ncoi restriction enzyme  (New England Biolabs)


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    Name:
    NcoI
    Description:
    NcoI 5 000 units
    Catalog Number:
    r0193l
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    261
    Size:
    5 000 units
    Category:
    Restriction Enzymes
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    New England Biolabs ncoi restriction enzyme
    NcoI
    NcoI 5 000 units
    https://www.bioz.com/result/ncoi restriction enzyme/product/New England Biolabs
    Average 90 stars, based on 497 article reviews
    Price from $9.99 to $1999.99
    ncoi restriction enzyme - by Bioz Stars, 2020-04
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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

    Related Articles

    Clone Assay:

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems
    Article Snippet: .. T4 polynucleotide kinase (PNK), NotI, NcoI, T4 DNA ligase and accompanying buffers purchased from New England Biolabs 100 mM ATP 100 µM CRISPR target oligonucleotides (designed as in ) and pACYC-GFP Gel purification kit (e.g. Qiagen QIAquick Gel Extraction kit or Promega Wizard SV Gel and PCR Clean-Up System) One Shot TOP10 Competent Cells (Thermo-Fisher) or similar cloning E. coli strain Miniprep kit (Qiagen or Promega) ..

    Article Title: Cloning, Transformation and Expression of Human Interferon ?2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi)
    Article Snippet: Paragraph title: 3.4. Cloning of INFα-2b Gene in pCAMBIA1304 ... So, this was digested with BstEII and NcoI (NEB Co).

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    Article Snippet: Three such plasmids, carrying expression DNA corresponding to each of the amino acid sequences compared in Fig. were produced as follows: Nampt-PET28a vector containing Nampt gene from Mus musculus strain C57BL/6 J, a gift from dr. Cynthia Wolberger (Addgene plasmid # 25630), was cloned in E . coli DH5α and maintained (as a glycerol stock) at −80 °C. pET28a-hdNadV and pET28a-soNadV plasmids carrying the nucleotide sequence corresponding to nadV gene from Haemophilus ducreyi (strain: ATCC 27722), respectively Shewanella oneidensis (strain: MR-1) were constructed as follows: The gene sequences were selected from the GenBank database (Gene ID: 2716561 respectively 1169740), optimized for expression in E . coli (using Genome Compiler software v. 2.2.55 (Genome Compiler Corporation 2015), considering the frequency of codon usage and GC content, eliminating unwanted restriction sequences), synthesized de novo (GenScript) and ligated into pET-28a(+) vector which was first double digested with NcoI and XhoI restriction enzymes. .. 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. ).

    Amplification:

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium
    Article Snippet: The sequence was then amplified using primers chloride-F (5′-CGAATTGAAGGAAGGCCG-3′) and chloride-R (5′-GCAGTGAAAGGAAGGCC-3′). .. The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen).

    Article Title: Cloning, Transformation and Expression of Human Interferon ?2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi)
    Article Snippet: The gene was amplified using mentioned primers, and then, PCR product was extracted from the gel using QIAGEN kit. .. So, this was digested with BstEII and NcoI (NEB Co).

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    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. ). .. The vectors presence was verified by 1.5% agarose gel electrophoresis and by PCR amplification with T7 forward and reverse primers (Supplementary Fig. ).

    Article Title: The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism
    Article Snippet: .. After 22 amplification cycles, the reactions were separated into four aliquots for digestion with NcoI, PstI (New England Biolabs), both, or neither. .. The products were analyzed on a 5% native polyacrylamide gel, visualized by autoradiography, and quantitated on a FUJIFILM FLA-5100 phosphoirmager (Fuji Medical Systems) using Multi Gauge software Version 2.3 (Fujifilm).

    Mass Spectrometry:

    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription
    Article Snippet: LC-MS/MS analysis In order to identify the transcription products of S -cdA and S -cdG using LC-MS/MS, RT-PCR products were treated with 50 U NcoI and 20 U shrimp alkaline phosphatase in 250 μL NEB buffer 3 at 37°C for 2 h, followed by heating at 80°C for 20 min. To the resulting solution was added 50 U of AseI, and the reaction mixture was incubated at 37°C for 1 h, followed by extraction once with phenol/chloroform/isoamyl alcohol (25:24:1, v/v). .. The ODN mixture was subjected to LC-MS/MS analysis, where the LTQ linear ion trap mass spectrometer was set up for monitoring the fragmentation of the [M-3H]3- ions of the 13mer [d(CATGGCGPGCTAT), where “P” designates A, T, C, or G, d(CATGGCTGGCTAT), d(CATGGCTAGCTAT)] and 12mer [d(CATGGCGGCTAT) and d(CATGGCAGCTAT)] ODNs, and the [M-4H]4- ion of the 16mer [i.e., d(CATGGCGATATGCTAT)] ODN.

    Synthesized:

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium
    Article Snippet: The chloride-inducible promoter (CIP) sequence used in this work was adapted from a CIP sequence of Sanders and coworkers (GenBank accession number ; base pairs 821 to 2,068) ( ) and synthesized by Geneart. .. The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen).

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    Article Snippet: For pET28a-soNadV plasmid, for technical reasons a restriction site for NcoI enzyme (5′C▼ CATGG3′) was added, assuming no effect on the enzyme activity. pUC57-Kan-prs plasmid was generated by ligation of Prs gene sequence (Gene ID: ) with L135I mutation (CTC to ATA) synthesized by GenScript and ligated into NcoI/XbaI restriction sites of pUC57-Kan plasmid. .. 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. ).

    Autoradiography:

    Article Title: The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism
    Article Snippet: After 22 amplification cycles, the reactions were separated into four aliquots for digestion with NcoI, PstI (New England Biolabs), both, or neither. .. The products were analyzed on a 5% native polyacrylamide gel, visualized by autoradiography, and quantitated on a FUJIFILM FLA-5100 phosphoirmager (Fuji Medical Systems) using Multi Gauge software Version 2.3 (Fujifilm).

    Construct:

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium
    Article Snippet: Plasmids were constructed using standard molecular cloning techniques. .. The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen).

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    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. ). .. Resulted bicistronic vector pET28a-baPrs-hdNadV was deposited at Addgene under the ID #91950.

    SYBR Green Assay:

    Article Title: Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology
    Article Snippet: Methylation-sensitive restriction enzyme-coupled qPCR assay Genomic DNA (200 ng) was digested at 37°C for 4 h with 10 U of the methylation-sensitive enzyme AciI , or NcoI (New England Biolabs) which does not have cutting sites in our regions of interest and served to normalize the data. .. The endonucleases were subsequently inactivated by incubation at 65°C for 20 min. Real-time PCR was carried out using the light cycler-DNA MasterPLUS SYBR Green I mix (Roche) supplemented with 0.5 μM specific primer pairs and with 2 μL of digested DNA.

    Incubation:

    Article Title: Mechanism of Action of a Distal NF-?B-Dependent Enhancer
    Article Snippet: To sequester the SDS, 2% Triton X-100 was added and the nuclei were incubated for 1 h at 37°C. .. The cross-linked DNA was digested overnight with 500 to 800 units NcoI restriction enzyme (New England Biolabs, Inc.).

    Article Title: Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology
    Article Snippet: Methylation-sensitive restriction enzyme-coupled qPCR assay Genomic DNA (200 ng) was digested at 37°C for 4 h with 10 U of the methylation-sensitive enzyme AciI , or NcoI (New England Biolabs) which does not have cutting sites in our regions of interest and served to normalize the data. .. The endonucleases were subsequently inactivated by incubation at 65°C for 20 min. Real-time PCR was carried out using the light cycler-DNA MasterPLUS SYBR Green I mix (Roche) supplemented with 0.5 μM specific primer pairs and with 2 μL of digested DNA.

    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription
    Article Snippet: PAGE analysis In order to analyze the transcription products of S -cdAand S -cdG using PAGE, a portion of the above RT-PCR fragments was treated with 10 U NcoI and 1 U shrimp alkaline phosphatase in 10 μL NEB buffer 3 at 37°C for 30 min, followed by heating at 80°C for 20 min to deactivate the phosphatase. .. To the reaction mixture was subsequently added 10 U AseI, and the solution was incubated at 37°C for 30 min, followed by quenching with 15 μL formamide gel loading buffer containing xylene cyanol FF and bromophenol blue dyes.

    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription
    Article Snippet: .. LC-MS/MS analysis In order to identify the transcription products of S -cdA and S -cdG using LC-MS/MS, RT-PCR products were treated with 50 U NcoI and 20 U shrimp alkaline phosphatase in 250 μL NEB buffer 3 at 37°C for 2 h, followed by heating at 80°C for 20 min. To the resulting solution was added 50 U of AseI, and the reaction mixture was incubated at 37°C for 1 h, followed by extraction once with phenol/chloroform/isoamyl alcohol (25:24:1, v/v). .. The aqueous portion was dried with Speed-vac, desalted with HPLC and dissolved in 12 μL water.

    Gel Extraction:

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems
    Article Snippet: .. T4 polynucleotide kinase (PNK), NotI, NcoI, T4 DNA ligase and accompanying buffers purchased from New England Biolabs 100 mM ATP 100 µM CRISPR target oligonucleotides (designed as in ) and pACYC-GFP Gel purification kit (e.g. Qiagen QIAquick Gel Extraction kit or Promega Wizard SV Gel and PCR Clean-Up System) One Shot TOP10 Competent Cells (Thermo-Fisher) or similar cloning E. coli strain Miniprep kit (Qiagen or Promega) ..

    Article Title: Cloning, Transformation and Expression of Human Interferon ?2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi)
    Article Snippet: So, this was digested with BstEII and NcoI (NEB Co). .. The digested PCR product was extracted from gel using gel extraction kit again and then ligated with digested pCAMBIA1304 vector by T4 DNA ligase in 16°C overnight and the results were transformed to E. coli.

    Article Title: Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo
    Article Snippet: Ethanol (Fisher Scientific, cat. no. BP2818) QIAquick gel extraction kit (Qiagen, cat. no. 28704) [γ-32 P]ATP (PerkinElmer, cat. no. NEG002H250UC) ! .. SfaNI (New England BioLabs, cat. no. R0172S) NcoI (New England BioLabs, cat. no. R0193S) NotI (New England BioLabs, cat. no. R0189S) T7 RNA polymerase, supplied with 5× transcription buffer (Promega, cat. no. P2075) 100 mM DTT (Promega, cat. no. P1171) 100 mM rATP (Promega, cat. no. E6011) 100 mM rUTP (Promega, cat. no. E6021) 100 mM rGTP (Promega, cat. no. E6031) 100 mM rCTP (Promega, cat. no. E6041) RNase inhibitor (New England BioLabs, cat. no. M0307S) HeLaScribe nuclear extract in vitro transcription system, supplied with HeLa nuclear extract, 1× transcription buffer and HeLa extract stop solution (Promega, cat. no. E3110) pGEM-T vector (Promega, cat. no. A3600) Lipofectamine 2000 transfection reagent (Invitrogen, cat. no. 11668-019) DMEM (Life Technologies, cat. no. 11995-073) FBS (Life Technologies, cat. no. 16000-044) Penicillin-streptomycin solution (American Type Culture Collection, cat. no. 30-2300) 0.25% (wt/vol) trypsin-EDTA (Life Technologies, cat. no. 25200-056) 10× PBS solution (VWR, cat. no. 97064-158) E.Z.N.A.

    Activity Assay:

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    Article Snippet: For pET28a-soNadV plasmid, for technical reasons a restriction site for NcoI enzyme (5′C▼ CATGG3′) was added, assuming no effect on the enzyme activity. pUC57-Kan-prs plasmid was generated by ligation of Prs gene sequence (Gene ID: ) with L135I mutation (CTC to ATA) synthesized by GenScript and ligated into NcoI/XbaI restriction sites of pUC57-Kan plasmid. .. 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. ).

    Expressing:

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    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. ). .. Resulted bicistronic vector pET28a-baPrs-hdNadV was deposited at Addgene under the ID #91950.

    Modification:

    Article Title: Mechanism of Action of a Distal NF-?B-Dependent Enhancer
    Article Snippet: A modified version of the 3C assay, adapted for mammalian cells ( , ), was used. .. The cross-linked DNA was digested overnight with 500 to 800 units NcoI restriction enzyme (New England Biolabs, Inc.).

    Transformation Assay:

    Article Title: Codon Usage Optimization and Construction of Plasmid Encoding Iranian Human Papillomavirus Type 16 E7 Oncogene for Lactococcus Lactis Subsp. Cremoris MG1363
    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). .. The subsequent plasmid (named pNZ8148-HPV 16-opti E7) was transformed to chemically competent E. coli MC1061 cells (MoBiTec, Germany).

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium
    Article Snippet: .. The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen). .. Successful transformants were identified by colony PCR using the primers pNZ8048-F (5′-GCCCCGTTAGTTGAAGAAGG-3′) and pNZ8048-R (5′-CAATTGAACGTTTCAAGCCTTGG-3′) and further verified by sequencing analysis.

    Article Title: Cloning, Transformation and Expression of Human Interferon ?2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi)
    Article Snippet: So, this was digested with BstEII and NcoI (NEB Co). .. The digested PCR product was extracted from gel using gel extraction kit again and then ligated with digested pCAMBIA1304 vector by T4 DNA ligase in 16°C overnight and the results were transformed to E. coli.

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    Article Snippet: The transformed kanamycin (km) resistant bacteria were selected on agar plates. .. 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. ).

    Gel Purification:

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems
    Article Snippet: .. T4 polynucleotide kinase (PNK), NotI, NcoI, T4 DNA ligase and accompanying buffers purchased from New England Biolabs 100 mM ATP 100 µM CRISPR target oligonucleotides (designed as in ) and pACYC-GFP Gel purification kit (e.g. Qiagen QIAquick Gel Extraction kit or Promega Wizard SV Gel and PCR Clean-Up System) One Shot TOP10 Competent Cells (Thermo-Fisher) or similar cloning E. coli strain Miniprep kit (Qiagen or Promega) ..

    High Performance Liquid Chromatography:

    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription
    Article Snippet: LC-MS/MS analysis In order to identify the transcription products of S -cdA and S -cdG using LC-MS/MS, RT-PCR products were treated with 50 U NcoI and 20 U shrimp alkaline phosphatase in 250 μL NEB buffer 3 at 37°C for 2 h, followed by heating at 80°C for 20 min. To the resulting solution was added 50 U of AseI, and the reaction mixture was incubated at 37°C for 1 h, followed by extraction once with phenol/chloroform/isoamyl alcohol (25:24:1, v/v). .. The aqueous portion was dried with Speed-vac, desalted with HPLC and dissolved in 12 μL water.

    Transfection:

    Article Title: Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells
    Article Snippet: The restriction enzymes of NcoI and XhoI were purchased from NEB (Ipswich, MA, USA). .. Competent cell NovaBlue, pTriEx-1.1 Hygro vector, and GeneJuice® transfection reagent were supplied by Novagen (Billerica, MA, USA).

    Article Title: Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo
    Article Snippet: .. SfaNI (New England BioLabs, cat. no. R0172S) NcoI (New England BioLabs, cat. no. R0193S) NotI (New England BioLabs, cat. no. R0189S) T7 RNA polymerase, supplied with 5× transcription buffer (Promega, cat. no. P2075) 100 mM DTT (Promega, cat. no. P1171) 100 mM rATP (Promega, cat. no. E6011) 100 mM rUTP (Promega, cat. no. E6021) 100 mM rGTP (Promega, cat. no. E6031) 100 mM rCTP (Promega, cat. no. E6041) RNase inhibitor (New England BioLabs, cat. no. M0307S) HeLaScribe nuclear extract in vitro transcription system, supplied with HeLa nuclear extract, 1× transcription buffer and HeLa extract stop solution (Promega, cat. no. E3110) pGEM-T vector (Promega, cat. no. A3600) Lipofectamine 2000 transfection reagent (Invitrogen, cat. no. 11668-019) DMEM (Life Technologies, cat. no. 11995-073) FBS (Life Technologies, cat. no. 16000-044) Penicillin-streptomycin solution (American Type Culture Collection, cat. no. 30-2300) 0.25% (wt/vol) trypsin-EDTA (Life Technologies, cat. no. 25200-056) 10× PBS solution (VWR, cat. no. 97064-158) E.Z.N.A. .. Total RNA kit I (Omega Bio-Tek, cat. no. 101319-240) Ambion DNA-free kit (Life Technologies, cat. no. AM1906) M-MLV reverse transcriptase, supplied with 5× reaction buffer and HeLa extract stop solution (Promega, cat. no. E3110) MluCI (New England BioLabs, cat. no. R0538S) Cac8I (New England BioLabs, cat. no. R0579L) 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP; Oakwood Chemical, cat. no. 003409) Methanol (Fisher Scientific, cat. no. A452-SK4) Sodium chloride (EMD, cat. no. 7710) Sodium hydroxide (Fisher Scientific, cat. no. S318-10) Agarose (Sigma-Aldrich, cat. no. A9539) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Tris base (Fisher Scientific, cat. no. BP152-5) EDTA (Teknova, cat. no. E5599) Hydrochloric acid (Fisher Scientific, cat. no. A144-500) !

    Sequencing:

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium
    Article Snippet: The sequence was then amplified using primers chloride-F (5′-CGAATTGAAGGAAGGCCG-3′) and chloride-R (5′-GCAGTGAAAGGAAGGCC-3′). .. The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen).

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    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. ). .. Resulted bicistronic vector pET28a-baPrs-hdNadV was deposited at Addgene under the ID #91950.

    Ligation:

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium
    Article Snippet: .. The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen). .. Successful transformants were identified by colony PCR using the primers pNZ8048-F (5′-GCCCCGTTAGTTGAAGAAGG-3′) and pNZ8048-R (5′-CAATTGAACGTTTCAAGCCTTGG-3′) and further verified by sequencing analysis.

    Article Title: Cloning, Transformation and Expression of Human Interferon ?2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi)
    Article Snippet: So, this was digested with BstEII and NcoI (NEB Co). .. Consequently, the INFα2b fragment was replaced in GUS- GFP region of pCAMBIA1304 under the control of the CaMV35s promoter and the NOS terminator by ligation process ( ).

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    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. ). .. Resulted bicistronic vector pET28a-baPrs-hdNadV was deposited at Addgene under the ID #91950.

    Generated:

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    Article Snippet: For pET28a-soNadV plasmid, for technical reasons a restriction site for NcoI enzyme (5′C▼ CATGG3′) was added, assuming no effect on the enzyme activity. pUC57-Kan-prs plasmid was generated by ligation of Prs gene sequence (Gene ID: ) with L135I mutation (CTC to ATA) synthesized by GenScript and ligated into NcoI/XbaI restriction sites of pUC57-Kan plasmid. .. 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. ).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells
    Article Snippet: Materials and reagents SV Total RNA Isolation System, Access reverse transcription polymerase chain reaction (RT-PCR) System, and T4 DNA ligase were purchased from Promega (Madison, WI, USA). .. The restriction enzymes of NcoI and XhoI were purchased from NEB (Ipswich, MA, USA).

    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription
    Article Snippet: .. PAGE analysis In order to analyze the transcription products of S -cdAand S -cdG using PAGE, a portion of the above RT-PCR fragments was treated with 10 U NcoI and 1 U shrimp alkaline phosphatase in 10 μL NEB buffer 3 at 37°C for 30 min, followed by heating at 80°C for 20 min to deactivate the phosphatase. ..

    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription
    Article Snippet: .. LC-MS/MS analysis In order to identify the transcription products of S -cdA and S -cdG using LC-MS/MS, RT-PCR products were treated with 50 U NcoI and 20 U shrimp alkaline phosphatase in 250 μL NEB buffer 3 at 37°C for 2 h, followed by heating at 80°C for 20 min. To the resulting solution was added 50 U of AseI, and the reaction mixture was incubated at 37°C for 1 h, followed by extraction once with phenol/chloroform/isoamyl alcohol (25:24:1, v/v). .. The aqueous portion was dried with Speed-vac, desalted with HPLC and dissolved in 12 μL water.

    Article Title: The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism
    Article Snippet: Paragraph title: RT-PCR Assays. ... After 22 amplification cycles, the reactions were separated into four aliquots for digestion with NcoI, PstI (New England Biolabs), both, or neither.

    Recombinant:

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    Article Snippet: Recombinant Nicotinamide phosphoribosyltransferase (Nampt) gene was cloned in pET-28a(+) expression vector from Novagen which permits protein expression under the control of T7 lac promoter . .. 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. ).

    Molecular Cloning:

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium
    Article Snippet: Plasmids were constructed using standard molecular cloning techniques. .. The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen).

    Methylation:

    Article Title: Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology
    Article Snippet: .. Methylation-sensitive restriction enzyme-coupled qPCR assay Genomic DNA (200 ng) was digested at 37°C for 4 h with 10 U of the methylation-sensitive enzyme AciI , or NcoI (New England Biolabs) which does not have cutting sites in our regions of interest and served to normalize the data. .. The endonucleases were subsequently inactivated by incubation at 65°C for 20 min. Real-time PCR was carried out using the light cycler-DNA MasterPLUS SYBR Green I mix (Roche) supplemented with 0.5 μM specific primer pairs and with 2 μL of digested DNA.

    Mutagenesis:

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    Article Snippet: For pET28a-soNadV plasmid, for technical reasons a restriction site for NcoI enzyme (5′C▼ CATGG3′) was added, assuming no effect on the enzyme activity. pUC57-Kan-prs plasmid was generated by ligation of Prs gene sequence (Gene ID: ) with L135I mutation (CTC to ATA) synthesized by GenScript and ligated into NcoI/XbaI restriction sites of pUC57-Kan plasmid. .. 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. ).

    Isolation:

    Article Title: Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells
    Article Snippet: Materials and reagents SV Total RNA Isolation System, Access reverse transcription polymerase chain reaction (RT-PCR) System, and T4 DNA ligase were purchased from Promega (Madison, WI, USA). .. The restriction enzymes of NcoI and XhoI were purchased from NEB (Ipswich, MA, USA).

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium
    Article Snippet: The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen). .. The resulting plasmid, pNZC, was isolated from E. coli using a QIAprep Miniprep kit (Qiagen) and then transformed into electrocompetent L. lactis NZ9000 ( ).

    Size-exclusion Chromatography:

    Article Title: Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology
    Article Snippet: Methylation-sensitive restriction enzyme-coupled qPCR assay Genomic DNA (200 ng) was digested at 37°C for 4 h with 10 U of the methylation-sensitive enzyme AciI , or NcoI (New England Biolabs) which does not have cutting sites in our regions of interest and served to normalize the data. .. Real-time quantification PCR were run on a light cycler rapid thermal system (LightCycler®480 2.0 Real time PCR system, Roche) with 20 sec of denaturation at 95°C, 20 sec of annealing at 65°C and 20 sec of extension at 72°C for all primers, and analyzed by the comparative CT (∆CT) method according to the formula: methylation (%) = E(∆CT) × 100 where E represents PCR efficiency and ∆CT = CTsample (AciI digest) - CTsample (NcoI digest).

    Purification:

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    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. ). .. Resulted bicistronic vector pET28a-baPrs-hdNadV was deposited at Addgene under the ID #91950.

    Polymerase Chain Reaction:

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems
    Article Snippet: .. T4 polynucleotide kinase (PNK), NotI, NcoI, T4 DNA ligase and accompanying buffers purchased from New England Biolabs 100 mM ATP 100 µM CRISPR target oligonucleotides (designed as in ) and pACYC-GFP Gel purification kit (e.g. Qiagen QIAquick Gel Extraction kit or Promega Wizard SV Gel and PCR Clean-Up System) One Shot TOP10 Competent Cells (Thermo-Fisher) or similar cloning E. coli strain Miniprep kit (Qiagen or Promega) ..

    Article Title: Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology
    Article Snippet: Methylation-sensitive restriction enzyme-coupled qPCR assay Genomic DNA (200 ng) was digested at 37°C for 4 h with 10 U of the methylation-sensitive enzyme AciI , or NcoI (New England Biolabs) which does not have cutting sites in our regions of interest and served to normalize the data. .. Real-time quantification PCR were run on a light cycler rapid thermal system (LightCycler®480 2.0 Real time PCR system, Roche) with 20 sec of denaturation at 95°C, 20 sec of annealing at 65°C and 20 sec of extension at 72°C for all primers, and analyzed by the comparative CT (∆CT) method according to the formula: methylation (%) = E(∆CT) × 100 where E represents PCR efficiency and ∆CT = CTsample (AciI digest) - CTsample (NcoI digest).

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium
    Article Snippet: .. The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen). .. Successful transformants were identified by colony PCR using the primers pNZ8048-F (5′-GCCCCGTTAGTTGAAGAAGG-3′) and pNZ8048-R (5′-CAATTGAACGTTTCAAGCCTTGG-3′) and further verified by sequencing analysis.

    Article Title: Cloning, Transformation and Expression of Human Interferon ?2b Gene in Tobacco Plant (Nicotiana tabacum cv. xanthi)
    Article Snippet: The gene was amplified using mentioned primers, and then, PCR product was extracted from the gel using QIAGEN kit. .. So, this was digested with BstEII and NcoI (NEB Co).

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    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. ). .. Resulted bicistronic vector pET28a-baPrs-hdNadV was deposited at Addgene under the ID #91950.

    Article Title: The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism
    Article Snippet: Semiquantitative PCR using AmpliTaq polymerase (Applied Biosystems) was performed by including [ α -32 P]-dCTP in the reactions. .. After 22 amplification cycles, the reactions were separated into four aliquots for digestion with NcoI, PstI (New England Biolabs), both, or neither.

    Polyacrylamide Gel Electrophoresis:

    Article Title: A Quantitative Assay for Assessing the Effects of DNA Lesions on Transcription
    Article Snippet: .. PAGE analysis In order to analyze the transcription products of S -cdAand S -cdG using PAGE, a portion of the above RT-PCR fragments was treated with 10 U NcoI and 1 U shrimp alkaline phosphatase in 10 μL NEB buffer 3 at 37°C for 30 min, followed by heating at 80°C for 20 min to deactivate the phosphatase. ..

    CRISPR:

    Article Title: Fluorescence-based methods for measuring target interference by CRISPR-Cas systems
    Article Snippet: .. T4 polynucleotide kinase (PNK), NotI, NcoI, T4 DNA ligase and accompanying buffers purchased from New England Biolabs 100 mM ATP 100 µM CRISPR target oligonucleotides (designed as in ) and pACYC-GFP Gel purification kit (e.g. Qiagen QIAquick Gel Extraction kit or Promega Wizard SV Gel and PCR Clean-Up System) One Shot TOP10 Competent Cells (Thermo-Fisher) or similar cloning E. coli strain Miniprep kit (Qiagen or Promega) ..

    Plasmid Preparation:

    Article Title: Codon Usage Optimization and Construction of Plasmid Encoding Iranian Human Papillomavirus Type 16 E7 Oncogene for Lactococcus Lactis Subsp. Cremoris MG1363
    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). .. The resultant DNA fragment was ligated into the NcoI/SacI site of pNZ8148 shuttle vector (MoBiTec, Germany).

    Article Title: Multiresidue Method for Analysis of β Agonists in Swine Urine by Enzyme Linked Receptor Assay Based on β2 Adrenergic Receptor Expressed in HEK293 Cells
    Article Snippet: The restriction enzymes of NcoI and XhoI were purchased from NEB (Ipswich, MA, USA). .. Competent cell NovaBlue, pTriEx-1.1 Hygro vector, and GeneJuice® transfection reagent were supplied by Novagen (Billerica, MA, USA).

    Article Title: Chloride-Inducible Expression Vector for Delivery of Antimicrobial Peptides Targeting Antibiotic-Resistant Enterococcus faecium
    Article Snippet: .. The PCR fragment and plasmid pNZ8048 were both digested with restriction enzymes BglII and NcoI (New England BioLabs) and ligated at 16°C for 16 h. The resulting ligation was then transformed into electrocompetent E. coli MC1061 F′ (Lucigen). .. Successful transformants were identified by colony PCR using the primers pNZ8048-F (5′-GCCCCGTTAGTTGAAGAAGG-3′) and pNZ8048-R (5′-CAATTGAACGTTTCAAGCCTTGG-3′) and further verified by sequencing analysis.

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    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. ). .. Resulted bicistronic vector pET28a-baPrs-hdNadV was deposited at Addgene under the ID #91950.

    Article Title: Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo
    Article Snippet: .. SfaNI (New England BioLabs, cat. no. R0172S) NcoI (New England BioLabs, cat. no. R0193S) NotI (New England BioLabs, cat. no. R0189S) T7 RNA polymerase, supplied with 5× transcription buffer (Promega, cat. no. P2075) 100 mM DTT (Promega, cat. no. P1171) 100 mM rATP (Promega, cat. no. E6011) 100 mM rUTP (Promega, cat. no. E6021) 100 mM rGTP (Promega, cat. no. E6031) 100 mM rCTP (Promega, cat. no. E6041) RNase inhibitor (New England BioLabs, cat. no. M0307S) HeLaScribe nuclear extract in vitro transcription system, supplied with HeLa nuclear extract, 1× transcription buffer and HeLa extract stop solution (Promega, cat. no. E3110) pGEM-T vector (Promega, cat. no. A3600) Lipofectamine 2000 transfection reagent (Invitrogen, cat. no. 11668-019) DMEM (Life Technologies, cat. no. 11995-073) FBS (Life Technologies, cat. no. 16000-044) Penicillin-streptomycin solution (American Type Culture Collection, cat. no. 30-2300) 0.25% (wt/vol) trypsin-EDTA (Life Technologies, cat. no. 25200-056) 10× PBS solution (VWR, cat. no. 97064-158) E.Z.N.A. .. Total RNA kit I (Omega Bio-Tek, cat. no. 101319-240) Ambion DNA-free kit (Life Technologies, cat. no. AM1906) M-MLV reverse transcriptase, supplied with 5× reaction buffer and HeLa extract stop solution (Promega, cat. no. E3110) MluCI (New England BioLabs, cat. no. R0538S) Cac8I (New England BioLabs, cat. no. R0579L) 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP; Oakwood Chemical, cat. no. 003409) Methanol (Fisher Scientific, cat. no. A452-SK4) Sodium chloride (EMD, cat. no. 7710) Sodium hydroxide (Fisher Scientific, cat. no. S318-10) Agarose (Sigma-Aldrich, cat. no. A9539) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Tris base (Fisher Scientific, cat. no. BP152-5) EDTA (Teknova, cat. no. E5599) Hydrochloric acid (Fisher Scientific, cat. no. A144-500) !

    Software:

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    Article Snippet: Three such plasmids, carrying expression DNA corresponding to each of the amino acid sequences compared in Fig. were produced as follows: Nampt-PET28a vector containing Nampt gene from Mus musculus strain C57BL/6 J, a gift from dr. Cynthia Wolberger (Addgene plasmid # 25630), was cloned in E . coli DH5α and maintained (as a glycerol stock) at −80 °C. pET28a-hdNadV and pET28a-soNadV plasmids carrying the nucleotide sequence corresponding to nadV gene from Haemophilus ducreyi (strain: ATCC 27722), respectively Shewanella oneidensis (strain: MR-1) were constructed as follows: The gene sequences were selected from the GenBank database (Gene ID: 2716561 respectively 1169740), optimized for expression in E . coli (using Genome Compiler software v. 2.2.55 (Genome Compiler Corporation 2015), considering the frequency of codon usage and GC content, eliminating unwanted restriction sequences), synthesized de novo (GenScript) and ligated into pET-28a(+) vector which was first double digested with NcoI and XhoI restriction enzymes. .. 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. ).

    Article Title: The alternative splicing repressors hnRNP A1/A2 and PTB influence pyruvate kinase isoform expression and cell metabolism
    Article Snippet: After 22 amplification cycles, the reactions were separated into four aliquots for digestion with NcoI, PstI (New England Biolabs), both, or neither. .. The products were analyzed on a 5% native polyacrylamide gel, visualized by autoradiography, and quantitated on a FUJIFILM FLA-5100 phosphoirmager (Fuji Medical Systems) using Multi Gauge software Version 2.3 (Fujifilm).

    Real-time Polymerase Chain Reaction:

    Article Title: Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology
    Article Snippet: .. Methylation-sensitive restriction enzyme-coupled qPCR assay Genomic DNA (200 ng) was digested at 37°C for 4 h with 10 U of the methylation-sensitive enzyme AciI , or NcoI (New England Biolabs) which does not have cutting sites in our regions of interest and served to normalize the data. .. The endonucleases were subsequently inactivated by incubation at 65°C for 20 min. Real-time PCR was carried out using the light cycler-DNA MasterPLUS SYBR Green I mix (Roche) supplemented with 0.5 μM specific primer pairs and with 2 μL of digested DNA.

    Positron Emission Tomography:

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    Article Snippet: Three such plasmids, carrying expression DNA corresponding to each of the amino acid sequences compared in Fig. were produced as follows: Nampt-PET28a vector containing Nampt gene from Mus musculus strain C57BL/6 J, a gift from dr. Cynthia Wolberger (Addgene plasmid # 25630), was cloned in E . coli DH5α and maintained (as a glycerol stock) at −80 °C. pET28a-hdNadV and pET28a-soNadV plasmids carrying the nucleotide sequence corresponding to nadV gene from Haemophilus ducreyi (strain: ATCC 27722), respectively Shewanella oneidensis (strain: MR-1) were constructed as follows: The gene sequences were selected from the GenBank database (Gene ID: 2716561 respectively 1169740), optimized for expression in E . coli (using Genome Compiler software v. 2.2.55 (Genome Compiler Corporation 2015), considering the frequency of codon usage and GC content, eliminating unwanted restriction sequences), synthesized de novo (GenScript) and ligated into pET-28a(+) vector which was first double digested with NcoI and XhoI restriction enzymes. .. 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. ).

    Agarose Gel Electrophoresis:

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    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. ). .. Resulted bicistronic vector pET28a-baPrs-hdNadV was deposited at Addgene under the ID #91950.

    In Vitro:

    Article Title: Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo
    Article Snippet: .. SfaNI (New England BioLabs, cat. no. R0172S) NcoI (New England BioLabs, cat. no. R0193S) NotI (New England BioLabs, cat. no. R0189S) T7 RNA polymerase, supplied with 5× transcription buffer (Promega, cat. no. P2075) 100 mM DTT (Promega, cat. no. P1171) 100 mM rATP (Promega, cat. no. E6011) 100 mM rUTP (Promega, cat. no. E6021) 100 mM rGTP (Promega, cat. no. E6031) 100 mM rCTP (Promega, cat. no. E6041) RNase inhibitor (New England BioLabs, cat. no. M0307S) HeLaScribe nuclear extract in vitro transcription system, supplied with HeLa nuclear extract, 1× transcription buffer and HeLa extract stop solution (Promega, cat. no. E3110) pGEM-T vector (Promega, cat. no. A3600) Lipofectamine 2000 transfection reagent (Invitrogen, cat. no. 11668-019) DMEM (Life Technologies, cat. no. 11995-073) FBS (Life Technologies, cat. no. 16000-044) Penicillin-streptomycin solution (American Type Culture Collection, cat. no. 30-2300) 0.25% (wt/vol) trypsin-EDTA (Life Technologies, cat. no. 25200-056) 10× PBS solution (VWR, cat. no. 97064-158) E.Z.N.A. .. Total RNA kit I (Omega Bio-Tek, cat. no. 101319-240) Ambion DNA-free kit (Life Technologies, cat. no. AM1906) M-MLV reverse transcriptase, supplied with 5× reaction buffer and HeLa extract stop solution (Promega, cat. no. E3110) MluCI (New England BioLabs, cat. no. R0538S) Cac8I (New England BioLabs, cat. no. R0579L) 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP; Oakwood Chemical, cat. no. 003409) Methanol (Fisher Scientific, cat. no. A452-SK4) Sodium chloride (EMD, cat. no. 7710) Sodium hydroxide (Fisher Scientific, cat. no. S318-10) Agarose (Sigma-Aldrich, cat. no. A9539) Sodium acetate (Sigma-Aldrich, cat. no. S2889) Tris base (Fisher Scientific, cat. no. BP152-5) EDTA (Teknova, cat. no. E5599) Hydrochloric acid (Fisher Scientific, cat. no. A144-500) !

    Produced:

    Article Title: β-nicotinamide mononucleotide (NMN) production in Escherichia coli
    Article Snippet: Three such plasmids, carrying expression DNA corresponding to each of the amino acid sequences compared in Fig. were produced as follows: Nampt-PET28a vector containing Nampt gene from Mus musculus strain C57BL/6 J, a gift from dr. Cynthia Wolberger (Addgene plasmid # 25630), was cloned in E . coli DH5α and maintained (as a glycerol stock) at −80 °C. pET28a-hdNadV and pET28a-soNadV plasmids carrying the nucleotide sequence corresponding to nadV gene from Haemophilus ducreyi (strain: ATCC 27722), respectively Shewanella oneidensis (strain: MR-1) were constructed as follows: The gene sequences were selected from the GenBank database (Gene ID: 2716561 respectively 1169740), optimized for expression in E . coli (using Genome Compiler software v. 2.2.55 (Genome Compiler Corporation 2015), considering the frequency of codon usage and GC content, eliminating unwanted restriction sequences), synthesized de novo (GenScript) and ligated into pET-28a(+) vector which was first double digested with NcoI and XhoI restriction enzymes. .. 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. ).

    Lysis:

    Article Title: Mechanism of Action of a Distal NF-?B-Dependent Enhancer
    Article Snippet: Cells were lysed in cold lysis buffer (0.34 M sucrose, 10 mM Tris, 10 mM NaCl, 1% NP-40) containing protease inhibitors, and the nuclei were collected. .. The cross-linked DNA was digested overnight with 500 to 800 units NcoI restriction enzyme (New England Biolabs, Inc.).

    Hood:

    Article Title: Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo
    Article Snippet: CAUTION Phenol:chloroform:isoamyl alcohol is toxic and corrosive; wear gloves and work in a fume hood. .. SfaNI (New England BioLabs, cat. no. R0172S) NcoI (New England BioLabs, cat. no. R0193S) NotI (New England BioLabs, cat. no. R0189S) T7 RNA polymerase, supplied with 5× transcription buffer (Promega, cat. no. P2075) 100 mM DTT (Promega, cat. no. P1171) 100 mM rATP (Promega, cat. no. E6011) 100 mM rUTP (Promega, cat. no. E6021) 100 mM rGTP (Promega, cat. no. E6031) 100 mM rCTP (Promega, cat. no. E6041) RNase inhibitor (New England BioLabs, cat. no. M0307S) HeLaScribe nuclear extract in vitro transcription system, supplied with HeLa nuclear extract, 1× transcription buffer and HeLa extract stop solution (Promega, cat. no. E3110) pGEM-T vector (Promega, cat. no. A3600) Lipofectamine 2000 transfection reagent (Invitrogen, cat. no. 11668-019) DMEM (Life Technologies, cat. no. 11995-073) FBS (Life Technologies, cat. no. 16000-044) Penicillin-streptomycin solution (American Type Culture Collection, cat. no. 30-2300) 0.25% (wt/vol) trypsin-EDTA (Life Technologies, cat. no. 25200-056) 10× PBS solution (VWR, cat. no. 97064-158) E.Z.N.A.

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    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: 90/100, based on 230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.00271-06

    Figure Lengend 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.

    Article Snippet: The cross-linked DNA was digested overnight with 500 to 800 units NcoI restriction enzyme (New England Biolabs, Inc.).

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

    PAGE analysis for determining the effects of DNA lesions on transcription. ( a ) Sample processing for restriction digestion using NcoI and SfaNI and postlabeling assay (p* indicates a 32 P-labeled phosphate group). The recognition sequences for restriction

    Journal: Nature protocols

    Article Title: Quantitative measurement of transcriptional inhibition and mutagenesis induced by site-specifically incorporated DNA lesions in vitro and in vivo

    doi: 10.1038/nprot.2015.094

    Figure Lengend Snippet: PAGE analysis for determining the effects of DNA lesions on transcription. ( a ) Sample processing for restriction digestion using NcoI and SfaNI and postlabeling assay (p* indicates a 32 P-labeled phosphate group). The recognition sequences for restriction

    Article Snippet: SfaNI (New England BioLabs, cat. no. R0172S) NcoI (New England BioLabs, cat. no. R0193S) NotI (New England BioLabs, cat. no. R0189S) T7 RNA polymerase, supplied with 5× transcription buffer (Promega, cat. no. P2075) 100 mM DTT (Promega, cat. no. P1171) 100 mM rATP (Promega, cat. no. E6011) 100 mM rUTP (Promega, cat. no. E6021) 100 mM rGTP (Promega, cat. no. E6031) 100 mM rCTP (Promega, cat. no. E6041) RNase inhibitor (New England BioLabs, cat. no. M0307S) HeLaScribe nuclear extract in vitro transcription system, supplied with HeLa nuclear extract, 1× transcription buffer and HeLa extract stop solution (Promega, cat. no. E3110) pGEM-T vector (Promega, cat. no. A3600) Lipofectamine 2000 transfection reagent (Invitrogen, cat. no. 11668-019) DMEM (Life Technologies, cat. no. 11995-073) FBS (Life Technologies, cat. no. 16000-044) Penicillin-streptomycin solution (American Type Culture Collection, cat. no. 30-2300) 0.25% (wt/vol) trypsin-EDTA (Life Technologies, cat. no. 25200-056) 10× PBS solution (VWR, cat. no. 97064-158) E.Z.N.A.

    Techniques: Polyacrylamide Gel Electrophoresis, Postlabeling Assay, Labeling