ncoi restriction enzyme  (New England Biolabs)


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    Structured Review

    New England Biolabs ncoi restriction enzyme
    An interaction between the distal and proximal regulatory regions of the MCP - 1 gene forms following TNF stimulation. (A) A schematic representation of the 3C assay and the architecture of the MCP - 1 promoter with cis regulatory sites with selected restriction sites and primer positions (P1 through P4) is shown. In the 3C assay, cells were fixed with formaldehyde and chromatin was isolated and digested with <t>NcoI.</t> Following inactivation of the restriction enzyme, the sample was diluted and subjected to <t>DNA</t> ligation such that only intramolecular ligations would be preferred. The cross-links were then reversed, and the DNA was purified. PCR primers P1 and P2 were used to detect the formation of the novel ligated 3C product. Primers P3 and P4 were used to verify that similar levels of MCP - 1 DNA were present in the assay and that the DNA was amplifiable. Products from the 3C assay were examined on agarose gels stained with ethidium bromide. (B) NIH 3T3 cells treated in the absence (−) (lanes 1 to 4) or presence (+) (lanes 5 to 8) of TNF for 2 h were processed in the above 3C assay with and without formaldehyde (CH 2 O) or DNA ligase, as indicated, to control for specificity of the protocol. M, DNA marker. (C) Purified mouse genomic DNA was processed in the 3C assay and demonstrates that the unique PCR product cannot form with naked DNA templates. (D) KpnI cleaves the PCR product generated by the 3C assay, producing the anticipated DNA fragments as shown on the agarose gel. (E) PCR amplification of the 3C product and the non-3C product display similar relative efficiencies. PCR products generated from undigested genomic DNA (1,207 bp) and DNA from a 3C assay (713 bp) were quantitated by fluorometry and used as templates in PCRs to determine if there were major differences in their PCR efficiency. DNA templates (40 pg, 8 pg, and 1.6 pg) were amplified by primers P1 and P2 under identical PCR conditions. (F) Chromatin isolated from TNF-treated or control cells fixed with formaldehyde was subjected to NcoI digestion as indicated. Following deproteination, the DNA was purified and analyzed by PCR using the indicated primers. Because the ligation step of the 3C assay was not performed, P1/P2, P1/P6, and P2/P5 PCR products can only be observed when NcoI was omitted from the reaction, indicating that the chromatin is accessible to restriction digestion even in the absence of TNF.
    Ncoi Restriction Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Mechanism of Action of a Distal NF-?B-Dependent Enhancer"

    Article Title: Mechanism of Action of a Distal NF-?B-Dependent Enhancer

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00271-06

    An interaction between the distal and proximal regulatory regions of the MCP - 1 gene forms following TNF stimulation. (A) A schematic representation of the 3C assay and the architecture of the MCP - 1 promoter with cis regulatory sites with selected restriction sites and primer positions (P1 through P4) is shown. In the 3C assay, cells were fixed with formaldehyde and chromatin was isolated and digested with NcoI. Following inactivation of the restriction enzyme, the sample was diluted and subjected to DNA ligation such that only intramolecular ligations would be preferred. The cross-links were then reversed, and the DNA was purified. PCR primers P1 and P2 were used to detect the formation of the novel ligated 3C product. Primers P3 and P4 were used to verify that similar levels of MCP - 1 DNA were present in the assay and that the DNA was amplifiable. Products from the 3C assay were examined on agarose gels stained with ethidium bromide. (B) NIH 3T3 cells treated in the absence (−) (lanes 1 to 4) or presence (+) (lanes 5 to 8) of TNF for 2 h were processed in the above 3C assay with and without formaldehyde (CH 2 O) or DNA ligase, as indicated, to control for specificity of the protocol. M, DNA marker. (C) Purified mouse genomic DNA was processed in the 3C assay and demonstrates that the unique PCR product cannot form with naked DNA templates. (D) KpnI cleaves the PCR product generated by the 3C assay, producing the anticipated DNA fragments as shown on the agarose gel. (E) PCR amplification of the 3C product and the non-3C product display similar relative efficiencies. PCR products generated from undigested genomic DNA (1,207 bp) and DNA from a 3C assay (713 bp) were quantitated by fluorometry and used as templates in PCRs to determine if there were major differences in their PCR efficiency. DNA templates (40 pg, 8 pg, and 1.6 pg) were amplified by primers P1 and P2 under identical PCR conditions. (F) Chromatin isolated from TNF-treated or control cells fixed with formaldehyde was subjected to NcoI digestion as indicated. Following deproteination, the DNA was purified and analyzed by PCR using the indicated primers. Because the ligation step of the 3C assay was not performed, P1/P2, P1/P6, and P2/P5 PCR products can only be observed when NcoI was omitted from the reaction, indicating that the chromatin is accessible to restriction digestion even in the absence of TNF.
    Figure Legend Snippet: An interaction between the distal and proximal regulatory regions of the MCP - 1 gene forms following TNF stimulation. (A) A schematic representation of the 3C assay and the architecture of the MCP - 1 promoter with cis regulatory sites with selected restriction sites and primer positions (P1 through P4) is shown. In the 3C assay, cells were fixed with formaldehyde and chromatin was isolated and digested with NcoI. Following inactivation of the restriction enzyme, the sample was diluted and subjected to DNA ligation such that only intramolecular ligations would be preferred. The cross-links were then reversed, and the DNA was purified. PCR primers P1 and P2 were used to detect the formation of the novel ligated 3C product. Primers P3 and P4 were used to verify that similar levels of MCP - 1 DNA were present in the assay and that the DNA was amplifiable. Products from the 3C assay were examined on agarose gels stained with ethidium bromide. (B) NIH 3T3 cells treated in the absence (−) (lanes 1 to 4) or presence (+) (lanes 5 to 8) of TNF for 2 h were processed in the above 3C assay with and without formaldehyde (CH 2 O) or DNA ligase, as indicated, to control for specificity of the protocol. M, DNA marker. (C) Purified mouse genomic DNA was processed in the 3C assay and demonstrates that the unique PCR product cannot form with naked DNA templates. (D) KpnI cleaves the PCR product generated by the 3C assay, producing the anticipated DNA fragments as shown on the agarose gel. (E) PCR amplification of the 3C product and the non-3C product display similar relative efficiencies. PCR products generated from undigested genomic DNA (1,207 bp) and DNA from a 3C assay (713 bp) were quantitated by fluorometry and used as templates in PCRs to determine if there were major differences in their PCR efficiency. DNA templates (40 pg, 8 pg, and 1.6 pg) were amplified by primers P1 and P2 under identical PCR conditions. (F) Chromatin isolated from TNF-treated or control cells fixed with formaldehyde was subjected to NcoI digestion as indicated. Following deproteination, the DNA was purified and analyzed by PCR using the indicated primers. Because the ligation step of the 3C assay was not performed, P1/P2, P1/P6, and P2/P5 PCR products can only be observed when NcoI was omitted from the reaction, indicating that the chromatin is accessible to restriction digestion even in the absence of TNF.

    Techniques Used: Isolation, DNA Ligation, Purification, Polymerase Chain Reaction, Staining, Marker, Generated, Agarose Gel Electrophoresis, Amplification, Ligation

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    New England Biolabs ncoi restriction enzymes
    pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with <t>NcoI</t> and <t>XbaI</t> restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.
    Ncoi Restriction Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs restriction enzymes ncoi
    The Double Digestion Patterns of the pNZ8148-HPV16-optiE7 Shuttle Plasmid via <t>NcoI</t> and <t>SacI</t> Restriction Endonuclease
    Restriction Enzymes Ncoi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    restriction enzymes ncoi - by Bioz Stars, 2022-09
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    pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Journal: Scientific Reports

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli

    doi: 10.1038/s41598-018-30792-0

    Figure Lengend Snippet: pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Article Snippet: For the simultaneous expression of Nampt and PRPP synthetase a bicistronic vector was constructed by PCR amplifying the baPrs sequence (Q5 High-Fidelity 2X Master Mix, NEB) from pUC57-Kan plasmid with M13 forward and reverse primers, double digestion with XbaI and NcoI restriction enzymes (NEB) of PCR product and pET28a-hdNAdV plasmid, followed by the separation of the desired DNA fragments on 1.5% agarose gel electrophoresis , purification from gel (using Wizard SV Gel and PCR Clean-Up System, Promega, Madison, USA) of desired bands (pET28a-hdNadV and baPrs) followed by ligation with T4 DNA ligase (NEB) (as shown in Fig. ).

    Techniques: Plasmid Preparation, Mutagenesis, Clone Assay, Polymerase Chain Reaction, Amplification, Purification, Construct, Transformation Assay, Agarose Gel Electrophoresis, Fluorimetry Assay

    The Double Digestion Patterns of the pNZ8148-HPV16-optiE7 Shuttle Plasmid via NcoI and SacI Restriction Endonuclease

    Journal: Asian Pacific Journal of Cancer Prevention : APJCP

    Article Title: Codon Usage Optimization and Construction of Plasmid Encoding Iranian Human Papillomavirus Type 16 E7 Oncogene for Lactococcus Lactis Subsp. Cremoris MG1363

    doi: 10.22034/APJCP.2017.18.3.783

    Figure Lengend Snippet: The Double Digestion Patterns of the pNZ8148-HPV16-optiE7 Shuttle Plasmid via NcoI and SacI Restriction Endonuclease

    Article Snippet: Construction of shuttle vector The optimized E7 gene, encoding the E7 oncoprotein from HPV 16, was obtained as a 291 bp DNA fragment by digesting plasmid PMD18 with restriction enzymes NcoI and SacI (New England Biolabs).

    Techniques: Plasmid Preparation

    Restriction fragment length polymorphism (RFLP) analysis of PCR products. (A) Representative ethidium bromide stained (15%) non-denaturing polyacrylamide gel (PAGE) showing amplified gene product of TNF-α (−238G/A) 118 bp uncut and RFLP pattern observed after digestion with Bgl II. (Lane M -20 bp ladder, Lane 2 and 8-GA genotype, Lane 3 to 7-GG Genotype. (B) Representative non-denaturing PAGE (15%) showing amplified gene segment of TNF-α (−308G/A) 118 bp uncut and RFLP pattern observed after digestion with Nco I. Lane 1-uncut 118 bp product, Lane 2 and 6-GG Genotype, Lane 3 and 5-GA genotype, Lane 4-AA Genotype, Lane M-20 bp Ladder. (C) Representative non-denaturing PAGE (15%) showing amplified gene segment of TNF-α (−863C/A) 126 bp uncut and RFLP pattern observed after digestion with Bsa AI. Lane 1-Negative Control, Lane 2-uncut 126 bp, Lane 3-AA Genotype, Lane 4-CA Genotype, Lane 5, 8 and 9-CC Genotype, Lane 6 and 7-CA Genotype, Lane M-50 bp ladder.

    Journal: PLoS ONE

    Article Title: High Producer Haplotype (CAG) of -863C/A, -308G/A and -238G/A Polymorphisms in the Promoter Region of TNF-α Gene Associate with Enhanced Apoptosis of Lymphocytes in HIV-1 Subtype C Infected Individuals from North India

    doi: 10.1371/journal.pone.0098020

    Figure Lengend Snippet: Restriction fragment length polymorphism (RFLP) analysis of PCR products. (A) Representative ethidium bromide stained (15%) non-denaturing polyacrylamide gel (PAGE) showing amplified gene product of TNF-α (−238G/A) 118 bp uncut and RFLP pattern observed after digestion with Bgl II. (Lane M -20 bp ladder, Lane 2 and 8-GA genotype, Lane 3 to 7-GG Genotype. (B) Representative non-denaturing PAGE (15%) showing amplified gene segment of TNF-α (−308G/A) 118 bp uncut and RFLP pattern observed after digestion with Nco I. Lane 1-uncut 118 bp product, Lane 2 and 6-GG Genotype, Lane 3 and 5-GA genotype, Lane 4-AA Genotype, Lane M-20 bp Ladder. (C) Representative non-denaturing PAGE (15%) showing amplified gene segment of TNF-α (−863C/A) 126 bp uncut and RFLP pattern observed after digestion with Bsa AI. Lane 1-Negative Control, Lane 2-uncut 126 bp, Lane 3-AA Genotype, Lane 4-CA Genotype, Lane 5, 8 and 9-CC Genotype, Lane 6 and 7-CA Genotype, Lane M-50 bp ladder.

    Article Snippet: Four micrograms of nested PCR product (118 bp) was digested at 37°C for 8 hours in a 10 µL reaction volume containing 2.5 units of restriction enzyme Nco I (NEB, USA) to detect -308G/A polymorphism and 2.5 units of restriction enzyme BglII (NEB, USA) to detect -238G/A polymorphism, and then analyzed on a 15% non-denaturing polyacrylamide gel (PAGE).

    Techniques: Polymerase Chain Reaction, Staining, Polyacrylamide Gel Electrophoresis, Amplification, Negative Control

    pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Journal: Scientific Reports

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli

    doi: 10.1038/s41598-018-30792-0

    Figure Lengend Snippet: pET28a-baPrs-hdNadV bicistronic vector construction. Prs gene from Bacillus amyloliquefaciens with L135I mutation (ctc to ata) was cloned in pUC57-kan vector (GenScript synthesis). pET28a-hdNadV Vector and PCR amplification of prs fragment were digested (with NcoI and XbaI restriction enzymes) and the fragments corresponding to 1030 bp and 6687 bp were purified and ligated. The DNA construct was chemically transformed in Escherichia coli DH5α, verified by agarose gel electrophoresis and transformed in strain BL21(DE3)pLysS. After transformation, cells were grown into a 500 mL bench-top bioreactor system and the NMN yield was determined by derivatization followed by fluorimetric assay.

    Article Snippet: For the simultaneous expression of Nampt and PRPP synthetase a bicistronic vector was constructed by PCR amplifying the baPrs sequence (Q5 High-Fidelity 2X Master Mix, NEB) from pUC57-Kan plasmid with M13 forward and reverse primers, double digestion with XbaI and NcoI restriction enzymes (NEB) of PCR product and pET28a-hdNAdV plasmid, followed by the separation of the desired DNA fragments on 1.5% agarose gel electrophoresis , purification from gel (using Wizard SV Gel and PCR Clean-Up System, Promega, Madison, USA) of desired bands (pET28a-hdNadV and baPrs) followed by ligation with T4 DNA ligase (NEB) (as shown in Fig. ).

    Techniques: Plasmid Preparation, Mutagenesis, Clone Assay, Polymerase Chain Reaction, Amplification, Purification, Construct, Transformation Assay, Agarose Gel Electrophoresis, Fluorimetry Assay