dpni enzyme  (New England Biolabs)


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  • 99
    Name:
    DpnI
    Description:
    DpnI 5 000 units
    Catalog Number:
    r0176l
    Price:
    269
    Size:
    5 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs dpni enzyme
    DpnI
    DpnI 5 000 units
    https://www.bioz.com/result/dpni enzyme/product/New England Biolabs
    Average 99 stars, based on 20 article reviews
    Price from $9.99 to $1999.99
    dpni enzyme - by Bioz Stars, 2020-07
    99/100 stars

    Images

    1) Product Images from "High-throughput mutagenesis using a two-fragment PCR approach"

    Article Title: High-throughput mutagenesis using a two-fragment PCR approach

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07010-4

    Overview of the mutagenesis technique. Two PCR reactions are done per mutant, in each of them approximately half of the vector is amplified. Two fragments containing one mutation are combined, followed by DpnI digestion at 37 °C overnight. Reaction clean-up is performed to purify DNA fragments, which are then assembled by Gibson assembly reaction. Bacteria are transformed with the resulting circular plasmid and plated on selective LB agar plates. One clone per mutant is sent for sequencing either on a selective LB agar 96-well plate or as purified DNA. All steps excluding the plating of the bacteria are done in 96-well plates.
    Figure Legend Snippet: Overview of the mutagenesis technique. Two PCR reactions are done per mutant, in each of them approximately half of the vector is amplified. Two fragments containing one mutation are combined, followed by DpnI digestion at 37 °C overnight. Reaction clean-up is performed to purify DNA fragments, which are then assembled by Gibson assembly reaction. Bacteria are transformed with the resulting circular plasmid and plated on selective LB agar plates. One clone per mutant is sent for sequencing either on a selective LB agar 96-well plate or as purified DNA. All steps excluding the plating of the bacteria are done in 96-well plates.

    Techniques Used: Mutagenesis, Polymerase Chain Reaction, Plasmid Preparation, Amplification, Transformation Assay, Sequencing, Purification

    Related Articles

    Methylation:

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli
    Article Snippet: .. Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites. ..

    Isolation:

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins
    Article Snippet: .. Bacterial genomic DNA was isolated from 3 ml LBamp cultures from individual colonies using the DNeasy Tissue kit (Qiagen, 69504). gDNA (1 µg) was digested with 10 units of Dpn I (NEB, R0176S) for 1 h at 37°C. ..

    Ligation:

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans
    Article Snippet: .. Ligation to Linker A was carried out in a 50 μl reaction using the following mix: 10 μl Dpn I product, 1.5 μl of 0.05 mM Linker A, 11.0 μl dH2 O, 25.0 μl 2× Quick Ligase Buffer, 2.5 μl Quick Ligase (NEB #M2200). .. To increase the number of ligated molecules, we added a second ligation step using the following mix: 10 μl Quick Ligase product, 7 μl dH2 O, 2 μl 10× ligase buffer, 1 μl T4 DNA ligase (2,000 U/μl; NEB #M0202).

    Construct:

    Article Title: Secondary structure formation and DNA instability at fragile site FRA16B
    Article Snippet: .. To determine replication efficiency of the constructs ( B), SV40-replicated DNAs were digested with HindIII and NdeI (New England Biolabs) to linearize the plasmids, and with DpnI (New England Biolabs) to remove unreplicated parental templates. ..

    other:

    Article Title: Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication ▿
    Article Snippet: HindIII, DpnI, and λ DNA were purchased from New England Biolabs.

    Plasmid Preparation:

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli
    Article Snippet: .. Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites. ..

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    New England Biolabs dpni restriction enzyme
    Real-time PCR analysis of viral <t>DNA</t> synthesis in Sf9 cells. Total DNA was isolated from Sf9 cells transfected with vAc ac76-KO-PH-GFP or vAc-GP64-KO at selected time points, digested with the restriction enzyme <t>DpnI</t> to eliminate input bacmid DNA, and assayed
    Dpni Restriction Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dpni restriction enzyme/product/New England Biolabs
    Average 99 stars, based on 62 article reviews
    Price from $9.99 to $1999.99
    dpni restriction enzyme - by Bioz Stars, 2020-07
    99/100 stars
      Buy from Supplier

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    Real-time PCR analysis of viral DNA synthesis in Sf9 cells. Total DNA was isolated from Sf9 cells transfected with vAc ac76-KO-PH-GFP or vAc-GP64-KO at selected time points, digested with the restriction enzyme DpnI to eliminate input bacmid DNA, and assayed

    Journal: Journal of Virology

    Article Title: Autographa californica Multiple Nucleopolyhedrovirus ac76 Is Involved in Intranuclear Microvesicle Formation ▿

    doi: 10.1128/JVI.02103-09

    Figure Lengend Snippet: Real-time PCR analysis of viral DNA synthesis in Sf9 cells. Total DNA was isolated from Sf9 cells transfected with vAc ac76-KO-PH-GFP or vAc-GP64-KO at selected time points, digested with the restriction enzyme DpnI to eliminate input bacmid DNA, and assayed

    Article Snippet: Prior to PCR, 5 μl of total DNA from each time point was digested with 20 units of DpnI restriction enzyme (NEB) overnight in a 50-μl reaction volume to eliminate input bacmid DNA.

    Techniques: Real-time Polymerase Chain Reaction, DNA Synthesis, Isolation, Transfection

    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

    Two-step derivation of mutations in E1B and E4. A. The mutant constructs were first amplified with two partially overlapping primers designed to incorporate the desired alteration (shown in green), using the corresponding wild type block as a template. The input template DNA was then eliminated by digestion with the restriction enzyme DpnI. In the second step, the PCR-derived plasmid was circularized in an assembly reaction, which merged and sealed the overlapping ends. B. Oligonucleotide pairs that were used to generate the mutants are illustrated. The mutant block 1-derived constructs were E1B19K-null, E1B55K-null or modified to express an endogenous E1B55K protein with a c-terminal FLAG epitope. Similarly, the E4ORF3 open reading frame was disrupted by a mutation at the initiation codon or the introduction of a c-terminal FLAG tag. The targeted codons are boxed, single base mutations are shown in red. At positions where the initiation codon of the target protein overlapped with a codon for a different protein, base substitutions were selected so that the mutation would be silent with respect to the second open reading frame. C. hTERT-RPE1 cells were infected with the synthetic Ad5 virus and the E1B mutant viruses generated in this study. Cells were lysed 16 h post-infection, and assessed by immunoblot with antibodies against p53 and the FLAG epitope, as indicated. D. hTERT-RPE1 cells were uninfected (no virus) or infected with wild type Ad5 and the E4 mutants. For all viruses, the MOI was 100. GAPDH was probed as a loading control.

    Journal: PLoS ONE

    Article Title: Seamless assembly of recombinant adenoviral genomes from high-copy plasmids

    doi: 10.1371/journal.pone.0199563

    Figure Lengend Snippet: Two-step derivation of mutations in E1B and E4. A. The mutant constructs were first amplified with two partially overlapping primers designed to incorporate the desired alteration (shown in green), using the corresponding wild type block as a template. The input template DNA was then eliminated by digestion with the restriction enzyme DpnI. In the second step, the PCR-derived plasmid was circularized in an assembly reaction, which merged and sealed the overlapping ends. B. Oligonucleotide pairs that were used to generate the mutants are illustrated. The mutant block 1-derived constructs were E1B19K-null, E1B55K-null or modified to express an endogenous E1B55K protein with a c-terminal FLAG epitope. Similarly, the E4ORF3 open reading frame was disrupted by a mutation at the initiation codon or the introduction of a c-terminal FLAG tag. The targeted codons are boxed, single base mutations are shown in red. At positions where the initiation codon of the target protein overlapped with a codon for a different protein, base substitutions were selected so that the mutation would be silent with respect to the second open reading frame. C. hTERT-RPE1 cells were infected with the synthetic Ad5 virus and the E1B mutant viruses generated in this study. Cells were lysed 16 h post-infection, and assessed by immunoblot with antibodies against p53 and the FLAG epitope, as indicated. D. hTERT-RPE1 cells were uninfected (no virus) or infected with wild type Ad5 and the E4 mutants. For all viruses, the MOI was 100. GAPDH was probed as a loading control.

    Article Snippet: Input plasmid DNA templates were digested with 20 U of the restriction enzyme DpnI (New England Biolabs) for 30 min at 37°C.

    Techniques: Mutagenesis, Construct, Amplification, Blocking Assay, Polymerase Chain Reaction, Derivative Assay, Plasmid Preparation, Modification, FLAG-tag, Infection, Generated

    Time course of viral replication in transfected cells. In total, Sf9 cells (1 × 10 6 ) were transfected with 1 μg of Ac-MK, Ac11KO, Ac11Re, or GP64KO bacmids. At the designated time points, total cellular DNA from Sf9 cells transfected with each bacmid DNA was isolated, digested with the restriction enzyme DpnI to eliminate the input bacmid, and analyzed by qPCR. The results are from three separate transfections, and the error bars represent standard deviations.

    Journal: Journal of Virology

    Article Title: Autographa californica Multiple Nucleopolyhedrovirus ORF11 Is Essential for Budded-Virus Production and Occlusion-Derived-Virus Envelopment

    doi: 10.1128/JVI.01742-14

    Figure Lengend Snippet: Time course of viral replication in transfected cells. In total, Sf9 cells (1 × 10 6 ) were transfected with 1 μg of Ac-MK, Ac11KO, Ac11Re, or GP64KO bacmids. At the designated time points, total cellular DNA from Sf9 cells transfected with each bacmid DNA was isolated, digested with the restriction enzyme DpnI to eliminate the input bacmid, and analyzed by qPCR. The results are from three separate transfections, and the error bars represent standard deviations.

    Article Snippet: Prior to PCR, 2 μl of total DNA from each time point was digested with 40 U of DpnI restriction enzyme (New England BioLabs, USA) overnight in a 20-μl total reaction mixture volume.

    Techniques: Transfection, Isolation, Real-time Polymerase Chain Reaction