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    Name:
    DpnII
    Description:
    DpnII 5 000 units
    Catalog Number:
    R0543L
    Price:
    285
    Size:
    5 000 units
    Category:
    Restriction Enzymes
    Score:
    85
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    Structured Review

    New England Biolabs dpnii
    DpnII
    DpnII 5 000 units
    https://www.bioz.com/result/dpnii/product/New England Biolabs
    Average 99 stars, based on 45 article reviews
    Price from $9.99 to $1999.99
    dpnii - by Bioz Stars, 2019-12
    99/100 stars

    Images

    1) Product Images from "A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips"

    Article Title: A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips

    Journal: Nature biotechnology

    doi: 10.1038/nbt.1716

    Scalable gene synthesis platform schematic for OLS Pool 2 Pre-designed oligonucleotides (no distinction is made between dsDNA and ssDNA in the figure) are synthesized on a DNA microchip ( a ) and then cleaved to make a pool of oligonucleotides (b) . Plate-specific primer sequences (yellow or brown) are used to amplify separate Plate Subpools (c) (only two are shown), which contain DNA to assemble different genes (only three are shown for each plate subpool). Assembly specific sequences (shades of blue) are used to amplify assembly subpools (d) that contain only the DNA required to make a single gene. The primer sequences are cleaved (e) using either Type IIS restriction enzymes (resulting in dsDNA) or by DpnII/USER/γ exonuclease processing (producing ssDNA). Construction primers (shown as white and black sites flanking the full assembly) are then used in an assembly PCR reaction to build a gene from each assembly subpool (f) . Depending on the downstream application the assembled products are then cloned either before or after an enzymatic error correction step.
    Figure Legend Snippet: Scalable gene synthesis platform schematic for OLS Pool 2 Pre-designed oligonucleotides (no distinction is made between dsDNA and ssDNA in the figure) are synthesized on a DNA microchip ( a ) and then cleaved to make a pool of oligonucleotides (b) . Plate-specific primer sequences (yellow or brown) are used to amplify separate Plate Subpools (c) (only two are shown), which contain DNA to assemble different genes (only three are shown for each plate subpool). Assembly specific sequences (shades of blue) are used to amplify assembly subpools (d) that contain only the DNA required to make a single gene. The primer sequences are cleaved (e) using either Type IIS restriction enzymes (resulting in dsDNA) or by DpnII/USER/γ exonuclease processing (producing ssDNA). Construction primers (shown as white and black sites flanking the full assembly) are then used in an assembly PCR reaction to build a gene from each assembly subpool (f) . Depending on the downstream application the assembled products are then cloned either before or after an enzymatic error correction step.

    Techniques Used: Synthesized, MicroChIP Assay, Polymerase Cycling Assembly, Clone Assay

    2) Product Images from "Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex"

    Article Title: Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex

    Journal: Nature Communications

    doi: 10.1038/ncomms12743

    Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.
    Figure Legend Snippet: Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.

    Techniques Used: Sequencing, Amplification, Real-time Polymerase Chain Reaction, Methylation, Polymerase Chain Reaction, Ligation, Injection, Chromatin Immunoprecipitation, Binding Assay, Mouse Assay

    3) Product Images from "Retroviral Integration Mutagenesis in Mice and Comparative Analysis in Human AML Identify Reduced PTP4A3 Expression as a Prognostic Indicator"

    Article Title: Retroviral Integration Mutagenesis in Mice and Comparative Analysis in Human AML Identify Reduced PTP4A3 Expression as a Prognostic Indicator

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0026537

    Identification of mVIS. Strategy outline for identification of regions flanking DNA methylated viral integration sites (mVIS) within murine leukemias. Genomic DNA was digested with DpnII (step 1), followed by methylated DNA immunoprecipitation (MeDIP, step 2). MeDIP enriched fragments were ligated (step 3) and amplified using primers within the LTR (step 4). These fragments were hybridized on a DNA promoter array (step 5). Hypergeometric Analysis of Tiling Arrays (HAT) was used to identify regions flanking mVIS (step 6).
    Figure Legend Snippet: Identification of mVIS. Strategy outline for identification of regions flanking DNA methylated viral integration sites (mVIS) within murine leukemias. Genomic DNA was digested with DpnII (step 1), followed by methylated DNA immunoprecipitation (MeDIP, step 2). MeDIP enriched fragments were ligated (step 3) and amplified using primers within the LTR (step 4). These fragments were hybridized on a DNA promoter array (step 5). Hypergeometric Analysis of Tiling Arrays (HAT) was used to identify regions flanking mVIS (step 6).

    Techniques Used: Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Amplification, HAT Assay

    4) Product Images from "Chromatin Remodeling Complexes Interact Dynamically with a Glucocorticoid Receptor–regulated Promoter"

    Article Title: Chromatin Remodeling Complexes Interact Dynamically with a Glucocorticoid Receptor–regulated Promoter

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E08-02-0123

    BRG1 is required for the hormone-dependent remodeling of MMTV chromatin and associated loading of RNA pol II at the MMTV promoter. 5555 and 3617 cells were untreated or treated with 100 nM dexamethasone for 30 min and tetracycline as indicated. Nuclei were isolated and digested with SacI and DpnII restriction enzymes. Digestion products were detected by linear amplification using a radiolabeled primer specific to the MMTV promoter region. Percent cleavage and the fractional change in the accessibility of MMTV promoter to restriction enzymes are indicators of nuclease hypersensitivity and chromatin remodeling of MMTV promoter region. (A) Intensity of SacI digestion product is divided by the sum of the intensities of SacI and DpnII digestion products and presented as percent cleavage at the bottom of each lane. (B) Bar graph shows dexamethasone- or dexamethasone-, GR-, and BRG1-K-R– induced change in percent cleavage in SacI hypersensitivity between 5555 and 3617 cells from A. (C) Expression of BRG1-K-R reduced the loading of RNA pol II to MMTV promoter. 5555 and 3617 cells were untreated or treated with 100 nM dexamethasone for 30 min and tetracycline as indicated. Chromatin was immunoprecipitated using an antibody specific for RNA pol II or no antibody (control). Immunoprecipitated and input DNA were amplified using primers specific to the MMTV promoter. (D) Expression of BRG1-K-R reduced the loading of RNA pol II to the dexamethasone-induced Rgs2 locus. 5555 cells were untreated or treated with 100 nM dexamethasone for 30 min and tetracycline as indicated. cDNA was prepared from RNAs isolated from the indicated conditions. Chromatin was immunoprecipitated using an antibody specific for RNA pol II or no antibody (control). Immunoprecipitated and input DNA or cDNA were amplified using primers specific to the Rgs2 coding region.
    Figure Legend Snippet: BRG1 is required for the hormone-dependent remodeling of MMTV chromatin and associated loading of RNA pol II at the MMTV promoter. 5555 and 3617 cells were untreated or treated with 100 nM dexamethasone for 30 min and tetracycline as indicated. Nuclei were isolated and digested with SacI and DpnII restriction enzymes. Digestion products were detected by linear amplification using a radiolabeled primer specific to the MMTV promoter region. Percent cleavage and the fractional change in the accessibility of MMTV promoter to restriction enzymes are indicators of nuclease hypersensitivity and chromatin remodeling of MMTV promoter region. (A) Intensity of SacI digestion product is divided by the sum of the intensities of SacI and DpnII digestion products and presented as percent cleavage at the bottom of each lane. (B) Bar graph shows dexamethasone- or dexamethasone-, GR-, and BRG1-K-R– induced change in percent cleavage in SacI hypersensitivity between 5555 and 3617 cells from A. (C) Expression of BRG1-K-R reduced the loading of RNA pol II to MMTV promoter. 5555 and 3617 cells were untreated or treated with 100 nM dexamethasone for 30 min and tetracycline as indicated. Chromatin was immunoprecipitated using an antibody specific for RNA pol II or no antibody (control). Immunoprecipitated and input DNA were amplified using primers specific to the MMTV promoter. (D) Expression of BRG1-K-R reduced the loading of RNA pol II to the dexamethasone-induced Rgs2 locus. 5555 cells were untreated or treated with 100 nM dexamethasone for 30 min and tetracycline as indicated. cDNA was prepared from RNAs isolated from the indicated conditions. Chromatin was immunoprecipitated using an antibody specific for RNA pol II or no antibody (control). Immunoprecipitated and input DNA or cDNA were amplified using primers specific to the Rgs2 coding region.

    Techniques Used: Isolation, Amplification, Expressing, Immunoprecipitation

    5) Product Images from "Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex"

    Article Title: Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex

    Journal: Nature Communications

    doi: 10.1038/ncomms12743

    Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.
    Figure Legend Snippet: Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.

    Techniques Used: Sequencing, Amplification, Real-time Polymerase Chain Reaction, Methylation, Polymerase Chain Reaction, Ligation, Injection, Chromatin Immunoprecipitation, Binding Assay, Mouse Assay

    6) Product Images from "Multiple Copies of the 16S rRNA Gene in Nocardia nova Isolates and Implications for Sequence-Based Identification Procedures"

    Article Title: Multiple Copies of the 16S rRNA Gene in Nocardia nova Isolates and Implications for Sequence-Based Identification Procedures

    Journal:

    doi: 10.1128/JCM.43.6.2881-2885.2005

    DpnII digests of a 999-bp amplified region of the 16S rRNA gene. Lane 1, base pair marker; lane 2, N. nova ATCC 33726 T ; lane 3, isolate A ( N. nova variant); lane 4, isolate B; lane 5, isolate C; lane 6, base pair marker.
    Figure Legend Snippet: DpnII digests of a 999-bp amplified region of the 16S rRNA gene. Lane 1, base pair marker; lane 2, N. nova ATCC 33726 T ; lane 3, isolate A ( N. nova variant); lane 4, isolate B; lane 5, isolate C; lane 6, base pair marker.

    Techniques Used: Amplification, Marker, Variant Assay

    7) Product Images from "Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing"

    Article Title: Antagonistic roles for the ubiquitin ligase Asr1 and the ubiquitin-specific protease Ubp3 in subtelomeric gene silencing

    Journal:

    doi: 10.1073/pnas.1518375113

    The ubiquitin ligase activity of Asr1 is required for subtelomeric gene silencing. ( A ) Asr1 is required for full silencing of a telomere-proximal URA3 reporter. Equal amounts of yeast ( WT , LPY4819; ∆ asr1 , LPY4819 ∆Asr1; ∆ sir2 , LPY4977) were plated on media with or without 5-FOA, and the relative number of FOA-resistant colonies was calculated. Error bars represent SEM ( n = 3). ( B ) The Ub-ligase activity of Asr1 is required for silencing of a telomere-proximal ADE2 reporter. Yeasts ( WT , UTAT TM4; ∆ asr1 , UTAT TM5; ∆ asr1/ASR1 , UTAT TM6; ∆ asr1/asr1 RINGm , UTAT TM7; ∆ asr1/ASR1 PHDm , UTAT TM8) were plated on nonselective media, and the ratio of white and red colonies was calculated. Error bars represent SEM ( n = 3). ( C ) The Ub-ligase activity of Asr1 is required for silencing telomere-proximal genes. Total RNA was extracted from yeast ( WT , BY4741; ∆ asr1 , YTM5; asr1 RINGm , YTM27; ∆ sir2 , ∆Sir2), and RT–QPCR was used to measure transcript levels from the indicated loci ( 57W , YFR057W; 77C , YNR077C; 74W , YCL074W; ALD6 ). Error bars represent SEM ( n = 6–8). ( D ) Asr1 associates with subtelomeric chromatin. DNA was isolated from congenic strains expressing either an Asr1–DAM (YTM38) or Asr1RINGm –DAM (YTM39) fusion, or unfused DAM expressed from the ASR1 promoter (YTM40). DNA was then either untreated, or cleaved with DpnII. QPCR was performed using primers flanking a DpnII site within each gene to determine the extent of cutting. Signals for each fusion strain were normalized to that from the strain expressing unfused DAM protein, and data were plotted as the ratio of methylated/nonmethylated DNA. Error bars represent SEM ( n = 5). ( E ) Asr1-dependent ubiquitylation sites on Rpb1 are required for silencing. The experiment was performed as in C , except on RNA isolated from WT yeast (BY4742) or yeast expressing the Rpb1–∆2KTM mutant (YTM29). Error bars represent SEM ( n = 4–5). ( F ) Loss of silencing in Asr1-mutant cells is not accompanied by an increase in H4K16 acetylation. ChIP was performed on chromatin isolated from the indicated strains ( WT , BY4741; ∆ asr1 , YTM5; asr1 RINGm , YTM27; Rpb1–∆2KTM , YTM29; ∆ sir2 , ∆Sir2), using an anti-H4K16 acetylation (H4K16Ac)-specific antibody. Coprecipitating DNAs were quantified by QPCR and normalized to the signal from a primer set that amplifies an intergenic portion on the left arm of chromosome V. Error bars represent SEM ( n = 4).
    Figure Legend Snippet: The ubiquitin ligase activity of Asr1 is required for subtelomeric gene silencing. ( A ) Asr1 is required for full silencing of a telomere-proximal URA3 reporter. Equal amounts of yeast ( WT , LPY4819; ∆ asr1 , LPY4819 ∆Asr1; ∆ sir2 , LPY4977) were plated on media with or without 5-FOA, and the relative number of FOA-resistant colonies was calculated. Error bars represent SEM ( n = 3). ( B ) The Ub-ligase activity of Asr1 is required for silencing of a telomere-proximal ADE2 reporter. Yeasts ( WT , UTAT TM4; ∆ asr1 , UTAT TM5; ∆ asr1/ASR1 , UTAT TM6; ∆ asr1/asr1 RINGm , UTAT TM7; ∆ asr1/ASR1 PHDm , UTAT TM8) were plated on nonselective media, and the ratio of white and red colonies was calculated. Error bars represent SEM ( n = 3). ( C ) The Ub-ligase activity of Asr1 is required for silencing telomere-proximal genes. Total RNA was extracted from yeast ( WT , BY4741; ∆ asr1 , YTM5; asr1 RINGm , YTM27; ∆ sir2 , ∆Sir2), and RT–QPCR was used to measure transcript levels from the indicated loci ( 57W , YFR057W; 77C , YNR077C; 74W , YCL074W; ALD6 ). Error bars represent SEM ( n = 6–8). ( D ) Asr1 associates with subtelomeric chromatin. DNA was isolated from congenic strains expressing either an Asr1–DAM (YTM38) or Asr1RINGm –DAM (YTM39) fusion, or unfused DAM expressed from the ASR1 promoter (YTM40). DNA was then either untreated, or cleaved with DpnII. QPCR was performed using primers flanking a DpnII site within each gene to determine the extent of cutting. Signals for each fusion strain were normalized to that from the strain expressing unfused DAM protein, and data were plotted as the ratio of methylated/nonmethylated DNA. Error bars represent SEM ( n = 5). ( E ) Asr1-dependent ubiquitylation sites on Rpb1 are required for silencing. The experiment was performed as in C , except on RNA isolated from WT yeast (BY4742) or yeast expressing the Rpb1–∆2KTM mutant (YTM29). Error bars represent SEM ( n = 4–5). ( F ) Loss of silencing in Asr1-mutant cells is not accompanied by an increase in H4K16 acetylation. ChIP was performed on chromatin isolated from the indicated strains ( WT , BY4741; ∆ asr1 , YTM5; asr1 RINGm , YTM27; Rpb1–∆2KTM , YTM29; ∆ sir2 , ∆Sir2), using an anti-H4K16 acetylation (H4K16Ac)-specific antibody. Coprecipitating DNAs were quantified by QPCR and normalized to the signal from a primer set that amplifies an intergenic portion on the left arm of chromosome V. Error bars represent SEM ( n = 4).

    Techniques Used: Activity Assay, Quantitative RT-PCR, Isolation, Expressing, Real-time Polymerase Chain Reaction, Methylation, Mutagenesis, Chromatin Immunoprecipitation

    Related Articles

    Clone Assay:

    Article Title: SadA, a novel adhesion receptor in Dictyostelium
    Article Snippet: In brief, a Bio-Rad Laboratories gene pulser set to 0.85 kV/25 μF was used to electroporate 10 μg BamHI-linearized pUCΔBamBsr ( ) together with 10 U DpnII enzyme (NEB) into 7 × 106 wild-type (AX3) cells. .. In brief, a Bio-Rad Laboratories gene pulser set to 0.85 kV/25 μF was used to electroporate 10 μg BamHI-linearized pUCΔBamBsr ( ) together with 10 U DpnII enzyme (NEB) into 7 × 106 wild-type (AX3) cells.

    Article Title: Adenine methylation may contribute to endosymbiont selection in a clonal aphid population
    Article Snippet: DNA fragments containing methylated adenines were isolated from the genomic DNA using the methylation-specific restriction enzyme DpnI together with the methyl-sensitive restriction enzyme DpnII (New England Biolabs, Ipswich, MA, USA). .. This was followed by PCR amplification using AdR_PCR (5′-GGTCGCGGCCGAGGATC-3′), as described previously [ ].

    Amplification:

    Article Title: A Distal CCAAT/NUCLEAR FACTOR Y Complex Promotes Chromatin Looping at the FLOWERING LOCUS T Promoter and Regulates the Timing of Flowering in Arabidopsis
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    Article Title: Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins
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    Article Snippet: A single PCR product was observed for each DNA isolate. .. A 10-μl aliquot of the amplified fragments (roughly 155 to 160 bp on gel) was further digested using either Sau3AI or DpnII according to the manufacturer's instructions (New England Biolabs, Beverly, MA, for DpnII and Roche-Biomedicals, Meylan, France for Sau3AI). .. These enzymes specifically cut at the GATC sequence overlapping the M. bovis -like −215C narGHJI promoter site, producing two bands (of approximately 90 and of 70 bp) in PCR-RFLP of the LC66-LC67 PCR fragment.

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    Mass Spectrometry:

    Article Title: Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA
    Article Snippet: The substrates were degraded enzymatically to deoxynucleosides or nucleosides and analyzed by LC/MS/MS using an Agilent HP1100 HPLC system interfaced an Applied Biosystems API4000 hybrid triple quadrupole/linear ion trap mass spectrometer, essentially as described ( ). .. The oligodeoxynucleotide was ethanol precipitated and resuspended in DpnII reaction buffer (New England Biolabs), annealed to the complementary oligodeoxynucleotide and treated with 10 units of DpnII at 37 °C for 1 h. Substrate and product bands (49- and 22-mer) were separated by PAGE and fluorescence visualized using a Typhoon TRIO GE scanner.

    Synthesized:

    Article Title: Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA
    Article Snippet: Fluorescent oligonucleotide substrates: 49-mer oligode-oxynucleotides containing 3-meC or 1-meA in the recognition site for the methylation sensitive restriction enzyme DpnII with the sequence as described in Ref. , were labeled with 6-carboxyfluorescein at the 5′ end, and synthesized with phosphorothioate linkages (5 bases) at the 3′ and 5′ ends. hABH1(Δ3) was incubated with 1 pmol of single-stranded or double-stranded substrate in 10 m m Tris-HCl, pH 6.6, 10 m m KCl, 100 μg/ml bovine serum albumin, 4 m m DTT, 100 μ m 2OG, and 10 μ m FeSO4 (NH4 )2 SO4 at 37 °C for 15 min. .. The oligodeoxynucleotide was ethanol precipitated and resuspended in DpnII reaction buffer (New England Biolabs), annealed to the complementary oligodeoxynucleotide and treated with 10 units of DpnII at 37 °C for 1 h. Substrate and product bands (49- and 22-mer) were separated by PAGE and fluorescence visualized using a Typhoon TRIO GE scanner.

    Construct:

    Article Title: A Distal CCAAT/NUCLEAR FACTOR Y Complex Promotes Chromatin Looping at the FLOWERING LOCUS T Promoter and Regulates the Timing of Flowering in Arabidopsis
    Article Snippet: The FT template was obtained using the above-mentioned FT construct (6.5-kb FT promoter plus FT genomic coding region in pDONR207). .. Five micrograms of pFT:FT construct was digested for 6 h with Dpn II (New England Biolabs), extracted with phenol-chloroform, and precipitated. .. Isolated DNA was ligated for 1 h at room temperature, followed by 5 h at 16°C (Fermentas; 100 μL 10× ligation buffer, 5 μL ligase [5 units/μL], and 895 μL water).

    Article Title: Regulation of the Protocadherin Celsr3 Gene and Its Role in Globus Pallidus Development and Connectivity
    Article Snippet: Circular chromosome confirmation capture (4C) libraries for high-throughput sequencing (4C-seq) were constructed as described previously , with some modifications. .. After phenol-chloroform extraction and ethanol precipitation, the purified DNA was digested with the secondary restriction enzyme DpnII (NEB) and ligated again.

    Real-time Polymerase Chain Reaction:

    Article Title: A Distal CCAAT/NUCLEAR FACTOR Y Complex Promotes Chromatin Looping at the FLOWERING LOCUS T Promoter and Regulates the Timing of Flowering in Arabidopsis
    Article Snippet: Paragraph title: - ... Five micrograms of pFT:FT construct was digested for 6 h with Dpn II (New England Biolabs), extracted with phenol-chloroform, and precipitated.

    Article Title: Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins
    Article Snippet: After digestion, double-stranded adapters comprising annealed oligonucleotide AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′) were ligated overnight with the digested DpnI fragments (T4 ligase 799009; Roche) followed by DPNII digestion (R0543S; New England Biolabs, Inc.) for 1 h. This material was amplified in ligation-mediated PCR using AdR_PCR primer (5′-GGTCGCGGCCGAGGATC-3′) and Advantage cDNA Polymerase (Takara Bio Inc.). .. After digestion, double-stranded adapters comprising annealed oligonucleotide AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′) were ligated overnight with the digested DpnI fragments (T4 ligase 799009; Roche) followed by DPNII digestion (R0543S; New England Biolabs, Inc.) for 1 h. This material was amplified in ligation-mediated PCR using AdR_PCR primer (5′-GGTCGCGGCCGAGGATC-3′) and Advantage cDNA Polymerase (Takara Bio Inc.).

    Article Title: Clock-dependent chromatin topology modulates circadian transcription and behavior
    Article Snippet: Nuclei were resuspended in 1 mL of a buffer containing 10 mM Tris-HCL (pH 8.0), 10 mM NaCl, 0.2% NP-40, and 1× protease inhibitor cocktail (Complete Mini EDTA-free protease inhibitor cocktail; Sigma-Aldrich); kept for 15 min on ice; and washed twice with 1× DpnII buffer (New England Biolabs). .. Nuclei were resuspended in 1 mL of a buffer containing 10 mM Tris-HCL (pH 8.0), 10 mM NaCl, 0.2% NP-40, and 1× protease inhibitor cocktail (Complete Mini EDTA-free protease inhibitor cocktail; Sigma-Aldrich); kept for 15 min on ice; and washed twice with 1× DpnII buffer (New England Biolabs).

    Incubation:

    Article Title: SadA, a novel adhesion receptor in Dictyostelium
    Article Snippet: In brief, a Bio-Rad Laboratories gene pulser set to 0.85 kV/25 μF was used to electroporate 10 μg BamHI-linearized pUCΔBamBsr ( ) together with 10 U DpnII enzyme (NEB) into 7 × 106 wild-type (AX3) cells. .. In brief, a Bio-Rad Laboratories gene pulser set to 0.85 kV/25 μF was used to electroporate 10 μg BamHI-linearized pUCΔBamBsr ( ) together with 10 U DpnII enzyme (NEB) into 7 × 106 wild-type (AX3) cells.

    Article Title: Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast
    Article Snippet: Precipitated chromatin was washed once with DpnII restriction enzyme incubation buffer (New England Biolabs), then resuspended in DpnII buffer and incubated at 65 ºC for 10 minutes with 0.1 % (w/v) of SDS. .. After addition of 1 % (w/v) Triton X-100, chromatin was digested overnight with DpnII (New England Biolabs) and RNase A (Sigma) at 37 ºC.

    Article Title: Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver
    Article Snippet: The supernatant was removed following the second wash, and cell pellets were resuspended in 8 ml of lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100, and 1X Complete Protease Inhibitor Cocktail) and incubated on ice for 40 min with occasional mixing. .. Primary digestion of 10 million nuclei with 50,000 U of DpnII (NEB: #R0543) was performed overnight at 37°C in 450 μl of NEBuffer 3 (NEB: #B7003S; 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) with agitation at 900 RPM.

    Article Title: Clock-dependent chromatin topology modulates circadian transcription and behavior
    Article Snippet: Thirty million nuclei were resuspended in 1× DpnII buffer (New England Biolabs) containing 0.1% SDS and incubated for 10 min at 65°C. .. Chromatin was digested overnight with 400 U of DpnII (New England Biolabs) at 37°C with shaking.

    Article Title:
    Article Snippet: DpnII (New England Biolabs) restriction enzyme was added to the remaining sample, and it was digested overnight at 37 °C. .. DpnII (New England Biolabs) restriction enzyme was added to the remaining sample, and it was digested overnight at 37 °C.

    Article Title: Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA
    Article Snippet: Fluorescent oligonucleotide substrates: 49-mer oligode-oxynucleotides containing 3-meC or 1-meA in the recognition site for the methylation sensitive restriction enzyme DpnII with the sequence as described in Ref. , were labeled with 6-carboxyfluorescein at the 5′ end, and synthesized with phosphorothioate linkages (5 bases) at the 3′ and 5′ ends. hABH1(Δ3) was incubated with 1 pmol of single-stranded or double-stranded substrate in 10 m m Tris-HCl, pH 6.6, 10 m m KCl, 100 μg/ml bovine serum albumin, 4 m m DTT, 100 μ m 2OG, and 10 μ m FeSO4 (NH4 )2 SO4 at 37 °C for 15 min. .. The oligodeoxynucleotide was ethanol precipitated and resuspended in DpnII reaction buffer (New England Biolabs), annealed to the complementary oligodeoxynucleotide and treated with 10 units of DpnII at 37 °C for 1 h. Substrate and product bands (49- and 22-mer) were separated by PAGE and fluorescence visualized using a Typhoon TRIO GE scanner.

    Article Title: Facultative CTCF sites moderate mammary super-enhancer activity and regulate juxtaposed gene in non-mammary cells
    Article Snippet: Pellets were lysed in lysis buffer (10 mM Tris HCI pH 8.0, 10 mM NaCl, 0.5% Nonidet P-40) containing phenylmethylsulfonyl fluoride and protease inhibitors, incubated on ice for 30 min, and dounced using a pre-chilled glass homogenizer, followed by another 10 min incubation on ice. .. After removal of supernatant, nuclei pellets were re-suspended in DpnII buffer (New England Biolabs).

    Expressing:

    Article Title: Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation
    Article Snippet: The DNA was digested with DpnII (R0543M; New England BioLabs) and Csp6I (FD0214; Fermentas, Thermo Scientific) as primary and secondary enzymes, respectively. .. Raw sequencing data were demultiplexed and aligned using the zebrafish Zv9/danRer7 reference genome.

    Article Title: Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins
    Article Snippet: In brief, DNA was isolated from cells expressing Dam or Dam-LMNB1 using DNA Mini kit (QIAGEN), precipitated, and resuspended to 1 µg/µl. .. After digestion, double-stranded adapters comprising annealed oligonucleotide AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′) were ligated overnight with the digested DpnI fragments (T4 ligase 799009; Roche) followed by DPNII digestion (R0543S; New England Biolabs, Inc.) for 1 h. This material was amplified in ligation-mediated PCR using AdR_PCR primer (5′-GGTCGCGGCCGAGGATC-3′) and Advantage cDNA Polymerase (Takara Bio Inc.).

    Ex Vivo:

    Article Title: Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins
    Article Snippet: Ex vivo expanded Rag2−/− pro–B cells were spinfected by centrifuging in 24- or 48-well plates at 500 g for 2 h with viral supernatant plus 3 ng/ml interleukin-7 (IL-7) and 4 µg polybrene at room temperature. .. After digestion, double-stranded adapters comprising annealed oligonucleotide AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′) were ligated overnight with the digested DpnI fragments (T4 ligase 799009; Roche) followed by DPNII digestion (R0543S; New England Biolabs, Inc.) for 1 h. This material was amplified in ligation-mediated PCR using AdR_PCR primer (5′-GGTCGCGGCCGAGGATC-3′) and Advantage cDNA Polymerase (Takara Bio Inc.).

    Hybridization:

    Article Title: Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins
    Article Snippet: After digestion, double-stranded adapters comprising annealed oligonucleotide AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′) were ligated overnight with the digested DpnI fragments (T4 ligase 799009; Roche) followed by DPNII digestion (R0543S; New England Biolabs, Inc.) for 1 h. This material was amplified in ligation-mediated PCR using AdR_PCR primer (5′-GGTCGCGGCCGAGGATC-3′) and Advantage cDNA Polymerase (Takara Bio Inc.). .. The resulting material was checked by agarose gel electrophoresis to ensure that specific amplification of methylated DNA fragments occurred and then column purified (QIAquick PCR purification kit; QIAGEN).

    High Performance Liquid Chromatography:

    Article Title: Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA
    Article Snippet: The substrates were degraded enzymatically to deoxynucleosides or nucleosides and analyzed by LC/MS/MS using an Agilent HP1100 HPLC system interfaced an Applied Biosystems API4000 hybrid triple quadrupole/linear ion trap mass spectrometer, essentially as described ( ). .. The oligodeoxynucleotide was ethanol precipitated and resuspended in DpnII reaction buffer (New England Biolabs), annealed to the complementary oligodeoxynucleotide and treated with 10 units of DpnII at 37 °C for 1 h. Substrate and product bands (49- and 22-mer) were separated by PAGE and fluorescence visualized using a Typhoon TRIO GE scanner.

    Ligation:

    Article Title: A Distal CCAAT/NUCLEAR FACTOR Y Complex Promotes Chromatin Looping at the FLOWERING LOCUS T Promoter and Regulates the Timing of Flowering in Arabidopsis
    Article Snippet: To control for differences in primer set efficiency during PCR amplification, a control template was required that contains all possible ligation products of the loci of interest ( FT and EF1α ) in equimolar amounts. .. Five micrograms of pFT:FT construct was digested for 6 h with Dpn II (New England Biolabs), extracted with phenol-chloroform, and precipitated.

    Article Title: A common copy-number variant within SIRPB1 correlates with human Out-of-Africa migration after genetic drift correction
    Article Snippet: Isolated cells were lysed (lysis buffer: 10 mM Tris-HCl pH 8, 10 mM NaCl, 0.3% Igepal CA-630 (Sigma-Aldrich, I8896) and 1× protease inhibitor cocktail (Complete, Roche, 11697498001)), and the DNA was digested with DpnII (New England BioLabs, R0543M) and Csp6I (Fermentas, Thermo Scientific, FD0214) as primary and secondary enzymes, respectively. .. T4 DNA ligase (Promega, M1804) was used for both ligation steps.

    Article Title: Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins
    Article Snippet: 2 µg of this genomic DNA was digested overnight with the restriction enzyme DpnI (R0176L; New England Biolabs, Inc.), which cuts at methylated GAme TC. .. After digestion, double-stranded adapters comprising annealed oligonucleotide AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′) were ligated overnight with the digested DpnI fragments (T4 ligase 799009; Roche) followed by DPNII digestion (R0543S; New England Biolabs, Inc.) for 1 h. This material was amplified in ligation-mediated PCR using AdR_PCR primer (5′-GGTCGCGGCCGAGGATC-3′) and Advantage cDNA Polymerase (Takara Bio Inc.). .. The resulting material was checked by agarose gel electrophoresis to ensure that specific amplification of methylated DNA fragments occurred and then column purified (QIAquick PCR purification kit; QIAGEN).

    Article Title: Epigenomic profiling of primary gastric adenocarcinoma reveals super-enhancer heterogeneity
    Article Snippet: Next, HindIII-digested DNA was subjected to proximity ligation using T4 DNA ligase (EL0013, Thermo Scientific), followed by cross-link removal using Proteinase K (AM2546, Ambion), yielding 3C libraries. .. The 3C libraries were then subjected to a second restriction enzyme digestion using DpnII (R0543L, NEB), followed by a circularization reaction using T4 DNA ligase.

    Article Title: Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver
    Article Snippet: Primary digestion of 10 million nuclei with 50,000 U of DpnII (NEB: #R0543) was performed overnight at 37°C in 450 μl of NEBuffer 3 (NEB: #B7003S; 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) with agitation at 900 RPM. .. After confirming digestion by agarose gel electrophoresis, DpnII was inactivated with SDS (2%, final concentration) and the samples then diluted 5-fold in 1X ligation buffer (Enzymatics #B6030).

    Article Title:
    Article Snippet: DpnII (New England Biolabs) restriction enzyme was added to the remaining sample, and it was digested overnight at 37 °C. .. DpnII (New England Biolabs) restriction enzyme was added to the remaining sample, and it was digested overnight at 37 °C.

    Protease Inhibitor:

    Article Title: A common copy-number variant within SIRPB1 correlates with human Out-of-Africa migration after genetic drift correction
    Article Snippet: Fixation was then quenched with glycine (0.15M final concentration).The rest of the 4C-seq assays were performed as previously reported13,21 . .. Isolated cells were lysed (lysis buffer: 10 mM Tris-HCl pH 8, 10 mM NaCl, 0.3% Igepal CA-630 (Sigma-Aldrich, I8896) and 1× protease inhibitor cocktail (Complete, Roche, 11697498001)), and the DNA was digested with DpnII (New England BioLabs, R0543M) and Csp6I (Fermentas, Thermo Scientific, FD0214) as primary and secondary enzymes, respectively. .. T4 DNA ligase (Promega, M1804) was used for both ligation steps.

    Article Title: Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver
    Article Snippet: The supernatant was removed following the second wash, and cell pellets were resuspended in 8 ml of lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100, and 1X Complete Protease Inhibitor Cocktail) and incubated on ice for 40 min with occasional mixing. .. Primary digestion of 10 million nuclei with 50,000 U of DpnII (NEB: #R0543) was performed overnight at 37°C in 450 μl of NEBuffer 3 (NEB: #B7003S; 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) with agitation at 900 RPM.

    Article Title: Clock-dependent chromatin topology modulates circadian transcription and behavior
    Article Snippet: Nuclei were resuspended in 1 mL of a buffer containing 10 mM Tris-HCL (pH 8.0), 10 mM NaCl, 0.2% NP-40, and 1× protease inhibitor cocktail (Complete Mini EDTA-free protease inhibitor cocktail; Sigma-Aldrich); kept for 15 min on ice; and washed twice with 1× DpnII buffer (New England Biolabs). .. Chromatin was digested overnight with 400 U of DpnII (New England Biolabs) at 37°C with shaking.

    Article Title: Clock-dependent chromatin topology modulates circadian transcription and behavior
    Article Snippet: 4C templates were prepared as in . .. Nuclei were resuspended in 1 mL of a buffer containing 10 mM Tris-HCL (pH 8.0), 10 mM NaCl, 0.2% NP-40, and 1× protease inhibitor cocktail (Complete Mini EDTA-free protease inhibitor cocktail; Sigma-Aldrich); kept for 15 min on ice; and washed twice with 1× DpnII buffer (New England Biolabs). .. Thirty million nuclei were resuspended in 1× DpnII buffer (New England Biolabs) containing 0.1% SDS and incubated for 10 min at 65°C.

    Cell Culture:

    Article Title: Epigenomic profiling of primary gastric adenocarcinoma reveals super-enhancer heterogeneity
    Article Snippet: In brief, cultured cells were diluted into single-cell suspensions, and chromatin was cross-linked with 1% formaldehyde for 10 min at room temperature. .. The 3C libraries were then subjected to a second restriction enzyme digestion using DpnII (R0543L, NEB), followed by a circularization reaction using T4 DNA ligase.

    Capture-C:

    Article Title: Impaired DNA demethylation of C/EBP sites causes premature aging
    Article Snippet: Paragraph title: NG Capture-C ... After snap-freezing in liquid nitrogen, cell pellets were resuspended in a total of 1.4 mL of 1× DpnII buffer (New England Biolabs) and homogenized on ice with 55 strokes of a 5-mL Dounce homogenizer.

    Sequencing:

    Article Title: Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation
    Article Snippet: The DNA was digested with DpnII (R0543M; New England BioLabs) and Csp6I (FD0214; Fermentas, Thermo Scientific) as primary and secondary enzymes, respectively. .. These libraries were purified with a PCR Product Purification kit (No. 11732668001; Roche), their concentrations were measured using the Qubit dsDNA BR assay kit (No. ; Molecular Probes), and they were sent for deep sequencing.

    Article Title: Molecular Evolutionary History of Tubercle Bacilli Assessed by Study of the Polymorphic Nucleotide within the Nitrate Reductase (narGHJI) Operon Promoter
    Article Snippet: A 10-μl aliquot of the amplified fragments (roughly 155 to 160 bp on gel) was further digested using either Sau3AI or DpnII according to the manufacturer's instructions (New England Biolabs, Beverly, MA, for DpnII and Roche-Biomedicals, Meylan, France for Sau3AI). .. These enzymes specifically cut at the GATC sequence overlapping the M. bovis -like −215C narGHJI promoter site, producing two bands (of approximately 90 and of 70 bp) in PCR-RFLP of the LC66-LC67 PCR fragment.

    Article Title: Allelic exclusion of the immunoglobulin heavy chain locus is independent of its nuclear localization in mature B cells
    Article Snippet: Cells were fixed and lysed as described ( ) using HindIII (Roche) as a first cutter and DpnII (New England Biolabs) as a second cutter. .. Cells were fixed and lysed as described ( ) using HindIII (Roche) as a first cutter and DpnII (New England Biolabs) as a second cutter.

    Article Title: Regulation of the Protocadherin Celsr3 Gene and Its Role in Globus Pallidus Development and Connectivity
    Article Snippet: Circular chromosome confirmation capture (4C) libraries for high-throughput sequencing (4C-seq) were constructed as described previously , with some modifications. .. After phenol-chloroform extraction and ethanol precipitation, the purified DNA was digested with the secondary restriction enzyme DpnII (NEB) and ligated again.

    Article Title: Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA
    Article Snippet: Fluorescent oligonucleotide substrates: 49-mer oligode-oxynucleotides containing 3-meC or 1-meA in the recognition site for the methylation sensitive restriction enzyme DpnII with the sequence as described in Ref. , were labeled with 6-carboxyfluorescein at the 5′ end, and synthesized with phosphorothioate linkages (5 bases) at the 3′ and 5′ ends. hABH1(Δ3) was incubated with 1 pmol of single-stranded or double-stranded substrate in 10 m m Tris-HCl, pH 6.6, 10 m m KCl, 100 μg/ml bovine serum albumin, 4 m m DTT, 100 μ m 2OG, and 10 μ m FeSO4 (NH4 )2 SO4 at 37 °C for 15 min. .. The oligodeoxynucleotide was ethanol precipitated and resuspended in DpnII reaction buffer (New England Biolabs), annealed to the complementary oligodeoxynucleotide and treated with 10 units of DpnII at 37 °C for 1 h. Substrate and product bands (49- and 22-mer) were separated by PAGE and fluorescence visualized using a Typhoon TRIO GE scanner.

    Article Title: Adenine methylation may contribute to endosymbiont selection in a clonal aphid population
    Article Snippet: The fragments were identified and isolated using a pair of restriction enzymes recognizing the same nucleotide sequence (GATC): the first, DpnI , digests while the adenine is methylated; the second, DpnII , is blocked by the presence of a methyl group on the adenine. .. DNA fragments containing methylated adenines were isolated from the genomic DNA using the methylation-specific restriction enzyme DpnI together with the methyl-sensitive restriction enzyme DpnII (New England Biolabs, Ipswich, MA, USA).

    Cellular Antioxidant Activity Assay:

    Article Title: Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation
    Article Snippet: The following primers were used: rfng_4C_R: 5′-CGA ATC TTA TAA ACT TGA TGA ATG TGA TC-3′and rfng_4C_NR: 5′-TCA TTG CAA AGC TGA CAA CG-3′. .. The DNA was digested with DpnII (R0543M; New England BioLabs) and Csp6I (FD0214; Fermentas, Thermo Scientific) as primary and secondary enzymes, respectively.

    Hi-C:

    Article Title: Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast
    Article Snippet: After addition of 1 % (w/v) Triton X-100, chromatin was digested overnight with DpnII (New England Biolabs) and RNase A (Sigma) at 37 ºC. .. After heat inactivation of DpnII at 65 ºC for 20 minutes, digested chromatin was split into 3 parts, one for 3C library preparation and the other two for Hi-C library preparation.

    Fluorescence:

    Article Title: Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA
    Article Snippet: Fluorescent oligonucleotide substrates: 49-mer oligode-oxynucleotides containing 3-meC or 1-meA in the recognition site for the methylation sensitive restriction enzyme DpnII with the sequence as described in Ref. , were labeled with 6-carboxyfluorescein at the 5′ end, and synthesized with phosphorothioate linkages (5 bases) at the 3′ and 5′ ends. hABH1(Δ3) was incubated with 1 pmol of single-stranded or double-stranded substrate in 10 m m Tris-HCl, pH 6.6, 10 m m KCl, 100 μg/ml bovine serum albumin, 4 m m DTT, 100 μ m 2OG, and 10 μ m FeSO4 (NH4 )2 SO4 at 37 °C for 15 min. .. The oligodeoxynucleotide was ethanol precipitated and resuspended in DpnII reaction buffer (New England Biolabs), annealed to the complementary oligodeoxynucleotide and treated with 10 units of DpnII at 37 °C for 1 h. Substrate and product bands (49- and 22-mer) were separated by PAGE and fluorescence visualized using a Typhoon TRIO GE scanner. .. hABH1 Is a Widely Expressed Mitochondrial Protein —Northern blot analysis revealed that hABH1 is widely expressed in different human tissues at the mRNA level ( ).

    Methylation:

    Article Title: Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins
    Article Snippet: 2 µg of this genomic DNA was digested overnight with the restriction enzyme DpnI (R0176L; New England Biolabs, Inc.), which cuts at methylated GAme TC. .. After digestion, double-stranded adapters comprising annealed oligonucleotide AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′) were ligated overnight with the digested DpnI fragments (T4 ligase 799009; Roche) followed by DPNII digestion (R0543S; New England Biolabs, Inc.) for 1 h. This material was amplified in ligation-mediated PCR using AdR_PCR primer (5′-GGTCGCGGCCGAGGATC-3′) and Advantage cDNA Polymerase (Takara Bio Inc.).

    Article Title: Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA
    Article Snippet: Fluorescent oligonucleotide substrates: 49-mer oligode-oxynucleotides containing 3-meC or 1-meA in the recognition site for the methylation sensitive restriction enzyme DpnII with the sequence as described in Ref. , were labeled with 6-carboxyfluorescein at the 5′ end, and synthesized with phosphorothioate linkages (5 bases) at the 3′ and 5′ ends. hABH1(Δ3) was incubated with 1 pmol of single-stranded or double-stranded substrate in 10 m m Tris-HCl, pH 6.6, 10 m m KCl, 100 μg/ml bovine serum albumin, 4 m m DTT, 100 μ m 2OG, and 10 μ m FeSO4 (NH4 )2 SO4 at 37 °C for 15 min. .. The oligodeoxynucleotide was ethanol precipitated and resuspended in DpnII reaction buffer (New England Biolabs), annealed to the complementary oligodeoxynucleotide and treated with 10 units of DpnII at 37 °C for 1 h. Substrate and product bands (49- and 22-mer) were separated by PAGE and fluorescence visualized using a Typhoon TRIO GE scanner.

    Article Title: Adenine methylation may contribute to endosymbiont selection in a clonal aphid population
    Article Snippet: Briefly, gDNA was extracted from 100 mg of A. pisum (green and orange variants) using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). .. DNA fragments containing methylated adenines were isolated from the genomic DNA using the methylation-specific restriction enzyme DpnI together with the methyl-sensitive restriction enzyme DpnII (New England Biolabs, Ipswich, MA, USA). .. The digested fragments were ligated to the adaptors: AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′).

    Isolation:

    Article Title: A common copy-number variant within SIRPB1 correlates with human Out-of-Africa migration after genetic drift correction
    Article Snippet: Fixation was then quenched with glycine (0.15M final concentration).The rest of the 4C-seq assays were performed as previously reported13,21 . .. Isolated cells were lysed (lysis buffer: 10 mM Tris-HCl pH 8, 10 mM NaCl, 0.3% Igepal CA-630 (Sigma-Aldrich, I8896) and 1× protease inhibitor cocktail (Complete, Roche, 11697498001)), and the DNA was digested with DpnII (New England BioLabs, R0543M) and Csp6I (Fermentas, Thermo Scientific, FD0214) as primary and secondary enzymes, respectively. .. T4 DNA ligase (Promega, M1804) was used for both ligation steps.

    Article Title: Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins
    Article Snippet: In brief, DNA was isolated from cells expressing Dam or Dam-LMNB1 using DNA Mini kit (QIAGEN), precipitated, and resuspended to 1 µg/µl. .. After digestion, double-stranded adapters comprising annealed oligonucleotide AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′) were ligated overnight with the digested DpnI fragments (T4 ligase 799009; Roche) followed by DPNII digestion (R0543S; New England Biolabs, Inc.) for 1 h. This material was amplified in ligation-mediated PCR using AdR_PCR primer (5′-GGTCGCGGCCGAGGATC-3′) and Advantage cDNA Polymerase (Takara Bio Inc.).

    Article Title: SadA, a novel adhesion receptor in Dictyostelium
    Article Snippet: In brief, a Bio-Rad Laboratories gene pulser set to 0.85 kV/25 μF was used to electroporate 10 μg BamHI-linearized pUCΔBamBsr ( ) together with 10 U DpnII enzyme (NEB) into 7 × 106 wild-type (AX3) cells. .. In brief, a Bio-Rad Laboratories gene pulser set to 0.85 kV/25 μF was used to electroporate 10 μg BamHI-linearized pUCΔBamBsr ( ) together with 10 U DpnII enzyme (NEB) into 7 × 106 wild-type (AX3) cells.

    Article Title: Adenine methylation may contribute to endosymbiont selection in a clonal aphid population
    Article Snippet: Briefly, gDNA was extracted from 100 mg of A. pisum (green and orange variants) using the DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). .. DNA fragments containing methylated adenines were isolated from the genomic DNA using the methylation-specific restriction enzyme DpnI together with the methyl-sensitive restriction enzyme DpnII (New England Biolabs, Ipswich, MA, USA). .. The digested fragments were ligated to the adaptors: AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′).

    Article Title: DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis
    Article Snippet: Isolation of genomic DNA (gDNA) from WIDs is described above. .. Ligated gDNA fragments were subsequently digested with DpnII (10 U, New England Biolabs) in DpnII buffer (New England Biolabs) in a total volume of 50 µl for 1 h at 37 °C.

    Labeling:

    Article Title: Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins
    Article Snippet: After digestion, double-stranded adapters comprising annealed oligonucleotide AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′) were ligated overnight with the digested DpnI fragments (T4 ligase 799009; Roche) followed by DPNII digestion (R0543S; New England Biolabs, Inc.) for 1 h. This material was amplified in ligation-mediated PCR using AdR_PCR primer (5′-GGTCGCGGCCGAGGATC-3′) and Advantage cDNA Polymerase (Takara Bio Inc.). .. For qPCR assays, we used the following primer sets: specific to a region of the TCIS insert (TCIS_ChIP_F1 , 5′-AGCTTGGCGTAATCATGGTC-3′, and ChIP_R5 , 5′-ATTAGGCACCCCAGGCTTTA-3′), specific to an internal Igh LAD region (J558 1 , 5′-AGTGCAGGGCTCACAGAAAA-3′, and J558 12 , 5′-CAGCTCCATCCCATGGTTAGA-3′), or specific to a lamina-negative region in a LAD (also called Dip; chr10:105245772_141bp_F , 5′-AGGGACAGCCGTGGAGGAGC-3′, and chr10:105245772_141bp_R , 5′-CCGCACCGTCCGGTTCTCAG-3′; ).

    Article Title: Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA
    Article Snippet: Fluorescent oligonucleotide substrates: 49-mer oligode-oxynucleotides containing 3-meC or 1-meA in the recognition site for the methylation sensitive restriction enzyme DpnII with the sequence as described in Ref. , were labeled with 6-carboxyfluorescein at the 5′ end, and synthesized with phosphorothioate linkages (5 bases) at the 3′ and 5′ ends. hABH1(Δ3) was incubated with 1 pmol of single-stranded or double-stranded substrate in 10 m m Tris-HCl, pH 6.6, 10 m m KCl, 100 μg/ml bovine serum albumin, 4 m m DTT, 100 μ m 2OG, and 10 μ m FeSO4 (NH4 )2 SO4 at 37 °C for 15 min. .. The oligodeoxynucleotide was ethanol precipitated and resuspended in DpnII reaction buffer (New England Biolabs), annealed to the complementary oligodeoxynucleotide and treated with 10 units of DpnII at 37 °C for 1 h. Substrate and product bands (49- and 22-mer) were separated by PAGE and fluorescence visualized using a Typhoon TRIO GE scanner.

    Purification:

    Article Title: A Distal CCAAT/NUCLEAR FACTOR Y Complex Promotes Chromatin Looping at the FLOWERING LOCUS T Promoter and Regulates the Timing of Flowering in Arabidopsis
    Article Snippet: Five micrograms of pFT:FT construct was digested for 6 h with Dpn II (New England Biolabs), extracted with phenol-chloroform, and precipitated. .. Five micrograms of pFT:FT construct was digested for 6 h with Dpn II (New England Biolabs), extracted with phenol-chloroform, and precipitated.

    Article Title: A common copy-number variant within SIRPB1 correlates with human Out-of-Africa migration after genetic drift correction
    Article Snippet: Periferal blood mononuclear cells were obtained from six different healthy donnors (two for each of the genotypes studied), purified using Ficoll and rapidly fixed using paraformaldehyde (2% final concentration) for 10’ at room temperature. .. Isolated cells were lysed (lysis buffer: 10 mM Tris-HCl pH 8, 10 mM NaCl, 0.3% Igepal CA-630 (Sigma-Aldrich, I8896) and 1× protease inhibitor cocktail (Complete, Roche, 11697498001)), and the DNA was digested with DpnII (New England BioLabs, R0543M) and Csp6I (Fermentas, Thermo Scientific, FD0214) as primary and secondary enzymes, respectively.

    Article Title: Clock-dependent chromatin topology modulates circadian transcription and behavior
    Article Snippet: Chromatin was digested overnight with 400 U of DpnII (New England Biolabs) at 37°C with shaking. .. The cross-linking reaction was reverted by the addition of 50 µL of 10 mg/mL proteinase K and incubation overnight at 65°C.

    Article Title:
    Article Snippet: DpnII (New England Biolabs) restriction enzyme was added to the remaining sample, and it was digested overnight at 37 °C. .. DpnII (New England Biolabs) restriction enzyme was added to the remaining sample, and it was digested overnight at 37 °C.

    Article Title: Regulation of the Protocadherin Celsr3 Gene and Its Role in Globus Pallidus Development and Connectivity
    Article Snippet: The cross-linked DNA was digested with HindIII (NEB) overnight with shaking at 900 rpm and then ligated using T4 DNA ligase (NEB). .. After phenol-chloroform extraction and ethanol precipitation, the purified DNA was digested with the secondary restriction enzyme DpnII (NEB) and ligated again. .. Finally, 4C-seq libraries were amplified from the religated DNA using PCR with inverse primers, including Illumina adapter sequences (human CELSR3 , AATGA TACGG CGACC ACCGA GATCT ACACT CTTTC CCTAC ACGAC GCTCT TCCGA TCTCA GGAAC AGCAG CTTAC TATGG AAG and CAAGC AGAAG ACGGC ATACG AGATC GTGAT GTGAC TGGAG TTCAG ACGTG TGCTC TTCCG ATCGA ACCTA AGCAG AGTCC TTGTG AG; mouse Celsr3 , AATGA TACGG CGACC ACCGA GATCT ACACT CTTTC CCTAC ACGAC GCTCT TCCGA TCTCT GGTGA AGGTG CTTGG TGTCC and CAAGC AGAAG ACGGC ATACG AGATC GTGAT GTGAC TGGAG TTCAG ACGTG TGCTC TTCCG ATCAG TGCTG CTCAG GCAAC TCGTA C).

    Article Title: Facultative CTCF sites moderate mammary super-enhancer activity and regulate juxtaposed gene in non-mammary cells
    Article Snippet: After removal of supernatant, nuclei pellets were re-suspended in DpnII buffer (New England Biolabs). .. After removal of supernatant, nuclei pellets were re-suspended in DpnII buffer (New England Biolabs).

    Polymerase Chain Reaction:

    Article Title: A Distal CCAAT/NUCLEAR FACTOR Y Complex Promotes Chromatin Looping at the FLOWERING LOCUS T Promoter and Regulates the Timing of Flowering in Arabidopsis
    Article Snippet: To control for differences in primer set efficiency during PCR amplification, a control template was required that contains all possible ligation products of the loci of interest ( FT and EF1α ) in equimolar amounts. .. Five micrograms of pFT:FT construct was digested for 6 h with Dpn II (New England Biolabs), extracted with phenol-chloroform, and precipitated.

    Article Title: A common copy-number variant within SIRPB1 correlates with human Out-of-Africa migration after genetic drift correction
    Article Snippet: Isolated cells were lysed (lysis buffer: 10 mM Tris-HCl pH 8, 10 mM NaCl, 0.3% Igepal CA-630 (Sigma-Aldrich, I8896) and 1× protease inhibitor cocktail (Complete, Roche, 11697498001)), and the DNA was digested with DpnII (New England BioLabs, R0543M) and Csp6I (Fermentas, Thermo Scientific, FD0214) as primary and secondary enzymes, respectively. .. Isolated cells were lysed (lysis buffer: 10 mM Tris-HCl pH 8, 10 mM NaCl, 0.3% Igepal CA-630 (Sigma-Aldrich, I8896) and 1× protease inhibitor cocktail (Complete, Roche, 11697498001)), and the DNA was digested with DpnII (New England BioLabs, R0543M) and Csp6I (Fermentas, Thermo Scientific, FD0214) as primary and secondary enzymes, respectively.

    Article Title: Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins
    Article Snippet: 2 µg of this genomic DNA was digested overnight with the restriction enzyme DpnI (R0176L; New England Biolabs, Inc.), which cuts at methylated GAme TC. .. After digestion, double-stranded adapters comprising annealed oligonucleotide AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′) were ligated overnight with the digested DpnI fragments (T4 ligase 799009; Roche) followed by DPNII digestion (R0543S; New England Biolabs, Inc.) for 1 h. This material was amplified in ligation-mediated PCR using AdR_PCR primer (5′-GGTCGCGGCCGAGGATC-3′) and Advantage cDNA Polymerase (Takara Bio Inc.). .. The resulting material was checked by agarose gel electrophoresis to ensure that specific amplification of methylated DNA fragments occurred and then column purified (QIAquick PCR purification kit; QIAGEN).

    Article Title: Epigenomic profiling of primary gastric adenocarcinoma reveals super-enhancer heterogeneity
    Article Snippet: The 3C libraries were then subjected to a second restriction enzyme digestion using DpnII (R0543L, NEB), followed by a circularization reaction using T4 DNA ligase. .. For each viewpoint, 3.2 μg of the resulting 4C templates was used to perform a scale-up inverse, nested PCR , of which 32 reactions (100 ng in each) were pooled and purified using the MinElute PCR Purification kit (Qiagen).

    Article Title: Molecular Evolutionary History of Tubercle Bacilli Assessed by Study of the Polymorphic Nucleotide within the Nitrate Reductase (narGHJI) Operon Promoter
    Article Snippet: A single PCR product was observed for each DNA isolate. .. A 10-μl aliquot of the amplified fragments (roughly 155 to 160 bp on gel) was further digested using either Sau3AI or DpnII according to the manufacturer's instructions (New England Biolabs, Beverly, MA, for DpnII and Roche-Biomedicals, Meylan, France for Sau3AI).

    Article Title: Adenine methylation may contribute to endosymbiont selection in a clonal aphid population
    Article Snippet: DNA fragments containing methylated adenines were isolated from the genomic DNA using the methylation-specific restriction enzyme DpnI together with the methyl-sensitive restriction enzyme DpnII (New England Biolabs, Ipswich, MA, USA). .. DNA fragments containing methylated adenines were isolated from the genomic DNA using the methylation-specific restriction enzyme DpnI together with the methyl-sensitive restriction enzyme DpnII (New England Biolabs, Ipswich, MA, USA).

    Article Title: DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis
    Article Snippet: Paragraph title: DamID sample processing, PCR and NGS library preparation ... Ligated gDNA fragments were subsequently digested with DpnII (10 U, New England Biolabs) in DpnII buffer (New England Biolabs) in a total volume of 50 µl for 1 h at 37 °C.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Epigenomic profiling of primary gastric adenocarcinoma reveals super-enhancer heterogeneity
    Article Snippet: The 3C libraries were then subjected to a second restriction enzyme digestion using DpnII (R0543L, NEB), followed by a circularization reaction using T4 DNA ligase. .. For each viewpoint, 3.2 μg of the resulting 4C templates was used to perform a scale-up inverse, nested PCR , of which 32 reactions (100 ng in each) were pooled and purified using the MinElute PCR Purification kit (Qiagen).

    Article Title: Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA
    Article Snippet: Fluorescent oligonucleotide substrates: 49-mer oligode-oxynucleotides containing 3-meC or 1-meA in the recognition site for the methylation sensitive restriction enzyme DpnII with the sequence as described in Ref. , were labeled with 6-carboxyfluorescein at the 5′ end, and synthesized with phosphorothioate linkages (5 bases) at the 3′ and 5′ ends. hABH1(Δ3) was incubated with 1 pmol of single-stranded or double-stranded substrate in 10 m m Tris-HCl, pH 6.6, 10 m m KCl, 100 μg/ml bovine serum albumin, 4 m m DTT, 100 μ m 2OG, and 10 μ m FeSO4 (NH4 )2 SO4 at 37 °C for 15 min. .. The oligodeoxynucleotide was ethanol precipitated and resuspended in DpnII reaction buffer (New England Biolabs), annealed to the complementary oligodeoxynucleotide and treated with 10 units of DpnII at 37 °C for 1 h. Substrate and product bands (49- and 22-mer) were separated by PAGE and fluorescence visualized using a Typhoon TRIO GE scanner. .. hABH1 Is a Widely Expressed Mitochondrial Protein —Northern blot analysis revealed that hABH1 is widely expressed in different human tissues at the mRNA level ( ).

    Staining:

    Article Title: Molecular Evolutionary History of Tubercle Bacilli Assessed by Study of the Polymorphic Nucleotide within the Nitrate Reductase (narGHJI) Operon Promoter
    Article Snippet: A 10-μl aliquot of the amplified fragments (roughly 155 to 160 bp on gel) was further digested using either Sau3AI or DpnII according to the manufacturer's instructions (New England Biolabs, Beverly, MA, for DpnII and Roche-Biomedicals, Meylan, France for Sau3AI). .. In the case of the M. tuberculosis -like −215T sequence, the restriction site is absent and as a result PCR-RFLP of the narGHJI amplicon yields a single uncut band.

    Sample Prep:

    Article Title: Condensin-mediated remodeling of the mitotic chromatin landscape in fission yeast
    Article Snippet: Paragraph title: Hi-C/3C sample preparation ... After addition of 1 % (w/v) Triton X-100, chromatin was digested overnight with DpnII (New England Biolabs) and RNase A (Sigma) at 37 ºC.

    Activated Clotting Time Assay:

    Article Title: Evolutionary emergence of the rac3b/rfng/sgca regulatory cluster refined mechanisms for hindbrain boundaries formation
    Article Snippet: The following primers were used: rfng_4C_R: 5′-CGA ATC TTA TAA ACT TGA TGA ATG TGA TC-3′and rfng_4C_NR: 5′-TCA TTG CAA AGC TGA CAA CG-3′. .. The DNA was digested with DpnII (R0543M; New England BioLabs) and Csp6I (FD0214; Fermentas, Thermo Scientific) as primary and secondary enzymes, respectively.

    Chromatin Immunoprecipitation:

    Article Title: Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins
    Article Snippet: After digestion, double-stranded adapters comprising annealed oligonucleotide AdRt (5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′) and AdRb (5′-TCCTCGGCCG-3′) were ligated overnight with the digested DpnI fragments (T4 ligase 799009; Roche) followed by DPNII digestion (R0543S; New England Biolabs, Inc.) for 1 h. This material was amplified in ligation-mediated PCR using AdR_PCR primer (5′-GGTCGCGGCCGAGGATC-3′) and Advantage cDNA Polymerase (Takara Bio Inc.). .. This material was used either for qPCR assays or subjected to array hybridization.

    Liquid Chromatography:

    Article Title: Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA
    Article Snippet: The substrates were degraded enzymatically to deoxynucleosides or nucleosides and analyzed by LC/MS/MS using an Agilent HP1100 HPLC system interfaced an Applied Biosystems API4000 hybrid triple quadrupole/linear ion trap mass spectrometer, essentially as described ( ). .. The oligodeoxynucleotide was ethanol precipitated and resuspended in DpnII reaction buffer (New England Biolabs), annealed to the complementary oligodeoxynucleotide and treated with 10 units of DpnII at 37 °C for 1 h. Substrate and product bands (49- and 22-mer) were separated by PAGE and fluorescence visualized using a Typhoon TRIO GE scanner.

    Software:

    Article Title: Molecular Evolutionary History of Tubercle Bacilli Assessed by Study of the Polymorphic Nucleotide within the Nitrate Reductase (narGHJI) Operon Promoter
    Article Snippet: A 10-μl aliquot of the amplified fragments (roughly 155 to 160 bp on gel) was further digested using either Sau3AI or DpnII according to the manufacturer's instructions (New England Biolabs, Beverly, MA, for DpnII and Roche-Biomedicals, Meylan, France for Sau3AI). .. The restriction digest was visualized by agarose gel electrophoresis on 3% Metaphor (FMC Bioproducts, Rockland, Maine) and run in Tris-borate-EDTA (1×) buffer, followed by ethidium-bromide staining.

    Agarose Gel Electrophoresis:

    Article Title: Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver
    Article Snippet: Primary digestion of 10 million nuclei with 50,000 U of DpnII (NEB: #R0543) was performed overnight at 37°C in 450 μl of NEBuffer 3 (NEB: #B7003S; 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) with agitation at 900 RPM. .. Primary digestion of 10 million nuclei with 50,000 U of DpnII (NEB: #R0543) was performed overnight at 37°C in 450 μl of NEBuffer 3 (NEB: #B7003S; 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) with agitation at 900 RPM.

    Article Title: Molecular Evolutionary History of Tubercle Bacilli Assessed by Study of the Polymorphic Nucleotide within the Nitrate Reductase (narGHJI) Operon Promoter
    Article Snippet: A 10-μl aliquot of the amplified fragments (roughly 155 to 160 bp on gel) was further digested using either Sau3AI or DpnII according to the manufacturer's instructions (New England Biolabs, Beverly, MA, for DpnII and Roche-Biomedicals, Meylan, France for Sau3AI). .. In the case of the M. tuberculosis -like −215T sequence, the restriction site is absent and as a result PCR-RFLP of the narGHJI amplicon yields a single uncut band.

    Article Title: DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis
    Article Snippet: Ligated gDNA fragments were subsequently digested with DpnII (10 U, New England Biolabs) in DpnII buffer (New England Biolabs) in a total volume of 50 µl for 1 h at 37 °C. .. PCR program: 10 min at 68 °C; 1 min at 94 °C, 5 min at 65 °C, 15 min at 68 °C; 1 min at 94 °C, 1 min at 65 °C, 10 min at 68 °C—repeated 3X; 1 min at 94 °C, 1 min at 65 °C, 2 min at 68 °C—repeated (17X).

    Double Knockout:

    Article Title: Impaired DNA demethylation of C/EBP sites causes premature aging
    Article Snippet: Three independent wild-type, Gadd45a −/− , Ing1 −/− , and Gadd45a–Ing1 double-knockout MEF cell lines were grown to 80% confluency. .. After snap-freezing in liquid nitrogen, cell pellets were resuspended in a total of 1.4 mL of 1× DpnII buffer (New England Biolabs) and homogenized on ice with 55 strokes of a 5-mL Dounce homogenizer.

    Ethanol Precipitation:

    Article Title: Regulation of the Protocadherin Celsr3 Gene and Its Role in Globus Pallidus Development and Connectivity
    Article Snippet: The cross-linked DNA was digested with HindIII (NEB) overnight with shaking at 900 rpm and then ligated using T4 DNA ligase (NEB). .. After phenol-chloroform extraction and ethanol precipitation, the purified DNA was digested with the secondary restriction enzyme DpnII (NEB) and ligated again. .. Finally, 4C-seq libraries were amplified from the religated DNA using PCR with inverse primers, including Illumina adapter sequences (human CELSR3 , AATGA TACGG CGACC ACCGA GATCT ACACT CTTTC CCTAC ACGAC GCTCT TCCGA TCTCA GGAAC AGCAG CTTAC TATGG AAG and CAAGC AGAAG ACGGC ATACG AGATC GTGAT GTGAC TGGAG TTCAG ACGTG TGCTC TTCCG ATCGA ACCTA AGCAG AGTCC TTGTG AG; mouse Celsr3 , AATGA TACGG CGACC ACCGA GATCT ACACT CTTTC CCTAC ACGAC GCTCT TCCGA TCTCT GGTGA AGGTG CTTGG TGTCC and CAAGC AGAAG ACGGC ATACG AGATC GTGAT GTGAC TGGAG TTCAG ACGTG TGCTC TTCCG ATCAG TGCTG CTCAG GCAAC TCGTA C).

    Next-Generation Sequencing:

    Article Title: Epigenomic profiling of primary gastric adenocarcinoma reveals super-enhancer heterogeneity
    Article Snippet: The 3C libraries were then subjected to a second restriction enzyme digestion using DpnII (R0543L, NEB), followed by a circularization reaction using T4 DNA ligase. .. On the gel, smears from 200 to 600 bp were excised and unwanted PCR product bands were removed.

    Article Title: DamID profiling of dynamic Polycomb-binding sites in Drosophila imaginal disc development and tumorigenesis
    Article Snippet: Paragraph title: DamID sample processing, PCR and NGS library preparation ... Ligated gDNA fragments were subsequently digested with DpnII (10 U, New England Biolabs) in DpnII buffer (New England Biolabs) in a total volume of 50 µl for 1 h at 37 °C.

    Concentration Assay:

    Article Title: A common copy-number variant within SIRPB1 correlates with human Out-of-Africa migration after genetic drift correction
    Article Snippet: Fixation was then quenched with glycine (0.15M final concentration).The rest of the 4C-seq assays were performed as previously reported13,21 . .. Isolated cells were lysed (lysis buffer: 10 mM Tris-HCl pH 8, 10 mM NaCl, 0.3% Igepal CA-630 (Sigma-Aldrich, I8896) and 1× protease inhibitor cocktail (Complete, Roche, 11697498001)), and the DNA was digested with DpnII (New England BioLabs, R0543M) and Csp6I (Fermentas, Thermo Scientific, FD0214) as primary and secondary enzymes, respectively.

    Article Title: Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver
    Article Snippet: 270 μl of 37% formaldehyde was added to give a final concentration of 1%, and crosslinking was carried out for 10 min with nutation at room temperature. .. Primary digestion of 10 million nuclei with 50,000 U of DpnII (NEB: #R0543) was performed overnight at 37°C in 450 μl of NEBuffer 3 (NEB: #B7003S; 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) with agitation at 900 RPM.

    Article Title: Clock-dependent chromatin topology modulates circadian transcription and behavior
    Article Snippet: Triton X-100 was added to 1% final concentration. .. Chromatin was digested overnight with 400 U of DpnII (New England Biolabs) at 37°C with shaking.

    Article Title:
    Article Snippet: For DpnII digestion, Triton X-100 (Sigma) was added to a final concentration of 1% to sequester excess SDS. .. DpnII (New England Biolabs) restriction enzyme was added to the remaining sample, and it was digested overnight at 37 °C.

    Article Title: Clock-dependent chromatin topology modulates circadian transcription and behavior
    Article Snippet: Nuclei were resuspended in 1 mL of a buffer containing 10 mM Tris-HCL (pH 8.0), 10 mM NaCl, 0.2% NP-40, and 1× protease inhibitor cocktail (Complete Mini EDTA-free protease inhibitor cocktail; Sigma-Aldrich); kept for 15 min on ice; and washed twice with 1× DpnII buffer (New England Biolabs). .. Thirty million nuclei were resuspended in 1× DpnII buffer (New England Biolabs) containing 0.1% SDS and incubated for 10 min at 65°C.

    Article Title: Impaired DNA demethylation of C/EBP sites causes premature aging
    Article Snippet: After snap-freezing in liquid nitrogen, cell pellets were resuspended in a total of 1.4 mL of 1× DpnII buffer (New England Biolabs) and homogenized on ice with 55 strokes of a 5-mL Dounce homogenizer. .. After snap-freezing in liquid nitrogen, cell pellets were resuspended in a total of 1.4 mL of 1× DpnII buffer (New England Biolabs) and homogenized on ice with 55 strokes of a 5-mL Dounce homogenizer.

    Lysis:

    Article Title: A common copy-number variant within SIRPB1 correlates with human Out-of-Africa migration after genetic drift correction
    Article Snippet: Fixation was then quenched with glycine (0.15M final concentration).The rest of the 4C-seq assays were performed as previously reported13,21 . .. Isolated cells were lysed (lysis buffer: 10 mM Tris-HCl pH 8, 10 mM NaCl, 0.3% Igepal CA-630 (Sigma-Aldrich, I8896) and 1× protease inhibitor cocktail (Complete, Roche, 11697498001)), and the DNA was digested with DpnII (New England BioLabs, R0543M) and Csp6I (Fermentas, Thermo Scientific, FD0214) as primary and secondary enzymes, respectively. .. T4 DNA ligase (Promega, M1804) was used for both ligation steps.

    Article Title: Computational prediction of CTCF/cohesin-based intra-TAD loops that insulate chromatin contacts and gene expression in mouse liver
    Article Snippet: The supernatant was removed following the second wash, and cell pellets were resuspended in 8 ml of lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.5% NP-40, 1% TX-100, and 1X Complete Protease Inhibitor Cocktail) and incubated on ice for 40 min with occasional mixing. .. Primary digestion of 10 million nuclei with 50,000 U of DpnII (NEB: #R0543) was performed overnight at 37°C in 450 μl of NEBuffer 3 (NEB: #B7003S; 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2 , 1 mM DTT, pH 7.9) with agitation at 900 RPM.

    Article Title:
    Article Snippet: The pellet was then subjected to cold lysis buffer with protease inhibitors (Roche Applied Science), homogenized with a Dounce homogenizer on ice, and centrifuged to pellet the nuclei. .. DpnII (New England Biolabs) restriction enzyme was added to the remaining sample, and it was digested overnight at 37 °C.

    Article Title: Facultative CTCF sites moderate mammary super-enhancer activity and regulate juxtaposed gene in non-mammary cells
    Article Snippet: Pellets were lysed in lysis buffer (10 mM Tris HCI pH 8.0, 10 mM NaCl, 0.5% Nonidet P-40) containing phenylmethylsulfonyl fluoride and protease inhibitors, incubated on ice for 30 min, and dounced using a pre-chilled glass homogenizer, followed by another 10 min incubation on ice. .. After removal of supernatant, nuclei pellets were re-suspended in DpnII buffer (New England Biolabs).

    Article Title: Impaired DNA demethylation of C/EBP sites causes premature aging
    Article Snippet: After two PBS washes, cells were lysed in 5 mL of ice-cold lysis buffer (10 mM Tris at pH 8, 10 mM NaCl, 0.2 % Igepal CA-630, PI [Sigma]). .. After snap-freezing in liquid nitrogen, cell pellets were resuspended in a total of 1.4 mL of 1× DpnII buffer (New England Biolabs) and homogenized on ice with 55 strokes of a 5-mL Dounce homogenizer.

    High Throughput Screening Assay:

    Article Title: Regulation of the Protocadherin Celsr3 Gene and Its Role in Globus Pallidus Development and Connectivity
    Article Snippet: Circular chromosome confirmation capture (4C) libraries for high-throughput sequencing (4C-seq) were constructed as described previously , with some modifications. .. After phenol-chloroform extraction and ethanol precipitation, the purified DNA was digested with the secondary restriction enzyme DpnII (NEB) and ligated again.

    FACS:

    Article Title: Allelic exclusion of the immunoglobulin heavy chain locus is independent of its nuclear localization in mature B cells
    Article Snippet: FACS-sorted cells were used for 4C template preparation. .. Cells were fixed and lysed as described ( ) using HindIII (Roche) as a first cutter and DpnII (New England Biolabs) as a second cutter.

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    New England Biolabs dpnii
    Scalable gene synthesis platform schematic for OLS Pool 2 Pre-designed oligonucleotides (no distinction is made between dsDNA and ssDNA in the figure) are synthesized on a DNA microchip ( a ) and then cleaved to make a pool of oligonucleotides (b) . Plate-specific primer sequences (yellow or brown) are used to amplify separate Plate Subpools (c) (only two are shown), which contain DNA to assemble different genes (only three are shown for each plate subpool). Assembly specific sequences (shades of blue) are used to amplify assembly subpools (d) that contain only the DNA required to make a single gene. The primer sequences are cleaved (e) using either Type IIS restriction enzymes (resulting in dsDNA) or by <t>DpnII/USER/γ</t> exonuclease processing (producing ssDNA). Construction primers (shown as white and black sites flanking the full assembly) are then used in an assembly PCR reaction to build a gene from each assembly subpool (f) . Depending on the downstream application the assembled products are then cloned either before or after an enzymatic error correction step.
    Dpnii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Scalable gene synthesis platform schematic for OLS Pool 2 Pre-designed oligonucleotides (no distinction is made between dsDNA and ssDNA in the figure) are synthesized on a DNA microchip ( a ) and then cleaved to make a pool of oligonucleotides (b) . Plate-specific primer sequences (yellow or brown) are used to amplify separate Plate Subpools (c) (only two are shown), which contain DNA to assemble different genes (only three are shown for each plate subpool). Assembly specific sequences (shades of blue) are used to amplify assembly subpools (d) that contain only the DNA required to make a single gene. The primer sequences are cleaved (e) using either Type IIS restriction enzymes (resulting in dsDNA) or by DpnII/USER/γ exonuclease processing (producing ssDNA). Construction primers (shown as white and black sites flanking the full assembly) are then used in an assembly PCR reaction to build a gene from each assembly subpool (f) . Depending on the downstream application the assembled products are then cloned either before or after an enzymatic error correction step.

    Journal: Nature biotechnology

    Article Title: A Scalable Gene Synthesis Platform Using High-Fidelity DNA Microchips

    doi: 10.1038/nbt.1716

    Figure Lengend Snippet: Scalable gene synthesis platform schematic for OLS Pool 2 Pre-designed oligonucleotides (no distinction is made between dsDNA and ssDNA in the figure) are synthesized on a DNA microchip ( a ) and then cleaved to make a pool of oligonucleotides (b) . Plate-specific primer sequences (yellow or brown) are used to amplify separate Plate Subpools (c) (only two are shown), which contain DNA to assemble different genes (only three are shown for each plate subpool). Assembly specific sequences (shades of blue) are used to amplify assembly subpools (d) that contain only the DNA required to make a single gene. The primer sequences are cleaved (e) using either Type IIS restriction enzymes (resulting in dsDNA) or by DpnII/USER/γ exonuclease processing (producing ssDNA). Construction primers (shown as white and black sites flanking the full assembly) are then used in an assembly PCR reaction to build a gene from each assembly subpool (f) . Depending on the downstream application the assembled products are then cloned either before or after an enzymatic error correction step.

    Article Snippet: PCR amplification was followed by γ exonuclease digestion of 5′ phosphorylated strands, hybridization of the 3′ primer site to its complement, and cleavage of the 5′ and 3′ primer sites using USER enzyme mix and DpnII (New England Biolabs), respectively.

    Techniques: Synthesized, MicroChIP Assay, Polymerase Cycling Assembly, Clone Assay

    Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.

    Journal: Nature Communications

    Article Title: Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex

    doi: 10.1038/ncomms12743

    Figure Lengend Snippet: Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.

    Article Snippet: Non-DpnI-restricted DNA was cut with DpnII (New England Biolabs) for 1 h at 37 °C.

    Techniques: Sequencing, Amplification, Real-time Polymerase Chain Reaction, Methylation, Polymerase Chain Reaction, Ligation, Injection, Chromatin Immunoprecipitation, Binding Assay, Mouse Assay

    Isolation of methyladenine genomic fragments using methyl-sensitive restriction enzymes, followed by PCR amplification. DNA fragments containing methylated adenines were isolated from samples of total genomic DNA of A. pisum using the methylation-site-specific restriction enzyme DpnI , adaptor ligation and the methyl-sensitive restriction enzyme DpnII . This was followed by PCR amplification as described by Steensel and Henikoff [ 59 ]. The resulting PCR products were cloned and sequenced.

    Journal: BMC Genomics

    Article Title: Adenine methylation may contribute to endosymbiont selection in a clonal aphid population

    doi: 10.1186/1471-2164-15-999

    Figure Lengend Snippet: Isolation of methyladenine genomic fragments using methyl-sensitive restriction enzymes, followed by PCR amplification. DNA fragments containing methylated adenines were isolated from samples of total genomic DNA of A. pisum using the methylation-site-specific restriction enzyme DpnI , adaptor ligation and the methyl-sensitive restriction enzyme DpnII . This was followed by PCR amplification as described by Steensel and Henikoff [ 59 ]. The resulting PCR products were cloned and sequenced.

    Article Snippet: DNA fragments containing methylated adenines were isolated from the genomic DNA using the methylation-specific restriction enzyme DpnI together with the methyl-sensitive restriction enzyme DpnII (New England Biolabs, Ipswich, MA, USA).

    Techniques: Isolation, Polymerase Chain Reaction, Amplification, Methylation, Ligation, Clone Assay