r3193  (New England Biolabs)


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    Name:
    NcoI HF
    Description:
    NcoI HF 5 000 units
    Catalog Number:
    R3193L
    Price:
    261
    Category:
    Restriction Enzymes
    Size:
    5 000 units
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    Structured Review

    New England Biolabs r3193
    NcoI HF
    NcoI HF 5 000 units
    https://www.bioz.com/result/r3193/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    r3193 - by Bioz Stars, 2021-06
    99/100 stars

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    Related Articles

    Polyacrylamide Gel Electrophoresis:

    Article Title: Impact of tobacco-specific nitrosamine–derived DNA adducts on the efficiency and fidelity of DNA replication in human cells
    Article Snippet: .. For PAGE analysis, 150 ng of the PCR fragments were treated with 5 units of NcoI-HF and 1 unit of shrimp alkaline phosphatase (SAP) at 37 °C in 10 μl of NEBuffer 3 (New England Biolabs) for 1 h, followed by heating at 80 °C for 20 min to deactivate the SAP. ..

    Polymerase Chain Reaction:

    Article Title: Impact of tobacco-specific nitrosamine–derived DNA adducts on the efficiency and fidelity of DNA replication in human cells
    Article Snippet: .. For PAGE analysis, 150 ng of the PCR fragments were treated with 5 units of NcoI-HF and 1 unit of shrimp alkaline phosphatase (SAP) at 37 °C in 10 μl of NEBuffer 3 (New England Biolabs) for 1 h, followed by heating at 80 °C for 20 min to deactivate the SAP. ..

    Purification:

    Article Title: Mutants of Cre recombinase with improved accuracy
    Article Snippet: The plasmids were isolated using the QIAprep Spin Miniprep kit, eluting into 50 μL10 mM Tris-HCl pH 8. .. The purified plasmids were digested for 20 min at 37 °C in 60 μL reactions containing all of the collected DNA and 20 U of both ScaI-HF and NcoI-HF (both from New England Biolabs) in NEBuffer 4 (1 mM DTT, 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, pH 7.9). .. The digests quantified on 1% agarose gels following purification using the QIAprep Spin Miniprep kit.

    Article Title: Site-Specific Incorporation of Quadricyclane into a Protein and Photocleavage of the Quadricyclane Ligation Adduct
    Article Snippet: .. The insert was isolated by agarose gel electrophoresis as described above, and the gene ends were digested for 1 h 45 min at 37 °C with XhoI and NcoI-HF (both from New England Biolabs) and purified using a Zymo DNA Clean & Concentrator Kit (Zymo Research). .. Vector psfGFP150TAGHis6 PylT was linearized with XhoI and NcoI-HF (both from New England Biolabs) at 37 °C for 1 h 40 min, and linearized vector was isolated by agarose gel electrophoresis and treated with Antarctic phosphatase (New England Biolabs) at 37 °C for 1 h followed by purification with a Zymo DNA Clean & Concentrator Kit.

    Isolation:

    Article Title: Site-Specific Incorporation of Quadricyclane into a Protein and Photocleavage of the Quadricyclane Ligation Adduct
    Article Snippet: .. The insert was isolated by agarose gel electrophoresis as described above, and the gene ends were digested for 1 h 45 min at 37 °C with XhoI and NcoI-HF (both from New England Biolabs) and purified using a Zymo DNA Clean & Concentrator Kit (Zymo Research). .. Vector psfGFP150TAGHis6 PylT was linearized with XhoI and NcoI-HF (both from New England Biolabs) at 37 °C for 1 h 40 min, and linearized vector was isolated by agarose gel electrophoresis and treated with Antarctic phosphatase (New England Biolabs) at 37 °C for 1 h followed by purification with a Zymo DNA Clean & Concentrator Kit.

    Agarose Gel Electrophoresis:

    Article Title: Site-Specific Incorporation of Quadricyclane into a Protein and Photocleavage of the Quadricyclane Ligation Adduct
    Article Snippet: .. The insert was isolated by agarose gel electrophoresis as described above, and the gene ends were digested for 1 h 45 min at 37 °C with XhoI and NcoI-HF (both from New England Biolabs) and purified using a Zymo DNA Clean & Concentrator Kit (Zymo Research). .. Vector psfGFP150TAGHis6 PylT was linearized with XhoI and NcoI-HF (both from New England Biolabs) at 37 °C for 1 h 40 min, and linearized vector was isolated by agarose gel electrophoresis and treated with Antarctic phosphatase (New England Biolabs) at 37 °C for 1 h followed by purification with a Zymo DNA Clean & Concentrator Kit.

    Plasmid Preparation:

    Article Title: Directed Evolution of Improved Zinc Finger Methyltransferases
    Article Snippet: After growth overnight at 37°C and 250 rpm, plasmid DNA was purified from the cultures with a QIAprep Spin Miniprep Kit. .. Plasmid DNA (500 ng) was digested with NcoI-HF (10 units) and either FspI (2.5 units) or SnaBI (2.5 units) in 1X NEBuffer 4 for over one hour at 37°C. ..

    Article Title: Brain somatic mutations in MTOR reveal translational dysregulations underlying intractable focal epilepsy
    Article Snippet: .. 5′-UTRs with WT, transversion, and deletion sequences (see also ) were synthesized by Macrogen and subcloned into the pGL3-promoter vector (Promega, E1761) between the SV40 promoter and the firefly luciferase open reading frame using HindIII (New England BioLabs, R3104) and NcoI (New England BioLabs, R3193). ..

    Recombinant:

    Article Title: Self-assembling thermostable chimeras as new platform for arsenic biosensing
    Article Snippet: Colonies of E. coli Top10F’ transformed with pET28b(+)/arsC-vmh2 and pET28b(+)/vmh2-arsC (Supplementary Fig. ) were screened by PCR colony using the primer pairs chArsC FW, chVmh2 RV for arsC-vmh2 and chVmh2 FW, chArsC RV for vmh2-arsC and Taq DNA Pol (Thermo Scientific) (Table ). .. To verify the appropriate insertions, plasmids were recovered from the recombinant colonies using the QIAprep spin Miniprep Kit (QIAGEN) and digested with NcoI -HF and HindIII -HF (New England Biolabs). .. For the protein expression, E. coli BL21 (DE3) was transformed with the recombinant vectors.

    Synthesized:

    Article Title: Brain somatic mutations in MTOR reveal translational dysregulations underlying intractable focal epilepsy
    Article Snippet: .. 5′-UTRs with WT, transversion, and deletion sequences (see also ) were synthesized by Macrogen and subcloned into the pGL3-promoter vector (Promega, E1761) between the SV40 promoter and the firefly luciferase open reading frame using HindIII (New England BioLabs, R3104) and NcoI (New England BioLabs, R3193). ..

    Luciferase:

    Article Title: Brain somatic mutations in MTOR reveal translational dysregulations underlying intractable focal epilepsy
    Article Snippet: .. 5′-UTRs with WT, transversion, and deletion sequences (see also ) were synthesized by Macrogen and subcloned into the pGL3-promoter vector (Promega, E1761) between the SV40 promoter and the firefly luciferase open reading frame using HindIII (New England BioLabs, R3104) and NcoI (New England BioLabs, R3193). ..

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    New England Biolabs ncoi hf
    Methylation assay for selected variants. (A) Relative locations of the target site and non-target site on a plasmid linearized by <t>NcoI</t> digestion. For sequences of the target and non-target sites, refer to Figures 1C and D (B) The restriction endonuclease protection assay for methylation at the target and non-target site uses digestion with NcoI to linearize the plasmid and either <t>FspI</t> or SnaBI to assess the target and off-target methylation, respectively. FspI and SnaBI cannot digest a methylated site. Shown are results from select variants as well as the ‘wildtype’ heterodimeric enzyme (i.e. no mutations to residues 297–301) with or without a catalytically inactivating (C141S), or a catalytically compromised (Q147L) mutation.
    Ncoi Hf, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ncoi hf/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
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    ncoi hf - by Bioz Stars, 2021-06
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    Methylation assay for selected variants. (A) Relative locations of the target site and non-target site on a plasmid linearized by NcoI digestion. For sequences of the target and non-target sites, refer to Figures 1C and D (B) The restriction endonuclease protection assay for methylation at the target and non-target site uses digestion with NcoI to linearize the plasmid and either FspI or SnaBI to assess the target and off-target methylation, respectively. FspI and SnaBI cannot digest a methylated site. Shown are results from select variants as well as the ‘wildtype’ heterodimeric enzyme (i.e. no mutations to residues 297–301) with or without a catalytically inactivating (C141S), or a catalytically compromised (Q147L) mutation.

    Journal: PLoS ONE

    Article Title: Directed Evolution of Improved Zinc Finger Methyltransferases

    doi: 10.1371/journal.pone.0096931

    Figure Lengend Snippet: Methylation assay for selected variants. (A) Relative locations of the target site and non-target site on a plasmid linearized by NcoI digestion. For sequences of the target and non-target sites, refer to Figures 1C and D (B) The restriction endonuclease protection assay for methylation at the target and non-target site uses digestion with NcoI to linearize the plasmid and either FspI or SnaBI to assess the target and off-target methylation, respectively. FspI and SnaBI cannot digest a methylated site. Shown are results from select variants as well as the ‘wildtype’ heterodimeric enzyme (i.e. no mutations to residues 297–301) with or without a catalytically inactivating (C141S), or a catalytically compromised (Q147L) mutation.

    Article Snippet: Plasmid DNA (500 ng) was digested with NcoI-HF (10 units) and either FspI (2.5 units) or SnaBI (2.5 units) in 1X NEBuffer 4 for over one hour at 37°C.

    Techniques: Methylation, Plasmid Preparation, Mutagenesis

    Substitution of new zinc fingers targets methylation towards a new site. ( A ) A schematic of the designed methyltransferase is shown assembled over the new, targeted CpG site. New cognate zinc finger recognition sequences flank a CpG site nested within an FspI site. Zinc fingers CD54-31Opt and CD54a have replaced the HS1 and HS2 zinc fingers. ( B ) The non-target site contains the HS1 and HS2 zinc finger recognition sites flanking a CpG site nested within a FspI restriction site (i.e. this was the target site in experiments in Figure 2 ). ( C ) The relative locations of the target site and non-target site are shown on a plasmid linearized by NcoI digestion. ( D ) The restriction endonuclease protection assay for methylation at the target and non-target site for the ‘wildtype’ heterodimeric enzyme (KFNSE) and two selected variants with mutations in the region 297–301.

    Journal: PLoS ONE

    Article Title: Directed Evolution of Improved Zinc Finger Methyltransferases

    doi: 10.1371/journal.pone.0096931

    Figure Lengend Snippet: Substitution of new zinc fingers targets methylation towards a new site. ( A ) A schematic of the designed methyltransferase is shown assembled over the new, targeted CpG site. New cognate zinc finger recognition sequences flank a CpG site nested within an FspI site. Zinc fingers CD54-31Opt and CD54a have replaced the HS1 and HS2 zinc fingers. ( B ) The non-target site contains the HS1 and HS2 zinc finger recognition sites flanking a CpG site nested within a FspI restriction site (i.e. this was the target site in experiments in Figure 2 ). ( C ) The relative locations of the target site and non-target site are shown on a plasmid linearized by NcoI digestion. ( D ) The restriction endonuclease protection assay for methylation at the target and non-target site for the ‘wildtype’ heterodimeric enzyme (KFNSE) and two selected variants with mutations in the region 297–301.

    Article Snippet: Plasmid DNA (500 ng) was digested with NcoI-HF (10 units) and either FspI (2.5 units) or SnaBI (2.5 units) in 1X NEBuffer 4 for over one hour at 37°C.

    Techniques: Zinc-Fingers, Methylation, Plasmid Preparation

    Restriction digestion and post-labeling method for determining the bypass efficiencies and mutation frequencies of the lesions in HEK293T cells and the isogenic TLS polymerase-deficient cells. A , restriction digestion of PCR products using NcoI and SfaNI and the post-labeling assay. B , representative gel image showing the NcoI/SfaNI-produced restriction fragments of interest. The standard synthetic ODN representing the restriction fragment arising from the competitor vector ( i.e. 5′-CATGGCGATATGCTGT-3′) is designated as 16-mer. 13-mer A , 13-mer G , 13-mer T , and 13-mer C represent the standard synthetic ODNs 5′-CATGGCGMGCTGT-3′, where M is A, G, T, and C, respectively. C , sample processing for restriction cleavage using MluCI and Cac8I and the post-labeling assay. D , representative gel image showing the MluCI/Cac8I-generated restriction fragments of PCR products amplified from the progenies of the O 4 -POB-dT-containing plasmid arising from replication in HEK293T cells (WT) and the isogenic polymerase-deficient cells (the five lanes on the right ). 10-mer A , 10-mer G , 10-mer T , and 10-mer C designate the standard synthetic ODN 5′-AATTACAGCN-3′, where N is A, G, T, and C, respectively. p * indicates a 32 P-labeled phosphate group.

    Journal: The Journal of Biological Chemistry

    Article Title: Impact of tobacco-specific nitrosamine–derived DNA adducts on the efficiency and fidelity of DNA replication in human cells

    doi: 10.1074/jbc.RA118.003477

    Figure Lengend Snippet: Restriction digestion and post-labeling method for determining the bypass efficiencies and mutation frequencies of the lesions in HEK293T cells and the isogenic TLS polymerase-deficient cells. A , restriction digestion of PCR products using NcoI and SfaNI and the post-labeling assay. B , representative gel image showing the NcoI/SfaNI-produced restriction fragments of interest. The standard synthetic ODN representing the restriction fragment arising from the competitor vector ( i.e. 5′-CATGGCGATATGCTGT-3′) is designated as 16-mer. 13-mer A , 13-mer G , 13-mer T , and 13-mer C represent the standard synthetic ODNs 5′-CATGGCGMGCTGT-3′, where M is A, G, T, and C, respectively. C , sample processing for restriction cleavage using MluCI and Cac8I and the post-labeling assay. D , representative gel image showing the MluCI/Cac8I-generated restriction fragments of PCR products amplified from the progenies of the O 4 -POB-dT-containing plasmid arising from replication in HEK293T cells (WT) and the isogenic polymerase-deficient cells (the five lanes on the right ). 10-mer A , 10-mer G , 10-mer T , and 10-mer C designate the standard synthetic ODN 5′-AATTACAGCN-3′, where N is A, G, T, and C, respectively. p * indicates a 32 P-labeled phosphate group.

    Article Snippet: For PAGE analysis, 150 ng of the PCR fragments were treated with 5 units of NcoI-HF and 1 unit of shrimp alkaline phosphatase (SAP) at 37 °C in 10 μl of NEBuffer 3 (New England Biolabs) for 1 h, followed by heating at 80 °C for 20 min to deactivate the SAP.

    Techniques: Labeling, Mutagenesis, Polymerase Chain Reaction, Postlabeling Assay, Produced, Plasmid Preparation, Generated, Amplification

    In vivo recombination of plasmids by mutants of Cre a) Plasmid architecture of the three expected recombination products. Recombination sites are shown as triangles. For simplicity, the map is in linear form, where the NcoI sites at the ends represent a single NcoI site on the circular plasmids. The numbers under each segment indicate distance in bp (map not drawn to scale). The numbers adjacent to each map are the sizes of the expected digestion products, with the asterisks indicating the product size that is unique to the particular configuration. b) Digest analysis of loxP x loxP (left five lanes) and ψlox h7q21 x ψCore h7q21 (right five lanes) recombination. (c d) Inversion and recombination frequency of (c) loxP x loxP and (d) ψCore h7q21 x ψlox h7q21 recombination obtained by quantifying band intensities. Error bars correspond to 95% C.I. (n= 2 independent experiment). NI: NcoI site; SI: ScaI site.

    Journal: Nature communications

    Article Title: Mutants of Cre recombinase with improved accuracy

    doi: 10.1038/ncomms3509

    Figure Lengend Snippet: In vivo recombination of plasmids by mutants of Cre a) Plasmid architecture of the three expected recombination products. Recombination sites are shown as triangles. For simplicity, the map is in linear form, where the NcoI sites at the ends represent a single NcoI site on the circular plasmids. The numbers under each segment indicate distance in bp (map not drawn to scale). The numbers adjacent to each map are the sizes of the expected digestion products, with the asterisks indicating the product size that is unique to the particular configuration. b) Digest analysis of loxP x loxP (left five lanes) and ψlox h7q21 x ψCore h7q21 (right five lanes) recombination. (c d) Inversion and recombination frequency of (c) loxP x loxP and (d) ψCore h7q21 x ψlox h7q21 recombination obtained by quantifying band intensities. Error bars correspond to 95% C.I. (n= 2 independent experiment). NI: NcoI site; SI: ScaI site.

    Article Snippet: The purified plasmids were digested for 20 min at 37 °C in 60 μL reactions containing all of the collected DNA and 20 U of both ScaI-HF and NcoI-HF (both from New England Biolabs) in NEBuffer 4 (1 mM DTT, 50 mM potassium acetate, 20 mM tris-acetate, 10 mM magnesium acetate, pH 7.9).

    Techniques: In Vivo, Plasmid Preparation

    Chromatin conformation capture identifies a tissue-specific enhancer that interacts with the AXUD1 promoter. (A-B) Whole mount in situ hybridization for AXUD1 , depicting the specific mRNA expression during neural crest specification stages HH8 ( A ) and HH9 ( B ). (C) Schematic representation of a Chromosome Conformation Capture-qPCR (3C-qPCR) experiment. Crosslinked neural crest cells were incubated with restriction enzyme NCOI and a DNA ligase. These steps allow the formation of hybrid DNA molecules combining restriction fragments that were in close proximity in nuclei of the cells. Primers spanning the AXUD1 locus were paired with a primer anchored in the AXUD1 promoter to amplify hybrid DNA junctions and quantify the interaction frequency with the promoter. (D-F) Identification of active enhancers in the AXUD1 locus. 3C-qPCR interaction map for the AXUD1 locus reveals regions of high interaction frequency with the promoter region (blue dotted line, D ). Gray dotted lines highlight the six elements tested in transient transgenesis assays (see below). Error bars represent ± SEM. Purple and blue lines in ( D ) represent two replicates of the same 3C experiment. ATAC-seq, H3K27ac and TFAP2A CUT RUN profiles at AXUD1 locus depict regions of accessibility and active chromatin regions ( E ). ( F ) eRNA quantification (RT-PCR, normalized to reference gene) for the regions numbered in ( E ) indicates the level of transcription in the promoter region and putative distal regulatory elements. Error bars represent ± SEM. Statistical significance determined via an unpaired t-test. (G ) Reporter vector used in transgenesis reporter assays. The construct consists of the candidate enhancer region cloned upstream of the HSV-tk minimal promoter driving eGFP expression. (H-J ) The Axud1E1 element is active in neural crest cells. In vivo activity of Axud1E1 as shown by eGFP expression in reporter assays ( H ). Axud1E1 recapitulates endogenous gene expression in dorsal neural folds (arrows), while the putative enhancer ax . 1 displayed no specific activity ( I ). Transverse cryosection from a HH10 embryo electroporated with the Axud1E1 enhancer illustrates eGFP expression in the dorsal neural tube and migratory neural crest cells ( J ). HH, Hamburger and Hamilton. Scale bars represent 500μm ( A-B ), 200μm ( H-I ) and 100μm ( J ). *p

    Journal: PLoS Genetics

    Article Title: A regulatory sub-circuit downstream of Wnt signaling controls developmental transitions in neural crest formation

    doi: 10.1371/journal.pgen.1009296

    Figure Lengend Snippet: Chromatin conformation capture identifies a tissue-specific enhancer that interacts with the AXUD1 promoter. (A-B) Whole mount in situ hybridization for AXUD1 , depicting the specific mRNA expression during neural crest specification stages HH8 ( A ) and HH9 ( B ). (C) Schematic representation of a Chromosome Conformation Capture-qPCR (3C-qPCR) experiment. Crosslinked neural crest cells were incubated with restriction enzyme NCOI and a DNA ligase. These steps allow the formation of hybrid DNA molecules combining restriction fragments that were in close proximity in nuclei of the cells. Primers spanning the AXUD1 locus were paired with a primer anchored in the AXUD1 promoter to amplify hybrid DNA junctions and quantify the interaction frequency with the promoter. (D-F) Identification of active enhancers in the AXUD1 locus. 3C-qPCR interaction map for the AXUD1 locus reveals regions of high interaction frequency with the promoter region (blue dotted line, D ). Gray dotted lines highlight the six elements tested in transient transgenesis assays (see below). Error bars represent ± SEM. Purple and blue lines in ( D ) represent two replicates of the same 3C experiment. ATAC-seq, H3K27ac and TFAP2A CUT RUN profiles at AXUD1 locus depict regions of accessibility and active chromatin regions ( E ). ( F ) eRNA quantification (RT-PCR, normalized to reference gene) for the regions numbered in ( E ) indicates the level of transcription in the promoter region and putative distal regulatory elements. Error bars represent ± SEM. Statistical significance determined via an unpaired t-test. (G ) Reporter vector used in transgenesis reporter assays. The construct consists of the candidate enhancer region cloned upstream of the HSV-tk minimal promoter driving eGFP expression. (H-J ) The Axud1E1 element is active in neural crest cells. In vivo activity of Axud1E1 as shown by eGFP expression in reporter assays ( H ). Axud1E1 recapitulates endogenous gene expression in dorsal neural folds (arrows), while the putative enhancer ax . 1 displayed no specific activity ( I ). Transverse cryosection from a HH10 embryo electroporated with the Axud1E1 enhancer illustrates eGFP expression in the dorsal neural tube and migratory neural crest cells ( J ). HH, Hamburger and Hamilton. Scale bars represent 500μm ( A-B ), 200μm ( H-I ) and 100μm ( J ). *p

    Article Snippet: DNA digestion was performed with 400UI NCOI (New England Biolabs, #R3193) at 37°C overnight.

    Techniques: In Situ Hybridization, Expressing, Real-time Polymerase Chain Reaction, Incubation, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation, Construct, Clone Assay, In Vivo, Activity Assay