dpnii  (New England Biolabs)


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    Name:
    DpnII
    Description:
    DpnII 5 000 units
    Catalog Number:
    r0543l
    Price:
    285
    Size:
    5 000 units
    Category:
    Restriction Enzymes
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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 90 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    dpnii - by Bioz Stars, 2020-01
    90/100 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 "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

    4) Product Images from "Lack of MTTP protein in pluripotent stem cell-derived hepatocytes/cardiomyocytes abolishes apoB secretion and increases cell stress"

    Article Title: Lack of MTTP protein in pluripotent stem cell-derived hepatocytes/cardiomyocytes abolishes apoB secretion and increases cell stress

    Journal: Cell reports

    doi: 10.1016/j.celrep.2017.04.064

    Correction of C136G in MTTP rescues the ABL phenotype (A) Schematic strategy for correction of C136G in MTTP by CRISPR/Cas9. (B) iPSC from ABL patient was transfected with plasmids containing guide RNA and Cas9. Genomic DNA was extracted from GFP + colonies and subjected to PCR amplification. Subsequent DpnII digestion was applied to identify the positively targeted clones. (C) The corrected iPSC lines were tested for expression of pluripotency markers by real-time PCR. (D) Expression of hepatic genes was analyzed by real-time PCR in hepatocytes derived from the corrected iPSC lines. (E–G) Amount of newly synthesized apoB in the cell or secreted in the medium was measured at the end of a 2-hour label with [ 35 S]methionine/cysteine. F is a Western blot for apoB in the media. (H) Cellular lipid accumulation by Oil Red O staining following rescue of the C136G MTTP mutation by CRISPR/Cas9. Scale bar: 400 μm ± S.D. *P
    Figure Legend Snippet: Correction of C136G in MTTP rescues the ABL phenotype (A) Schematic strategy for correction of C136G in MTTP by CRISPR/Cas9. (B) iPSC from ABL patient was transfected with plasmids containing guide RNA and Cas9. Genomic DNA was extracted from GFP + colonies and subjected to PCR amplification. Subsequent DpnII digestion was applied to identify the positively targeted clones. (C) The corrected iPSC lines were tested for expression of pluripotency markers by real-time PCR. (D) Expression of hepatic genes was analyzed by real-time PCR in hepatocytes derived from the corrected iPSC lines. (E–G) Amount of newly synthesized apoB in the cell or secreted in the medium was measured at the end of a 2-hour label with [ 35 S]methionine/cysteine. F is a Western blot for apoB in the media. (H) Cellular lipid accumulation by Oil Red O staining following rescue of the C136G MTTP mutation by CRISPR/Cas9. Scale bar: 400 μm ± S.D. *P

    Techniques Used: CRISPR, Transfection, Polymerase Chain Reaction, Amplification, Expressing, Real-time Polymerase Chain Reaction, Derivative Assay, Synthesized, Western Blot, Staining, Mutagenesis

    Related Articles

    Clone Assay:

    Article Title: Genomic profiling distinguishes familial multiple and sporadic multiple meningiomas
    Article Snippet: These arrays contain 14,160 cDNA clones (Agilent Technologies, Palo Alto, CA) with a median interval between mapped elements of 100 kb, with 92.8% of intervals < 1 Mb and 98.6% < 3 Mb [ ]. .. Briefly, one μg of tumor or normal genomic DNA (male or female) was digested with Dpn II (New England Biolabs, Beverly, MA, USA), purified with DNA Clean & Concentrator (Zymo Research, Orange, CA, USA) and labeled with Cy3-dCTP or Cy5-dCTP (Amersham Bioscience, UK) using the Bioprime DNA Labeling System (Invitrogen Life Technologies, Carlsbad, CA, USA).

    Amplification:

    Article Title: Differentiation of Porcine Brachyspira Species by a Novel nox PCR-Based Restriction Fragment Length Polymorphism Analysis
    Article Snippet: After initial denaturation at 94°C for 3 min, each cycle involved denaturation at 94°C for 30 s, annealing at 59°C for 40 s, and extension at 72°C for 54 s. The amplification was finished after a final extension step at 72°C for 10 min. An aliquot of 10 μl of the PCR product was visualized after separation by electrophoresis in a 1.5% agarose gel and staining with ethidium bromide. .. The buffer composition of the remaining PCR product was modified to meet the conditions for restriction digestion with Dpn II (New England Biolabs, Frankfurt, Germany) and Bfm I (MBI Fermentas, St. Leon-Rot, Germany).

    Article Title: The T-Cell Oncogenic Protein HOX11 Activates Aldh1 Expression in NIH 3T3 Cells but Represses Its Expression in Mouse Spleen Development
    Article Snippet: Double-stranded cDNA (approximately 2 μg) was digested with Dpn II (NEB), extracted with phenol-chloroform, and ethanol precipitated. .. The ligations were diluted to 200 μl with TE and amplified by PCR with the RBgl24 oligonucleotide as the primer and 5 U of Amplitaq (Perkin-Elmer Cetus) in a 200-μl volume.

    Article Title: RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed genes
    Article Snippet: The cDNAs then were digested with Eco RII (Sigma) or Dpn II (New England Biolabs) at 37°C for 3 h followed by phenol/chloroform extraction and ethanol precipitation. .. The mixtures were diluted to 100 μl with 10 mM Tris/1 mM EDTA, pH 7.0, and at least 40 μl of the mixtures was used for PCR amplification.

    Article Title: Targeted DamID reveals differential binding of mammalian pluripotency factors
    Article Snippet: The ligated DNA was then digested with Dpn II to cleave any unmethylated GATC sites (New England Biolabs) and purified with a 1:1 ratio of Seramag beads. .. Adaptor-ligated fragments were then amplified with MyTaq (Bioline) and PCR purified.

    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 [W] [W] [OPEN]
    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: Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans
    Article Snippet: .. After inactivation of the ligase (65°C, 10 minutes), DNA fragments were digested with 5 units Dpn II (New England Biolabs) in a final volume of 80 μl to cut non-methylated GATC sites, thereby preventing PCR amplification of non-methylated gDNA. .. Methylated DNA was amplified using adaptor-specific primers.

    Article Title: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: .. To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer. .. Next, amplification was performed by using 0.5 μg of Dpn II-cut DNA, 1 μl of Advantage cDNA PCR polymerase mix (CLONTECH), 10 nmol of each dATP, dCTP, dGTP, and dTTP, and 62.5 pmol of primer (5′-GGTCGCGGCCGAGGATC-3′) in 50 μl total volume of Advantage PCR buffer, under the following cycling conditions: activation of the polymerase and nick translation for 10 min at 68°C, followed by one cycle of 1 min at 94°C, 5 min at 65°C and 15 min at 68°C; 3 cycles of 1 min at 94°C, 1 min at 65°C and 10 min at 68°C; and 14 cycles of 1 min at 94°C, 1 min at 65°C and 2 min at 68°C.

    Article Title: 15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain
    Article Snippet: Purified DNA was digested using Dpn II (NEB), re-ligated with T4 ligase (NEB), re-precipitated and then re-digested with Mnl 1 (NEB) overnight at 37°C. .. 4C libraries were created by linear amplification of DNA fragments with Expand Long Template PCR System (Roche Applied Sciences, Indianapolis, IN, USA).

    Blocking Assay:

    Article Title: Genomic profiling distinguishes familial multiple and sporadic multiple meningiomas
    Article Snippet: Briefly, one μg of tumor or normal genomic DNA (male or female) was digested with Dpn II (New England Biolabs, Beverly, MA, USA), purified with DNA Clean & Concentrator (Zymo Research, Orange, CA, USA) and labeled with Cy3-dCTP or Cy5-dCTP (Amersham Bioscience, UK) using the Bioprime DNA Labeling System (Invitrogen Life Technologies, Carlsbad, CA, USA). .. Labeled DNA was precipitated using isopropanol together with Cot-1 DNA (Invitrogen Life Technologies, Carlsbad, CA, USA), which was used to block repetitive sequences.

    Electrophoresis:

    Article Title: Differentiation of Porcine Brachyspira Species by a Novel nox PCR-Based Restriction Fragment Length Polymorphism Analysis
    Article Snippet: After initial denaturation at 94°C for 3 min, each cycle involved denaturation at 94°C for 30 s, annealing at 59°C for 40 s, and extension at 72°C for 54 s. The amplification was finished after a final extension step at 72°C for 10 min. An aliquot of 10 μl of the PCR product was visualized after separation by electrophoresis in a 1.5% agarose gel and staining with ethidium bromide. .. The buffer composition of the remaining PCR product was modified to meet the conditions for restriction digestion with Dpn II (New England Biolabs, Frankfurt, Germany) and Bfm I (MBI Fermentas, St. Leon-Rot, Germany).

    Microarray:

    Article Title: 15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain
    Article Snippet: Purified DNA was digested using Dpn II (NEB), re-ligated with T4 ligase (NEB), re-precipitated and then re-digested with Mnl 1 (NEB) overnight at 37°C. .. A custom DNA microarray tiling 26.3 Mb of NCBI34/hg17 human genome sequence including 13.3 Mb of 15q11.2–13.3, 2.6 Mb of 11p15.5, 3.8 Mb of 7q21.3, 2.9 Mb of 19p13.2, 0.3 Mb of 20q11.21, 1.5 Mb of 2p25.1, 0.4 Mb of 1p36.12, 2.9 Mb of 6p22.3 and 0.2 Mb of 11p14.1 with repetitive sequences removed was designed and produced by Roche-NimbleGen, Madison, WI, USA ( ).

    Incubation:

    Article Title: Transcriptional repression and DNA looping associated with a novel regulatory element in the final exon of the lymphotoxin-? gene
    Article Snippet: .. Briefly, 1 × 107 Jurkat T cells or HeLa cells were collected for a given condition, crosslinked for 10 min using 2% formaldehyde and lysed using ice-cold lysis buffer (10 m Tris pH 8, 10 m NaCl, 0.2% NP-40, 1 × complete protease inhibitor (Roche, Burgess Hill, UK)) and nuclei resuspended in restriction enzyme buffer, incubated at 37 °C with SDS (0.3% final) for 1 h then Triton-X 100 (2% final) for 1 h before restriction enzyme digestion with 400 U Dpn II (NEB, Hitchin, Hertfordshire, UK) overnight at 37 °C. .. Samples were then incubated with SDS (1.3% final) for 20 min at 65 °C, placed in ligation buffer with Triton-X 100 (Roche) (1% final) for 1 h at 37 °C before ligation using high-concentration T4 ligase (Fermentas UK, York, UK) at 16 °C for 4 h followed by 30 min at room temperature.

    Article Title: Promoter RNA links transcriptional regulation of inflammatory pathway genes
    Article Snippet: Three million nuclei were resuspended in 500 µl of 1.2×restriction enzyme buffer containing 0.1% (w/v) SDS and incubated at 65°C for 8 min to loosen chromatin. .. Samples were treated with 250 U of Dpn II (NEB) at 37°C overnight.

    Article Title: Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development
    Article Snippet: Capture C sample preparation 3C libraries were prepared from sorted populations from the thymus and spleen/lymph nodes as described previously with the following changes: Dpn II enzymes were used for restriction digest, with a total of 500 U fresh restriction enzyme added every 5–7 h, for a total incubation of 16–24 h after initial addition of restriction enzyme. .. After digest, Dpn II was heat inactivated and a ligation reaction with 200 U of T4 DNA ligase (NEB) was set up overnight at room temperature with gentle rotation.

    Article Title: Hepatitis B virus genotypes and hepatocellular carcinoma in Thailand
    Article Snippet: PCR products were subjected to RFLP analysis, using restriction endonuclease Ava II and Dpn II (New England Biolabs, Beverly, MA) to determine the HBV genotype. .. Briefly, 10 μL of PCR product was mixed with 1.5 μL of 10×buffer, 3 μL of sterile water and 0.5 μL (5U) of Ava II and Dpn II, respectively, in separate reactions and incubated at 37 °C for 3.5 h. After incubation, the samples were run on a composite gel containing 2% NuSieve agarose (FMC BioProducts, Rockland, ME) and 1% standard agarose.

    Article Title: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: The solution was mixed by inversion and the DNA precipitate was recovered, rinsed in 70% ethanol, and dissolved in 50 μl of T10 E10 by incubation at 37°C for several hours. .. To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer.

    Modification:

    Article Title: Differentiation of Porcine Brachyspira Species by a Novel nox PCR-Based Restriction Fragment Length Polymorphism Analysis
    Article Snippet: .. The buffer composition of the remaining PCR product was modified to meet the conditions for restriction digestion with Dpn II (New England Biolabs, Frankfurt, Germany) and Bfm I (MBI Fermentas, St. Leon-Rot, Germany). .. We prepared 10× PCR restriction buffer (230 mM Tris acetate [pH 7.6], 41 mM potassium acetate, 9.25 mM magnesium acetate, 30 mM spermidine [Sigma, Taufkirchen, Germany], 1 mg of bovine serum albumin/ml).

    Article Title: Targeted DamID reveals differential binding of mammalian pluripotency factors
    Article Snippet: The ligated DNA was then digested with Dpn II to cleave any unmethylated GATC sites (New England Biolabs) and purified with a 1:1 ratio of Seramag beads. .. The fragments were then prepared for Illumina sequencing according to the modified TruSeq protocol described by Marshall et al. (2017).

    Transformation Assay:

    Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
    Article Snippet: .. Approximately 1.5 × 108 cells were transformed with 10 μg of pmBsr linearized with Bam HI, along with 4 U of Dpn II (New England Biolabs, Beverly, MA) by electroporation. .. The resultant transformants were cultured on plastic dishes in HL-5 medium containing 10 μg/ml blasticidin S. After 5-6 d, the colonies formed by the transformed cells were observed under a microscope; those showing large numbers of multinucleated cells were isolated from the dish, recloned on bacterial lawns, and used for further analyses.

    Hybridization:

    Article Title: Genomic profiling distinguishes familial multiple and sporadic multiple meningiomas
    Article Snippet: Briefly, one μg of tumor or normal genomic DNA (male or female) was digested with Dpn II (New England Biolabs, Beverly, MA, USA), purified with DNA Clean & Concentrator (Zymo Research, Orange, CA, USA) and labeled with Cy3-dCTP or Cy5-dCTP (Amersham Bioscience, UK) using the Bioprime DNA Labeling System (Invitrogen Life Technologies, Carlsbad, CA, USA). .. The probe pellet was then washed with 70% ethanol, dried and dissolved in hybridization buffer consisting of 50% formamide, 2 × SSC, 10% dextran sulphate, 2% SDS and 10 μg/μl yeast tRNA.

    Electroporation:

    Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
    Article Snippet: .. Approximately 1.5 × 108 cells were transformed with 10 μg of pmBsr linearized with Bam HI, along with 4 U of Dpn II (New England Biolabs, Beverly, MA) by electroporation. .. The resultant transformants were cultured on plastic dishes in HL-5 medium containing 10 μg/ml blasticidin S. After 5-6 d, the colonies formed by the transformed cells were observed under a microscope; those showing large numbers of multinucleated cells were isolated from the dish, recloned on bacterial lawns, and used for further analyses.

    Transfection:

    Article Title: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer. .. Finally, labeled experimental (Dam–protein fusion) and reference (Dam) DNA samples were mixed and hybridized to microarrays in 3× SSC (450 mM sodium chloride/45 mM sodium citrate, pH 7.0) supplemented with 0.22% SDS, 20 μg of poly(dA–dT), 100 μg of yeast tRNA, and 25 μg of unlabeled Dpn I-digested plasmid encoding the fusion protein used for transfection.

    Ligation:

    Article Title: The T-Cell Oncogenic Protein HOX11 Activates Aldh1 Expression in NIH 3T3 Cells but Represses Its Expression in Mouse Spleen Development
    Article Snippet: Double-stranded cDNA (approximately 2 μg) was digested with Dpn II (NEB), extracted with phenol-chloroform, and ethanol precipitated. .. Prior to addition of DNA ligase, the partially complementary RBgl oligonucleotides were annealed by heating the ligation mixture to 50°C for 1 min and slowly cooling it to 10°C.

    Article Title: Transcriptional repression and DNA looping associated with a novel regulatory element in the final exon of the lymphotoxin-? gene
    Article Snippet: Briefly, 1 × 107 Jurkat T cells or HeLa cells were collected for a given condition, crosslinked for 10 min using 2% formaldehyde and lysed using ice-cold lysis buffer (10 m Tris pH 8, 10 m NaCl, 0.2% NP-40, 1 × complete protease inhibitor (Roche, Burgess Hill, UK)) and nuclei resuspended in restriction enzyme buffer, incubated at 37 °C with SDS (0.3% final) for 1 h then Triton-X 100 (2% final) for 1 h before restriction enzyme digestion with 400 U Dpn II (NEB, Hitchin, Hertfordshire, UK) overnight at 37 °C. .. Samples were then incubated with SDS (1.3% final) for 20 min at 65 °C, placed in ligation buffer with Triton-X 100 (Roche) (1% final) for 1 h at 37 °C before ligation using high-concentration T4 ligase (Fermentas UK, York, UK) at 16 °C for 4 h followed by 30 min at room temperature.

    Article Title: Promoter RNA links transcriptional regulation of inflammatory pathway genes
    Article Snippet: Samples were treated with 250 U of Dpn II (NEB) at 37°C overnight. .. For ligation, samples were incubated with 4000 cohesive end units of T4 DNA ligase (NEB) at 16°C for 5 h. Samples were then treated with 200 mM NaCl and 300 µg of proteinase K at 65°C overnight for reverse cross-linking.

    Article Title: Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development
    Article Snippet: .. After digest, Dpn II was heat inactivated and a ligation reaction with 200 U of T4 DNA ligase (NEB) was set up overnight at room temperature with gentle rotation. .. Two aliquots of 5 µg of 3C library was then sonicated to 200 bp using a Covaris LE220 instrument (190 s shearing time; 30% duty factor; 450PIP, 200 cycles per burst).

    Article Title: RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed genes
    Article Snippet: The cDNAs then were digested with Eco RII (Sigma) or Dpn II (New England Biolabs) at 37°C for 3 h followed by phenol/chloroform extraction and ethanol precipitation. .. The digested cDNAs were mixed with primers XE-14/XEA-13/XET-13 (final concentration, 20 μM) or XDPN-14/XDPN-12 (final concentration, 20 μM) in 30 μl of 1× ligation buffer (GIBCO/BRL), heated at 55°C for 1 min, and cooled down to 14°C within 1 h. After adding 3 μl of T4 ligase (5 units/μl) to the mixtures individually, ligation was carried out at 14°C overnight.

    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 [W] [W] [OPEN]
    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: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: After inactivation of Dpn I at 80°C for 20 min, 4 μg of the Dpn I-digested genomic DNA was ligated to 40 pmol of a double-stranded unphosphorylated adaptor (top strand: 5′-CTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGA-3′, bottom strand: 5′-TCCTCGGCCG-3′) for 2 h at 16°C with 5 units of T4-Ligase (Roche Molecular Biochemicals) in a total volume of 20 μl of ligation buffer. .. To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer.

    Article Title: 15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain
    Article Snippet: Cell nuclei were resuspended in restriction buffer and digested overnight with Bgl II (NEB, Ipswich, MA, USA) at 37°C followed by ligation using T4 ligase (Promega, Madison, WI, USA). .. Purified DNA was digested using Dpn II (NEB), re-ligated with T4 ligase (NEB), re-precipitated and then re-digested with Mnl 1 (NEB) overnight at 37°C.

    Protease Inhibitor:

    Article Title: Transcriptional repression and DNA looping associated with a novel regulatory element in the final exon of the lymphotoxin-? gene
    Article Snippet: .. Briefly, 1 × 107 Jurkat T cells or HeLa cells were collected for a given condition, crosslinked for 10 min using 2% formaldehyde and lysed using ice-cold lysis buffer (10 m Tris pH 8, 10 m NaCl, 0.2% NP-40, 1 × complete protease inhibitor (Roche, Burgess Hill, UK)) and nuclei resuspended in restriction enzyme buffer, incubated at 37 °C with SDS (0.3% final) for 1 h then Triton-X 100 (2% final) for 1 h before restriction enzyme digestion with 400 U Dpn II (NEB, Hitchin, Hertfordshire, UK) overnight at 37 °C. .. Samples were then incubated with SDS (1.3% final) for 20 min at 65 °C, placed in ligation buffer with Triton-X 100 (Roche) (1% final) for 1 h at 37 °C before ligation using high-concentration T4 ligase (Fermentas UK, York, UK) at 16 °C for 4 h followed by 30 min at room temperature.

    Nick Translation:

    Article Title: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer. .. Next, amplification was performed by using 0.5 μg of Dpn II-cut DNA, 1 μl of Advantage cDNA PCR polymerase mix (CLONTECH), 10 nmol of each dATP, dCTP, dGTP, and dTTP, and 62.5 pmol of primer (5′-GGTCGCGGCCGAGGATC-3′) in 50 μl total volume of Advantage PCR buffer, under the following cycling conditions: activation of the polymerase and nick translation for 10 min at 68°C, followed by one cycle of 1 min at 94°C, 5 min at 65°C and 15 min at 68°C; 3 cycles of 1 min at 94°C, 1 min at 65°C and 10 min at 68°C; and 14 cycles of 1 min at 94°C, 1 min at 65°C and 2 min at 68°C.

    Cell Culture:

    Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
    Article Snippet: Approximately 1.5 × 108 cells were transformed with 10 μg of pmBsr linearized with Bam HI, along with 4 U of Dpn II (New England Biolabs, Beverly, MA) by electroporation. .. The resultant transformants were cultured on plastic dishes in HL-5 medium containing 10 μg/ml blasticidin S. After 5-6 d, the colonies formed by the transformed cells were observed under a microscope; those showing large numbers of multinucleated cells were isolated from the dish, recloned on bacterial lawns, and used for further analyses.

    DNA Labeling:

    Article Title: Genomic profiling distinguishes familial multiple and sporadic multiple meningiomas
    Article Snippet: .. Briefly, one μg of tumor or normal genomic DNA (male or female) was digested with Dpn II (New England Biolabs, Beverly, MA, USA), purified with DNA Clean & Concentrator (Zymo Research, Orange, CA, USA) and labeled with Cy3-dCTP or Cy5-dCTP (Amersham Bioscience, UK) using the Bioprime DNA Labeling System (Invitrogen Life Technologies, Carlsbad, CA, USA). .. Labeled DNA was precipitated using isopropanol together with Cot-1 DNA (Invitrogen Life Technologies, Carlsbad, CA, USA), which was used to block repetitive sequences.

    Polymerase Chain Reaction:

    Article Title: Differentiation of Porcine Brachyspira Species by a Novel nox PCR-Based Restriction Fragment Length Polymorphism Analysis
    Article Snippet: .. The buffer composition of the remaining PCR product was modified to meet the conditions for restriction digestion with Dpn II (New England Biolabs, Frankfurt, Germany) and Bfm I (MBI Fermentas, St. Leon-Rot, Germany). .. We prepared 10× PCR restriction buffer (230 mM Tris acetate [pH 7.6], 41 mM potassium acetate, 9.25 mM magnesium acetate, 30 mM spermidine [Sigma, Taufkirchen, Germany], 1 mg of bovine serum albumin/ml).

    Article Title: The T-Cell Oncogenic Protein HOX11 Activates Aldh1 Expression in NIH 3T3 Cells but Represses Its Expression in Mouse Spleen Development
    Article Snippet: Double-stranded cDNA (approximately 2 μg) was digested with Dpn II (NEB), extracted with phenol-chloroform, and ethanol precipitated. .. The ligations were diluted to 200 μl with TE and amplified by PCR with the RBgl24 oligonucleotide as the primer and 5 U of Amplitaq (Perkin-Elmer Cetus) in a 200-μl volume.

    Article Title: Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development
    Article Snippet: After digest, Dpn II was heat inactivated and a ligation reaction with 200 U of T4 DNA ligase (NEB) was set up overnight at room temperature with gentle rotation. .. We indexed libraries with Illumina Truseq indexed sequencing adaptors using NEBnext reagents (E6000, E6040, E7335, or E7500) for end repair, dA labeling, adaptor ligation, and PCR indexing, primarily following the manufacturer’s instructions.

    Article Title: RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed genes
    Article Snippet: Paragraph title: Preparation of PCR-Based cDNA Libraries. ... The cDNAs then were digested with Eco RII (Sigma) or Dpn II (New England Biolabs) at 37°C for 3 h followed by phenol/chloroform extraction and ethanol precipitation.

    Article Title: Targeted DamID reveals differential binding of mammalian pluripotency factors
    Article Snippet: The DNA was then digested overnight at 37°C with Dpn I to cut methylated GATC sites (New England Biolabs) and purified with a QIAquick PCR Purification Kit. .. The ligated DNA was then digested with Dpn II to cleave any unmethylated GATC sites (New England Biolabs) and purified with a 1:1 ratio of Seramag beads.

    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 [W] [W] [OPEN]
    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: MDR-1 gene polymorphisms G2677T and C3435T in a case of Hodgkin's variant of Richter's syndrome
    Article Snippet: The PCR products were evaluated on a 1.5% agarose gel, photographed using Polaroid film and subjected to RFLP analysis. .. To distinguish the single nucleotide polymorphisms (SNPs), the restriction enzyme Ban I (New England BioLabs, Ipswich, MA, USA) was used for G2677T and Dpn II (New England BioLabs) was used for C3435T.

    Article Title: Hepatitis B virus genotypes and hepatocellular carcinoma in Thailand
    Article Snippet: .. PCR products were subjected to RFLP analysis, using restriction endonuclease Ava II and Dpn II (New England Biolabs, Beverly, MA) to determine the HBV genotype. .. Briefly, 10 μL of PCR product was mixed with 1.5 μL of 10×buffer, 3 μL of sterile water and 0.5 μL (5U) of Ava II and Dpn II, respectively, in separate reactions and incubated at 37 °C for 3.5 h. After incubation, the samples were run on a composite gel containing 2% NuSieve agarose (FMC BioProducts, Rockland, ME) and 1% standard agarose.

    Article Title: Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans
    Article Snippet: .. After inactivation of the ligase (65°C, 10 minutes), DNA fragments were digested with 5 units Dpn II (New England Biolabs) in a final volume of 80 μl to cut non-methylated GATC sites, thereby preventing PCR amplification of non-methylated gDNA. .. Methylated DNA was amplified using adaptor-specific primers.

    Article Title: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: For selective PCR amplication of methylated DNA fragments, 40 μg of the isolated genomic DNA was digested for 16 h at 37°C with 40 units of Dpn I (New England Biolabs) in the presence of 12.5 ng of DNase-free RNase A (Roche Molecular Biochemicals) in a total volume of 50 μl of buffer 4 (New England Biolabs). .. To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer.

    Article Title: 15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain
    Article Snippet: Purified DNA was digested using Dpn II (NEB), re-ligated with T4 ligase (NEB), re-precipitated and then re-digested with Mnl 1 (NEB) overnight at 37°C. .. 4C libraries were created by linear amplification of DNA fragments with Expand Long Template PCR System (Roche Applied Sciences, Indianapolis, IN, USA).

    Sonication:

    Article Title: Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development
    Article Snippet: After digest, Dpn II was heat inactivated and a ligation reaction with 200 U of T4 DNA ligase (NEB) was set up overnight at room temperature with gentle rotation. .. Two aliquots of 5 µg of 3C library was then sonicated to 200 bp using a Covaris LE220 instrument (190 s shearing time; 30% duty factor; 450PIP, 200 cycles per burst).

    Antiviral Assay:

    Article Title: Hepatitis B virus genotypes and hepatocellular carcinoma in Thailand
    Article Snippet: .. PCR products were subjected to RFLP analysis, using restriction endonuclease Ava II and Dpn II (New England Biolabs, Beverly, MA) to determine the HBV genotype. .. Briefly, 10 μL of PCR product was mixed with 1.5 μL of 10×buffer, 3 μL of sterile water and 0.5 μL (5U) of Ava II and Dpn II, respectively, in separate reactions and incubated at 37 °C for 3.5 h. After incubation, the samples were run on a composite gel containing 2% NuSieve agarose (FMC BioProducts, Rockland, ME) and 1% standard agarose.

    Cellular Antioxidant Activity Assay:

    Article Title: MDR-1 gene polymorphisms G2677T and C3435T in a case of Hodgkin's variant of Richter's syndrome
    Article Snippet: Those for the C3435T polymorphism in exon 26 were: 5′-GCT GCT TGA TGG CAA AGA AA-3′ and 5′-ATT AGG CAG TGA CTC GAT GAT GA-3′, producing a 208-bp fragment. .. To distinguish the single nucleotide polymorphisms (SNPs), the restriction enzyme Ban I (New England BioLabs, Ipswich, MA, USA) was used for G2677T and Dpn II (New England BioLabs) was used for C3435T.

    Nucleic Acid Electrophoresis:

    Article Title: 15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain
    Article Snippet: Purified DNA was digested using Dpn II (NEB), re-ligated with T4 ligase (NEB), re-precipitated and then re-digested with Mnl 1 (NEB) overnight at 37°C. .. Amplified 4C library fragments were evaluated by gel electrophoresis and column-purified using a PCR clean-up kit (Qiagen, Valencia, CA, USA).

    Methylation:

    Article Title: Targeted DamID reveals differential binding of mammalian pluripotency factors
    Article Snippet: The DNA was then digested overnight at 37°C with Dpn I to cut methylated GATC sites (New England Biolabs) and purified with a QIAquick PCR Purification Kit. .. The ligated DNA was then digested with Dpn II to cleave any unmethylated GATC sites (New England Biolabs) and purified with a 1:1 ratio of Seramag beads.

    Article Title: Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans
    Article Snippet: Methylated genomic DNA (gDNA) was purified and amplified from 30 mg nematodes using a protocol based on [ ] with some modifications. .. After inactivation of the ligase (65°C, 10 minutes), DNA fragments were digested with 5 units Dpn II (New England Biolabs) in a final volume of 80 μl to cut non-methylated GATC sites, thereby preventing PCR amplification of non-methylated gDNA.

    Article Title: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: For selective PCR amplication of methylated DNA fragments, 40 μg of the isolated genomic DNA was digested for 16 h at 37°C with 40 units of Dpn I (New England Biolabs) in the presence of 12.5 ng of DNase-free RNase A (Roche Molecular Biochemicals) in a total volume of 50 μl of buffer 4 (New England Biolabs). .. To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer.

    Mutagenesis:

    Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
    Article Snippet: Paragraph title: Restriction Enzyme-mediated Insertional (REMI) Mutagenesis ... Approximately 1.5 × 108 cells were transformed with 10 μg of pmBsr linearized with Bam HI, along with 4 U of Dpn II (New England Biolabs, Beverly, MA) by electroporation.

    Isolation:

    Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
    Article Snippet: Approximately 1.5 × 108 cells were transformed with 10 μg of pmBsr linearized with Bam HI, along with 4 U of Dpn II (New England Biolabs, Beverly, MA) by electroporation. .. The resultant transformants were cultured on plastic dishes in HL-5 medium containing 10 μg/ml blasticidin S. After 5-6 d, the colonies formed by the transformed cells were observed under a microscope; those showing large numbers of multinucleated cells were isolated from the dish, recloned on bacterial lawns, and used for further analyses.

    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 [W] [W] [OPEN]
    Article Snippet: Isolated chromatin was digested using Dpn . .. 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: Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans
    Article Snippet: Briefly, 2.5 μg of gDNA isolated with DNAeasy kit (QIAGEN, Venlo, Limburg, Netherlands), was digested with 10 units Dpn I (New England Biolabs, Ipswich, MA, USA) overnight in 10 μl to cut at Gm ATC sites. .. After inactivation of the ligase (65°C, 10 minutes), DNA fragments were digested with 5 units Dpn II (New England Biolabs) in a final volume of 80 μl to cut non-methylated GATC sites, thereby preventing PCR amplification of non-methylated gDNA.

    Article Title: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: For selective PCR amplication of methylated DNA fragments, 40 μg of the isolated genomic DNA was digested for 16 h at 37°C with 40 units of Dpn I (New England Biolabs) in the presence of 12.5 ng of DNase-free RNase A (Roche Molecular Biochemicals) in a total volume of 50 μl of buffer 4 (New England Biolabs). .. To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer.

    Capture-C:

    Article Title: Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development
    Article Snippet: Paragraph title: Capture C sample preparation ... After digest, Dpn II was heat inactivated and a ligation reaction with 200 U of T4 DNA ligase (NEB) was set up overnight at room temperature with gentle rotation.

    Size-exclusion Chromatography:

    Article Title: MDR-1 gene polymorphisms G2677T and C3435T in a case of Hodgkin's variant of Richter's syndrome
    Article Snippet: The PCR program for exon 26 consisted of 35 cycles at 94°C for 30 sec, 54°C for 30 sec, 72°C for 30 sec, and a final elongation step at 72°C for 10 min. .. To distinguish the single nucleotide polymorphisms (SNPs), the restriction enzyme Ban I (New England BioLabs, Ipswich, MA, USA) was used for G2677T and Dpn II (New England BioLabs) was used for C3435T.

    Microscopy:

    Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
    Article Snippet: Approximately 1.5 × 108 cells were transformed with 10 μg of pmBsr linearized with Bam HI, along with 4 U of Dpn II (New England Biolabs, Beverly, MA) by electroporation. .. The resultant transformants were cultured on plastic dishes in HL-5 medium containing 10 μg/ml blasticidin S. After 5-6 d, the colonies formed by the transformed cells were observed under a microscope; those showing large numbers of multinucleated cells were isolated from the dish, recloned on bacterial lawns, and used for further analyses.

    Purification:

    Article Title: Promoter RNA links transcriptional regulation of inflammatory pathway genes
    Article Snippet: Samples were treated with 250 U of Dpn II (NEB) at 37°C overnight. .. After treating samples with 40 ng/µl RNase A (300 µg) at 37°C for 40 min, DNA fragments were purified by phenol-chloroform extraction and ethanol precipitation.

    Article Title: Genomic profiling distinguishes familial multiple and sporadic multiple meningiomas
    Article Snippet: .. Briefly, one μg of tumor or normal genomic DNA (male or female) was digested with Dpn II (New England Biolabs, Beverly, MA, USA), purified with DNA Clean & Concentrator (Zymo Research, Orange, CA, USA) and labeled with Cy3-dCTP or Cy5-dCTP (Amersham Bioscience, UK) using the Bioprime DNA Labeling System (Invitrogen Life Technologies, Carlsbad, CA, USA). .. Labeled DNA was precipitated using isopropanol together with Cot-1 DNA (Invitrogen Life Technologies, Carlsbad, CA, USA), which was used to block repetitive sequences.

    Article Title: Targeted DamID reveals differential binding of mammalian pluripotency factors
    Article Snippet: .. The ligated DNA was then digested with Dpn II to cleave any unmethylated GATC sites (New England Biolabs) and purified with a 1:1 ratio of Seramag beads. .. Adaptor-ligated fragments were then amplified with MyTaq (Bioline) and PCR purified.

    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 [W] [W] [OPEN]
    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. .. The ligated DNA was purified by phenol-chloroform extraction, precipitated, washed, and dissolved in water.

    Article Title: Genome-wide analysis links emerin to neuromuscular junction activity in Caenorhabditis elegans
    Article Snippet: Methylated genomic DNA (gDNA) was purified and amplified from 30 mg nematodes using a protocol based on [ ] with some modifications. .. After inactivation of the ligase (65°C, 10 minutes), DNA fragments were digested with 5 units Dpn II (New England Biolabs) in a final volume of 80 μl to cut non-methylated GATC sites, thereby preventing PCR amplification of non-methylated gDNA.

    Article Title: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer. .. The PCR products were purified by using the QIAquick PCR purification kit (Qiagen) and labeled with Cy3 or Cy5 as described ( ).

    Article Title: 15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain
    Article Snippet: .. Purified DNA was digested using Dpn II (NEB), re-ligated with T4 ligase (NEB), re-precipitated and then re-digested with Mnl 1 (NEB) overnight at 37°C. .. 4C libraries were created by linear amplification of DNA fragments with Expand Long Template PCR System (Roche Applied Sciences, Indianapolis, IN, USA).

    Sequencing:

    Article Title: Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development
    Article Snippet: After digest, Dpn II was heat inactivated and a ligation reaction with 200 U of T4 DNA ligase (NEB) was set up overnight at room temperature with gentle rotation. .. We indexed libraries with Illumina Truseq indexed sequencing adaptors using NEBnext reagents (E6000, E6040, E7335, or E7500) for end repair, dA labeling, adaptor ligation, and PCR indexing, primarily following the manufacturer’s instructions.

    Article Title: Targeted DamID reveals differential binding of mammalian pluripotency factors
    Article Snippet: The ligated DNA was then digested with Dpn II to cleave any unmethylated GATC sites (New England Biolabs) and purified with a 1:1 ratio of Seramag beads. .. The fragments were then prepared for Illumina sequencing according to the modified TruSeq protocol described by Marshall et al. (2017).

    Article Title: 15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain
    Article Snippet: Purified DNA was digested using Dpn II (NEB), re-ligated with T4 ligase (NEB), re-precipitated and then re-digested with Mnl 1 (NEB) overnight at 37°C. .. A custom DNA microarray tiling 26.3 Mb of NCBI34/hg17 human genome sequence including 13.3 Mb of 15q11.2–13.3, 2.6 Mb of 11p15.5, 3.8 Mb of 7q21.3, 2.9 Mb of 19p13.2, 0.3 Mb of 20q11.21, 1.5 Mb of 2p25.1, 0.4 Mb of 1p36.12, 2.9 Mb of 6p22.3 and 0.2 Mb of 11p14.1 with repetitive sequences removed was designed and produced by Roche-NimbleGen, Madison, WI, USA ( ).

    Labeling:

    Article Title: Genomic profiling distinguishes familial multiple and sporadic multiple meningiomas
    Article Snippet: .. Briefly, one μg of tumor or normal genomic DNA (male or female) was digested with Dpn II (New England Biolabs, Beverly, MA, USA), purified with DNA Clean & Concentrator (Zymo Research, Orange, CA, USA) and labeled with Cy3-dCTP or Cy5-dCTP (Amersham Bioscience, UK) using the Bioprime DNA Labeling System (Invitrogen Life Technologies, Carlsbad, CA, USA). .. Labeled DNA was precipitated using isopropanol together with Cot-1 DNA (Invitrogen Life Technologies, Carlsbad, CA, USA), which was used to block repetitive sequences.

    Article Title: Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development
    Article Snippet: After digest, Dpn II was heat inactivated and a ligation reaction with 200 U of T4 DNA ligase (NEB) was set up overnight at room temperature with gentle rotation. .. We indexed libraries with Illumina Truseq indexed sequencing adaptors using NEBnext reagents (E6000, E6040, E7335, or E7500) for end repair, dA labeling, adaptor ligation, and PCR indexing, primarily following the manufacturer’s instructions.

    Article Title: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer. .. The PCR products were purified by using the QIAquick PCR purification kit (Qiagen) and labeled with Cy3 or Cy5 as described ( ).

    Lysis:

    Article Title: Transcriptional repression and DNA looping associated with a novel regulatory element in the final exon of the lymphotoxin-? gene
    Article Snippet: .. Briefly, 1 × 107 Jurkat T cells or HeLa cells were collected for a given condition, crosslinked for 10 min using 2% formaldehyde and lysed using ice-cold lysis buffer (10 m Tris pH 8, 10 m NaCl, 0.2% NP-40, 1 × complete protease inhibitor (Roche, Burgess Hill, UK)) and nuclei resuspended in restriction enzyme buffer, incubated at 37 °C with SDS (0.3% final) for 1 h then Triton-X 100 (2% final) for 1 h before restriction enzyme digestion with 400 U Dpn II (NEB, Hitchin, Hertfordshire, UK) overnight at 37 °C. .. Samples were then incubated with SDS (1.3% final) for 20 min at 65 °C, placed in ligation buffer with Triton-X 100 (Roche) (1% final) for 1 h at 37 °C before ligation using high-concentration T4 ligase (Fermentas UK, York, UK) at 16 °C for 4 h followed by 30 min at room temperature.

    Article Title: 15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain
    Article Snippet: Briefly, SH-SY5Y neurons were treated with formaldehyde to crosslink chromatin, followed by quenching of the reaction with 1 m glycine and cell lysis by resuspension in hypotonic buffer and dounce homogenization. .. Purified DNA was digested using Dpn II (NEB), re-ligated with T4 ligase (NEB), re-precipitated and then re-digested with Mnl 1 (NEB) overnight at 37°C.

    Concentration Assay:

    Article Title: Differentiation of Porcine Brachyspira Species by a Novel nox PCR-Based Restriction Fragment Length Polymorphism Analysis
    Article Snippet: Ten microliters of lysate was added to 40 μl of a PCR premixture to yield final concentrations of 1.5 mM MgCl2 (Gibco BRL, Karlsruhe, Germany), PCR buffer (Gibco BRL), a 0.2 mM concentration of each deoxynucleoside triphosphate (Roth, Karlsruhe, Germany), 0.5 μM Bnoxf, 0.5 μM Bnoxr, and 2.5 U of Taq polymerase (Gibco BRL). .. The buffer composition of the remaining PCR product was modified to meet the conditions for restriction digestion with Dpn II (New England Biolabs, Frankfurt, Germany) and Bfm I (MBI Fermentas, St. Leon-Rot, Germany).

    Article Title: RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed genes
    Article Snippet: The cDNAs then were digested with Eco RII (Sigma) or Dpn II (New England Biolabs) at 37°C for 3 h followed by phenol/chloroform extraction and ethanol precipitation. .. The digested cDNAs were mixed with primers XE-14/XEA-13/XET-13 (final concentration, 20 μM) or XDPN-14/XDPN-12 (final concentration, 20 μM) in 30 μl of 1× ligation buffer (GIBCO/BRL), heated at 55°C for 1 min, and cooled down to 14°C within 1 h. After adding 3 μl of T4 ligase (5 units/μl) to the mixtures individually, ligation was carried out at 14°C overnight.

    Agarose Gel Electrophoresis:

    Article Title: Differentiation of Porcine Brachyspira Species by a Novel nox PCR-Based Restriction Fragment Length Polymorphism Analysis
    Article Snippet: After initial denaturation at 94°C for 3 min, each cycle involved denaturation at 94°C for 30 s, annealing at 59°C for 40 s, and extension at 72°C for 54 s. The amplification was finished after a final extension step at 72°C for 10 min. An aliquot of 10 μl of the PCR product was visualized after separation by electrophoresis in a 1.5% agarose gel and staining with ethidium bromide. .. The buffer composition of the remaining PCR product was modified to meet the conditions for restriction digestion with Dpn II (New England Biolabs, Frankfurt, Germany) and Bfm I (MBI Fermentas, St. Leon-Rot, Germany).

    Article Title: MDR-1 gene polymorphisms G2677T and C3435T in a case of Hodgkin's variant of Richter's syndrome
    Article Snippet: The PCR products were evaluated on a 1.5% agarose gel, photographed using Polaroid film and subjected to RFLP analysis. .. To distinguish the single nucleotide polymorphisms (SNPs), the restriction enzyme Ban I (New England BioLabs, Ipswich, MA, USA) was used for G2677T and Dpn II (New England BioLabs) was used for C3435T.

    Plasmid Preparation:

    Article Title: DWWA, a Novel Protein Containing Two WW Domains and an IQ Motif, Is Required for Scission of the Residual Cytoplasmic Bridge during Cytokinesis in Dictyostelium
    Article Snippet: Bsr-REMI mutagenesis was carried out as described previously ( ; ), except that we used pmBsr, a miniaturized tagging vector with improved mutagenesis efficiency ( ). .. Approximately 1.5 × 108 cells were transformed with 10 μg of pmBsr linearized with Bam HI, along with 4 U of Dpn II (New England Biolabs, Beverly, MA) by electroporation.

    Article Title: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer. .. Finally, labeled experimental (Dam–protein fusion) and reference (Dam) DNA samples were mixed and hybridized to microarrays in 3× SSC (450 mM sodium chloride/45 mM sodium citrate, pH 7.0) supplemented with 0.22% SDS, 20 μg of poly(dA–dT), 100 μg of yeast tRNA, and 25 μg of unlabeled Dpn I-digested plasmid encoding the fusion protein used for transfection.

    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 [W] [W] [OPEN]
    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.

    Sample Prep:

    Article Title: Stage-specific epigenetic regulation of CD4 expression by coordinated enhancer elements during T cell development
    Article Snippet: Paragraph title: Capture C sample preparation ... After digest, Dpn II was heat inactivated and a ligation reaction with 200 U of T4 DNA ligase (NEB) was set up overnight at room temperature with gentle rotation.

    Homogenization:

    Article Title: 15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain
    Article Snippet: Briefly, SH-SY5Y neurons were treated with formaldehyde to crosslink chromatin, followed by quenching of the reaction with 1 m glycine and cell lysis by resuspension in hypotonic buffer and dounce homogenization. .. Purified DNA was digested using Dpn II (NEB), re-ligated with T4 ligase (NEB), re-precipitated and then re-digested with Mnl 1 (NEB) overnight at 37°C.

    Ethanol Precipitation:

    Article Title: Promoter RNA links transcriptional regulation of inflammatory pathway genes
    Article Snippet: Samples were treated with 250 U of Dpn II (NEB) at 37°C overnight. .. After treating samples with 40 ng/µl RNase A (300 µg) at 37°C for 40 min, DNA fragments were purified by phenol-chloroform extraction and ethanol precipitation.

    Article Title: RaSH, a rapid subtraction hybridization approach for identifying and cloning differentially expressed genes
    Article Snippet: .. The cDNAs then were digested with Eco RII (Sigma) or Dpn II (New England Biolabs) at 37°C for 3 h followed by phenol/chloroform extraction and ethanol precipitation. .. The digested cDNAs were mixed with primers XE-14/XEA-13/XET-13 (final concentration, 20 μM) or XDPN-14/XDPN-12 (final concentration, 20 μM) in 30 μl of 1× ligation buffer (GIBCO/BRL), heated at 55°C for 1 min, and cooled down to 14°C within 1 h. After adding 3 μl of T4 ligase (5 units/μl) to the mixtures individually, ligation was carried out at 14°C overnight.

    Produced:

    Article Title: 15q11.2-13.3 chromatin analysis reveals epigenetic regulation of CHRNA7 with deficiencies in Rett and autism brain
    Article Snippet: Purified DNA was digested using Dpn II (NEB), re-ligated with T4 ligase (NEB), re-precipitated and then re-digested with Mnl 1 (NEB) overnight at 37°C. .. A custom DNA microarray tiling 26.3 Mb of NCBI34/hg17 human genome sequence including 13.3 Mb of 15q11.2–13.3, 2.6 Mb of 11p15.5, 3.8 Mb of 7q21.3, 2.9 Mb of 19p13.2, 0.3 Mb of 20q11.21, 1.5 Mb of 2p25.1, 0.4 Mb of 1p36.12, 2.9 Mb of 6p22.3 and 0.2 Mb of 11p14.1 with repetitive sequences removed was designed and produced by Roche-NimbleGen, Madison, WI, USA ( ).

    Activation Assay:

    Article Title: Protein-DNA interaction mapping using genomic tiling path microarrays in Drosophila
    Article Snippet: To prevent amplification of DNA fragments containing unmethylated GATCs, 1 μg of the adaptor-ligated DNA was cut with 2 units of Dpn II (New England Biolabs) for1hat37°C in a total volume of 20 μl of Dpn II buffer. .. Next, amplification was performed by using 0.5 μg of Dpn II-cut DNA, 1 μl of Advantage cDNA PCR polymerase mix (CLONTECH), 10 nmol of each dATP, dCTP, dGTP, and dTTP, and 62.5 pmol of primer (5′-GGTCGCGGCCGAGGATC-3′) in 50 μl total volume of Advantage PCR buffer, under the following cycling conditions: activation of the polymerase and nick translation for 10 min at 68°C, followed by one cycle of 1 min at 94°C, 5 min at 65°C and 15 min at 68°C; 3 cycles of 1 min at 94°C, 1 min at 65°C and 10 min at 68°C; and 14 cycles of 1 min at 94°C, 1 min at 65°C and 2 min at 68°C.

    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 [W] [W] [OPEN]
    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. .. 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).

    Staining:

    Article Title: Differentiation of Porcine Brachyspira Species by a Novel nox PCR-Based Restriction Fragment Length Polymorphism Analysis
    Article Snippet: After initial denaturation at 94°C for 3 min, each cycle involved denaturation at 94°C for 30 s, annealing at 59°C for 40 s, and extension at 72°C for 54 s. The amplification was finished after a final extension step at 72°C for 10 min. An aliquot of 10 μl of the PCR product was visualized after separation by electrophoresis in a 1.5% agarose gel and staining with ethidium bromide. .. The buffer composition of the remaining PCR product was modified to meet the conditions for restriction digestion with Dpn II (New England Biolabs, Frankfurt, Germany) and Bfm I (MBI Fermentas, St. Leon-Rot, Germany).

    Article Title: Hepatitis B virus genotypes and hepatocellular carcinoma in Thailand
    Article Snippet: PCR products were subjected to RFLP analysis, using restriction endonuclease Ava II and Dpn II (New England Biolabs, Beverly, MA) to determine the HBV genotype. .. The sizes of the RFLP products, visible under UV light as a result of prior ethidium bromide staining, served to identify the various HBV genotypes based on the polymorphism patterns[ ].

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    New England Biolabs dpn ii
    Dpn Ii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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