in PWK but not in 129S1, due to SNP rs32640412)), and the strain-specific resulting restriction fragments (see Methods). B) MS-RFLP results of four mouse liver samples. The matrix above the gel shows the different conditions for each individual lane. The plus sign (+) indicates addition, while the minus sign (−) indicated no addition of each corresponding endonuclease. C) Percent maternal methylation of an individual CpG (targeted by the BsaAI endonuclease) within different tissues. Circles represent individual (PWK×129S1)F 1 mice, while triangles represent individual (129S1×PWK)F 1 mice. Horizontal bars represent percent maternal methylation averages.
    Figure Legend Snippet: Maternal methylation of a novel DMR at the Actn1 gene in diverse mouse tissues. A) A detailed map of the novel maternal Actn 1 DMR is shown in the lower part. The diagram directly above shows the design for the MS-RFLP and bisulfite sequencing validation assays. Also included in this diagram are the locations of the methylation-sensitive enzyme restriction sites tested with MS-RFLP ( BsaAI, EagI and HpaII) , the strain-specific cut sites ( AhdI (present in 129S1 but not in PWK, due to SNP rs32640406) and StyI (present in PWK but not in 129S1, due to SNP rs32640412)), and the strain-specific resulting restriction fragments (see Methods). B) MS-RFLP results of four mouse liver samples. The matrix above the gel shows the different conditions for each individual lane. The plus sign (+) indicates addition, while the minus sign (−) indicated no addition of each corresponding endonuclease. C) Percent maternal methylation of an individual CpG (targeted by the BsaAI endonuclease) within different tissues. Circles represent individual (PWK×129S1)F 1 mice, while triangles represent individual (129S1×PWK)F 1 mice. Horizontal bars represent percent maternal methylation averages.

    Techniques Used: Methylation, Mass Spectrometry, Methylation Sequencing, Mouse Assay

    5) Product Images from "Development and validation of a multiplex-PCR assay for X-linked intellectual disability"

    Article Title: Development and validation of a multiplex-PCR assay for X-linked intellectual disability

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-14-80

    Examples of Southern blot results. Genotyping using FMR1 probe: After EcoRI /EagI double digestion, normal females exhibit two fragments, a ~2.8 kb (active X) and a ~5.2 kb (inactive X), while in normal males only a ~2.8 kb fragment is seen: (a) FXS male mosaic for a 51 CGG allele and a full mutation; (b) Premutated FMR1 male (110 CGGs); (c) Klinefelter with two FMR1 normal sized-alleles, showing the active and the inactive X-chromosome; (d) Normal FMR1 male (19 CGGs); (e) Premutated FMR1 male (58 CGGs). Genotyping using AFF2 probe: Following AflIII /NotI double digestion, normal females present two fragments, a ~2.2 kb (active X) and a ~4.8 kb (inactive X), whereas normal males exhibit only a ~2.2 kb fragment: (f) Intermediate AFF2 male (47 CCGs); (g) Premutated AFF2 male (68 CCGs); (h) Homoallelic female [(CCG) 14 /(CCG) 14 ].
    Figure Legend Snippet: Examples of Southern blot results. Genotyping using FMR1 probe: After EcoRI /EagI double digestion, normal females exhibit two fragments, a ~2.8 kb (active X) and a ~5.2 kb (inactive X), while in normal males only a ~2.8 kb fragment is seen: (a) FXS male mosaic for a 51 CGG allele and a full mutation; (b) Premutated FMR1 male (110 CGGs); (c) Klinefelter with two FMR1 normal sized-alleles, showing the active and the inactive X-chromosome; (d) Normal FMR1 male (19 CGGs); (e) Premutated FMR1 male (58 CGGs). Genotyping using AFF2 probe: Following AflIII /NotI double digestion, normal females present two fragments, a ~2.2 kb (active X) and a ~4.8 kb (inactive X), whereas normal males exhibit only a ~2.2 kb fragment: (f) Intermediate AFF2 male (47 CCGs); (g) Premutated AFF2 male (68 CCGs); (h) Homoallelic female [(CCG) 14 /(CCG) 14 ].

    Techniques Used: Southern Blot, Mutagenesis

    6) Product Images from "Methicillin-Resistant Staphylococcus aureus Outbreak in a Veterinary Teaching Hospital: Potential Human-to-Animal Transmission"

    Article Title: Methicillin-Resistant Staphylococcus aureus Outbreak in a Veterinary Teaching Hospital: Potential Human-to-Animal Transmission

    Journal: Journal of Clinical Microbiology

    doi:

    Fingerprints of MRSA obtained by PFGE with Sma I and Eag I digestion. Lane Kb, a 48.5-kb bacteriophage lambda ladder. For the Sma I digestion, lanes 1 to 15 represent both equine and human isolates associated with the MRSA outbreak. For the Eag I digestion, the isolates associated with the MRSA outbreak are represented in lanes 1 to 11, 14, and 15.
    Figure Legend Snippet: Fingerprints of MRSA obtained by PFGE with Sma I and Eag I digestion. Lane Kb, a 48.5-kb bacteriophage lambda ladder. For the Sma I digestion, lanes 1 to 15 represent both equine and human isolates associated with the MRSA outbreak. For the Eag I digestion, the isolates associated with the MRSA outbreak are represented in lanes 1 to 11, 14, and 15.

    Techniques Used:

    7) Product Images from "End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis"

    Article Title: End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis

    Journal: Journal of Biological Engineering

    doi: 10.1186/s13036-016-0024-5

    Construction in yeast of a promoter library. a Promoter library schematic. b Gel electrophoresis image of amplified promoters: lane 1, Gal-250; lane 2, Gal-100; lane 3, Leu-250; lane 4, spo-250; lane 5, spo-100; lane 6, tef-250; lane 7, tef-100. c Gel electrophoresis image of: lane 1, pRS426; lane 2, pRS426-yeGFP EagI digest. M is GeneRuler 1 kb Plus DNA Ladder (Thermo Scientific). d Schematic of reagent transfers through the microfluidic chip. The green input wells contain a mixture of different promoters and digested plasmid pRS426 with Salomon sperm DNA as a carrier. The yellow input wells contain S. cerevisiae competent cells. Arrows show pathways of reagent transfer on-chip according to the automated protocol
    Figure Legend Snippet: Construction in yeast of a promoter library. a Promoter library schematic. b Gel electrophoresis image of amplified promoters: lane 1, Gal-250; lane 2, Gal-100; lane 3, Leu-250; lane 4, spo-250; lane 5, spo-100; lane 6, tef-250; lane 7, tef-100. c Gel electrophoresis image of: lane 1, pRS426; lane 2, pRS426-yeGFP EagI digest. M is GeneRuler 1 kb Plus DNA Ladder (Thermo Scientific). d Schematic of reagent transfers through the microfluidic chip. The green input wells contain a mixture of different promoters and digested plasmid pRS426 with Salomon sperm DNA as a carrier. The yellow input wells contain S. cerevisiae competent cells. Arrows show pathways of reagent transfer on-chip according to the automated protocol

    Techniques Used: Nucleic Acid Electrophoresis, Amplification, Chromatin Immunoprecipitation, Plasmid Preparation

    8) Product Images from "The New Apolipoprotein A-I Variant Leu174 - > Ser Causes Hereditary Cardiac Amyloidosis, and the Amyloid Fibrils Are Constituted by the 93-Residue N-Terminal Polypeptide"

    Article Title: The New Apolipoprotein A-I Variant Leu174 - > Ser Causes Hereditary Cardiac Amyloidosis, and the Amyloid Fibrils Are Constituted by the 93-Residue N-Terminal Polypeptide

    Journal: The American Journal of Pathology

    doi:

    RFLP analysis of a 134-bp PCR product from exon 4 of the apoA-1 gene. The samples were digested with Eag I. Lane M , marker φX174 Hae III; Lanes 1, 3, 5 , proband’s healthy siblings; Lane 2 proband’s maternal uncle; Lane 4 , proband. The PCR product of 134 bp is split into two fragments of 81 and 53 bp only in the presence of the transition T to C in position 2069.
    Figure Legend Snippet: RFLP analysis of a 134-bp PCR product from exon 4 of the apoA-1 gene. The samples were digested with Eag I. Lane M , marker φX174 Hae III; Lanes 1, 3, 5 , proband’s healthy siblings; Lane 2 proband’s maternal uncle; Lane 4 , proband. The PCR product of 134 bp is split into two fragments of 81 and 53 bp only in the presence of the transition T to C in position 2069.

    Techniques Used: Polymerase Chain Reaction, Marker

    Related Articles

    Amplification:

    Article Title: The New Apolipoprotein A-I Variant Leu174 - > Ser Causes Hereditary Cardiac Amyloidosis, and the Amyloid Fibrils Are Constituted by the 93-Residue N-Terminal Polypeptide
    Article Snippet: .. To confirm the presence of the mutation, amplified products of exon 4 from the proband and other family members, as well as from 100 controls, were digested with Eag I (New England Biolabs), because the mutation creates a new restriction site. .. Digested products were electrophoresed in a 2.5% agarose gel, stained with ethidium bromide, and visualized with UV light.

    Agarose Gel Electrophoresis:

    Article Title: Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA
    Article Snippet: .. This linear DNA was digested with restriction enzymes NcoI, EagI, SacI or XmnI under the recommended conditions (New England Biolabs) for 1 hour at 37°C and analyzed by agarose gel electrophoresis on a 0.8% agarose, TAE gel. .. Plasmid pDIMC8-rbsB DNA was incubated at 37°C for 20 minutes as above in 25 separate reactions, each containing 2 μg of pDIMC8-rbsB and 10 units S1 Nuclease.

    Southern Blot:

    Article Title: Development and validation of a multiplex-PCR assay for X-linked intellectual disability
    Article Snippet: .. Southern blot genotyping was done using ~10 μg of gDNA double-digested with the restriction enzymes EcoRI and EagI for FMR1 gene or AflIII and NotI for AFF2 gene (New England Biolabs, Ipswich, MA, USA). .. Further, the digested gDNA products were electrophoresed in parallel with a 1:1 mixture of the standard size markers, DNA Molecular Weight Marker II and III digoxigenin-labeled (Roche Applied Science, Indianapolis, IN, USA), blotted and hybridized using the FMR1 or the AFF2 digoxigenin-labeled specific probes, GLFXDIG1 or AJ31Dig1, as appropriate (Gene Link™, Hawthorne, NY, USA).

    Methylation:

    Article Title: Intronic Parent-of-Origin Dependent Differential Methylation at the Actn1 Gene Is Conserved in Rodents but Is Not Associated with Imprinted Expression
    Article Snippet: .. Samples were then either digested with methylation-sensitive enzymes BsaAI, EagI, HpaII (NEB), or mock treated (buffer only). .. The cut sites of BsaAI, EagI and HpaII include one or more of the CpGs targeted for PCR amplification.

    Mutagenesis:

    Article Title: The New Apolipoprotein A-I Variant Leu174 - > Ser Causes Hereditary Cardiac Amyloidosis, and the Amyloid Fibrils Are Constituted by the 93-Residue N-Terminal Polypeptide
    Article Snippet: .. To confirm the presence of the mutation, amplified products of exon 4 from the proband and other family members, as well as from 100 controls, were digested with Eag I (New England Biolabs), because the mutation creates a new restriction site. .. Digested products were electrophoresed in a 2.5% agarose gel, stained with ethidium bromide, and visualized with UV light.

    Purification:

    Article Title: End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis
    Article Snippet: .. Digestion of pRS426-yeGFP plasmid by EagI 50 μL EagI (NEB) digestion reactions consisting of 20 μL purified plasmid pRS426 (100 ng/μL), 5 μL CutSmart™ Buffer, EagI-HF (20 units/μL) 1 μL and 24 μL deionized water. ..

    Plasmid Preparation:

    Article Title: End-to-end automated microfluidic platform for synthetic biology: from design to functional analysis
    Article Snippet: .. Digestion of pRS426-yeGFP plasmid by EagI 50 μL EagI (NEB) digestion reactions consisting of 20 μL purified plasmid pRS426 (100 ng/μL), 5 μL CutSmart™ Buffer, EagI-HF (20 units/μL) 1 μL and 24 μL deionized water. ..

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    New England Biolabs eagi
    Validation of an intact M. genitalium genome by restriction analysis. ( A ) Diagram of the <t>EagI</t> (red), <t>BssHII</t> (blue), and AatII (green) restriction fragments expected for a complete and proper assembly of the synthetic JCVI-1.1 M. genitalium genome. The
    Eagi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/eagi/product/New England Biolabs
    Average 98 stars, based on 37 article reviews
    Price from $9.99 to $1999.99
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    98/100 stars
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    Validation of an intact M. genitalium genome by restriction analysis. ( A ) Diagram of the EagI (red), BssHII (blue), and AatII (green) restriction fragments expected for a complete and proper assembly of the synthetic JCVI-1.1 M. genitalium genome. The

    Journal:

    Article Title: One-step assembly in yeast of 25 overlapping DNA fragments to form a complete synthetic Mycoplasma genitalium genome

    doi: 10.1073/pnas.0811011106

    Figure Lengend Snippet: Validation of an intact M. genitalium genome by restriction analysis. ( A ) Diagram of the EagI (red), BssHII (blue), and AatII (green) restriction fragments expected for a complete and proper assembly of the synthetic JCVI-1.1 M. genitalium genome. The

    Article Snippet: Plugs were digested according to the Bio-Rad manual with EagI, BssHII, or AatII; restriction enzymes and buffers were supplied by New England Biolabs.

    Techniques:

    S1 nuclease converts supercoiled plasmid DNA to nicked circular and linear forms through digestion at a variety of locations in the plasmid. (A) Two μg of supercoiled pRH04.152-rbsB was incubated at 22°C (top) or 37°C (bottom) with different amounts of S1 nuclease for different lengths of time and then analyzed by agarose gel electrophoresis. The sixth and seventh lanes contain undigested supercoiled control and SpeI-digested linear control, respectively. The band that runs slower than the linear band was presumed to correspond to nicked circular plasmid. In the thirteenth lane, after the DNA was digested with 10 units for 24 hours an additional 10 units of S1 nuclease was added and the DNA was incubated for an additional hour. The first and last lanes contain the λDNA/HindIII molecular weight standards. (B) Plasmids with or without the f1 origin (pDIMC8-pfMBP and pRH04-pfMBP, respectively) were incubated with or without S1 nuclease, followed by incubation with or without NcoI, EagI, and SacII and then analyzed by agarose gel electrophoresis. (C) Plasmids with or without the f1 origin (pDIMC8-rbsb and pRH04-rbsb, respectively) were incubated with S1 nuclease or DNaseI, followed by incubation with XmnI and analyzed by agarose gel electrophoresis.

    Journal: Biotechnology and bioengineering

    Article Title: Protein switches identified from diverse insertion libraries created using S1 nuclease digestion of supercoiled-form plasmid DNA

    doi: 10.1002/bit.23224

    Figure Lengend Snippet: S1 nuclease converts supercoiled plasmid DNA to nicked circular and linear forms through digestion at a variety of locations in the plasmid. (A) Two μg of supercoiled pRH04.152-rbsB was incubated at 22°C (top) or 37°C (bottom) with different amounts of S1 nuclease for different lengths of time and then analyzed by agarose gel electrophoresis. The sixth and seventh lanes contain undigested supercoiled control and SpeI-digested linear control, respectively. The band that runs slower than the linear band was presumed to correspond to nicked circular plasmid. In the thirteenth lane, after the DNA was digested with 10 units for 24 hours an additional 10 units of S1 nuclease was added and the DNA was incubated for an additional hour. The first and last lanes contain the λDNA/HindIII molecular weight standards. (B) Plasmids with or without the f1 origin (pDIMC8-pfMBP and pRH04-pfMBP, respectively) were incubated with or without S1 nuclease, followed by incubation with or without NcoI, EagI, and SacII and then analyzed by agarose gel electrophoresis. (C) Plasmids with or without the f1 origin (pDIMC8-rbsb and pRH04-rbsb, respectively) were incubated with S1 nuclease or DNaseI, followed by incubation with XmnI and analyzed by agarose gel electrophoresis.

    Article Snippet: This linear DNA was digested with restriction enzymes NcoI, EagI, SacI or XmnI under the recommended conditions (New England Biolabs) for 1 hour at 37°C and analyzed by agarose gel electrophoresis on a 0.8% agarose, TAE gel.

    Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Molecular Weight

    Overall strategy for cloning methylated CpG islands. In step 1, genomic DNA was digested with Mse I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.

    Journal: Genome Research

    Article Title: A Genome-Wide Screen for Normally Methylated Human CpG Islands That Can Identify Novel Imprinted Genes

    doi: 10.1101/gr.224102

    Figure Lengend Snippet: Overall strategy for cloning methylated CpG islands. In step 1, genomic DNA was digested with Mse I (red), which cuts between CpG islands, and Hpa II (blue), which cuts unmethylated CpG islands. Mse I fragments containing methylated CpG islands then are transformed into a bacterial strain that does not cut methylated DNA. However, brief bacterial passage leads to loss of methylation of these previously methylated sequences. In step 2, the library DNA is pooled and digested with Eag I (green), which cuts relatively large fragments within CpG islands, and these fragments are then subcloned.

    Article Snippet: In the second step, 100 μg of the Mse I library DNA were digested with 1,000 U of Eag I (NEB) according to the manufacturer’s conditions.

    Techniques: Clone Assay, Methylation, Transformation Assay

    Maternal methylation of a novel DMR at the Actn1 gene in diverse mouse tissues. A) A detailed map of the novel maternal Actn 1 DMR is shown in the lower part. The diagram directly above shows the design for the MS-RFLP and bisulfite sequencing validation assays. Also included in this diagram are the locations of the methylation-sensitive enzyme restriction sites tested with MS-RFLP ( BsaAI, EagI and HpaII) , the strain-specific cut sites ( AhdI (present in 129S1 but not in PWK, due to SNP rs32640406) and StyI (present in PWK but not in 129S1, due to SNP rs32640412)), and the strain-specific resulting restriction fragments (see Methods). B) MS-RFLP results of four mouse liver samples. The matrix above the gel shows the different conditions for each individual lane. The plus sign (+) indicates addition, while the minus sign (−) indicated no addition of each corresponding endonuclease. C) Percent maternal methylation of an individual CpG (targeted by the BsaAI endonuclease) within different tissues. Circles represent individual (PWK×129S1)F 1 mice, while triangles represent individual (129S1×PWK)F 1 mice. Horizontal bars represent percent maternal methylation averages.

    Journal: PLoS ONE

    Article Title: Intronic Parent-of-Origin Dependent Differential Methylation at the Actn1 Gene Is Conserved in Rodents but Is Not Associated with Imprinted Expression

    doi: 10.1371/journal.pone.0048936

    Figure Lengend Snippet: Maternal methylation of a novel DMR at the Actn1 gene in diverse mouse tissues. A) A detailed map of the novel maternal Actn 1 DMR is shown in the lower part. The diagram directly above shows the design for the MS-RFLP and bisulfite sequencing validation assays. Also included in this diagram are the locations of the methylation-sensitive enzyme restriction sites tested with MS-RFLP ( BsaAI, EagI and HpaII) , the strain-specific cut sites ( AhdI (present in 129S1 but not in PWK, due to SNP rs32640406) and StyI (present in PWK but not in 129S1, due to SNP rs32640412)), and the strain-specific resulting restriction fragments (see Methods). B) MS-RFLP results of four mouse liver samples. The matrix above the gel shows the different conditions for each individual lane. The plus sign (+) indicates addition, while the minus sign (−) indicated no addition of each corresponding endonuclease. C) Percent maternal methylation of an individual CpG (targeted by the BsaAI endonuclease) within different tissues. Circles represent individual (PWK×129S1)F 1 mice, while triangles represent individual (129S1×PWK)F 1 mice. Horizontal bars represent percent maternal methylation averages.

    Article Snippet: Samples were then either digested with methylation-sensitive enzymes BsaAI, EagI, HpaII (NEB), or mock treated (buffer only).

    Techniques: Methylation, Mass Spectrometry, Methylation Sequencing, Mouse Assay