endonuclease iii  (New England Biolabs)


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    Name:
    Endonuclease III Nth
    Description:
    Endonuclease III Nth 1 000 units
    Catalog Number:
    m0268s
    Price:
    72
    Size:
    1 000 units
    Category:
    Other Endonucleases
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    Structured Review

    New England Biolabs endonuclease iii
    Endonuclease III Nth
    Endonuclease III Nth 1 000 units
    https://www.bioz.com/result/endonuclease iii/product/New England Biolabs
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    endonuclease iii - by Bioz Stars, 2020-01
    90/100 stars

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    Related Articles

    Amplification:

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: .. For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. DNA fragments with a gap have slower mobility on 6% native PAGE; and this was used to monitor completion of digestion.

    Article Title: Defective Repair of Oxidative Base Lesions by the DNA Glycosylase Nth1 Associates with Multiple Telomere Defects
    Article Snippet: 400 ng of duplex oligomer or genomic DNA were incubated overnight with 12 units of Endonuclease III (New England Biolabs) in 1× NEB Endonuclease III buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM Dithiothreitol, pH 8.0). .. All samples were set up on ice, then incubated at 37°C overnight to allow complete digestion and followed by the quantitative Real-Time amplification (qPCR) .

    Positive Control:

    Article Title: Antimicrobial photodynamic therapy with fulleropyrrolidine: photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies
    Article Snippet: As a positive control, S. aureus was treated with 100 μM TMPyP and illuminated with 40 J/cm2 of red light (630 nm). .. Post irradiation, the genomic DNA (10 ng) was extracted from the S. aureus cells using Extract ME DNA Bacteria purification kit (Blirt S.A.) and treated with endonuclease III (New England, BioLabs).

    Real-time Polymerase Chain Reaction:

    Article Title: NEIL1 and NEIL2 DNA glycosylases protect neural crest development against mitochondrial oxidative stress
    Article Snippet: Quantitative real time PCR as described above was used to calculate the enrichment of mtDNA over gDNA by the ΔΔCp method ( ). .. After phenol/chloroform extraction, DNA was further incubated with 20 units Endonuclease III (Nth, NEB, M0268) in a 50 µl reaction volume for 2 hr at 37°C, phenol/chloroform extracted, ethanol precipitated and resuspended in H2 O supplemented with 40 µM BHT and DFOM.

    Article Title: Defective Repair of Oxidative Base Lesions by the DNA Glycosylase Nth1 Associates with Multiple Telomere Defects
    Article Snippet: 400 ng of duplex oligomer or genomic DNA were incubated overnight with 12 units of Endonuclease III (New England Biolabs) in 1× NEB Endonuclease III buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM Dithiothreitol, pH 8.0). .. All samples were set up on ice, then incubated at 37°C overnight to allow complete digestion and followed by the quantitative Real-Time amplification (qPCR) .

    Incubation:

    Article Title: Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets
    Article Snippet: The cleavage of the target strand was performed with 1 M NaOH incubated for 20 min at 95°C. .. For comparison of the AP-cleaving enzymes, 0.05 U/μl UDG (Fermentas) was combined pair wise with 0.05 U/μl of the following AP-cleaving enzymes: Ape 1 (New England Biolabs), Endonuclease III (New England Biolabs), Endonuclease IV (Fermentas), Endonuclease V (Fermentas), Exonuclease III (Fermentas) and Fpg (New England Biolabs).

    Article Title: NEIL1 and NEIL2 DNA glycosylases protect neural crest development against mitochondrial oxidative stress
    Article Snippet: .. After phenol/chloroform extraction, DNA was further incubated with 20 units Endonuclease III (Nth, NEB, M0268) in a 50 µl reaction volume for 2 hr at 37°C, phenol/chloroform extracted, ethanol precipitated and resuspended in H2 O supplemented with 40 µM BHT and DFOM. ..

    Article Title: Defective Repair of Oxidative Base Lesions by the DNA Glycosylase Nth1 Associates with Multiple Telomere Defects
    Article Snippet: .. 400 ng of duplex oligomer or genomic DNA were incubated overnight with 12 units of Endonuclease III (New England Biolabs) in 1× NEB Endonuclease III buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM Dithiothreitol, pH 8.0). .. A duplicate digestion was also set up for each corresponding sample in mock digestion buffer (i.e. enzyme was excluded and substituted with H2 O).

    Article Title: Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Repairs DNA Damage Induced by Topoisomerases I and II and Base Alkylation in Vertebrate Cells
    Article Snippet: .. Annealed DNA was incubated with uracil-DNA glycosylase for 1 h at 37 °C, and then Endonuclease III (New England BioLabs) was added for 1 h at 37 °C to generate the 3′-dRP at a nicked DNA site. .. Unincorporated radioactive nucleotides were removed using a mini Quick Spin Oligo column (Roche Diagnostics).

    Article Title:
    Article Snippet: The quantitated total cellular DNA was heated to 70 °C for 15 min, cooled to room temperature for 10 min, and treated for 30 min with 5 units/μg endonuclease III and 5 units/μg formamidopyrimidine glycosylase (New England Biolabs). .. Then, NaOH was added to a final concentration of 0.1 n , and the samples were incubated at 37 °C for 20 min. Alkaline loading dye (10 m m EDTA, 25% Ficoll, 0.25% bromcresol purple, 500 m m NaOH in H2 O) was added to each sample, and the samples were loaded on a 0.6% alkaline-agarose gel (0.6% agarose, 1 m m EDTA, pH 8.0, 30 m m NaOH) in alkaline running buffer (1 m m EDTA, pH 8.0, 45 m m NaOH).

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase
    Article Snippet: .. Afterwards, 1μg of TcNTH1 native recombinant protein was incubated with 2 pg of the oligo substrate in Endonuclease III (Endo III) buffer solution (New England Biolabs, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM DTT) for 1 h at 37°C. ..

    Single Cell Gel Electrophoresis:

    Article Title: DNA damage and repair in Fuchs endothelial corneal dystrophy
    Article Snippet: .. To assess the level of oxidative DNA damage, the modified comet assay with human 8-oxoguanine DNA glycosylase (hOGG1; New England Biolabs, Ipswich, MA, USA) or Endonuclease III (EndoIII; New England Biolabs, Ipswich, MA, USA) was conducted according to the procedure described earlier [ ]. hOGG1 recognizes and removes mainly 7,8-dihydro-8-oxoguanine (8-oxoguanine) when paired with cytosine, 8-oxoadenine when paired with cytosine, foramidopyrimidine (fapy)-guanine and methy-fapy-guanine [ , ]. ..

    Modification:

    Article Title: DNA damage and repair in Fuchs endothelial corneal dystrophy
    Article Snippet: .. To assess the level of oxidative DNA damage, the modified comet assay with human 8-oxoguanine DNA glycosylase (hOGG1; New England Biolabs, Ipswich, MA, USA) or Endonuclease III (EndoIII; New England Biolabs, Ipswich, MA, USA) was conducted according to the procedure described earlier [ ]. hOGG1 recognizes and removes mainly 7,8-dihydro-8-oxoguanine (8-oxoguanine) when paired with cytosine, 8-oxoadenine when paired with cytosine, foramidopyrimidine (fapy)-guanine and methy-fapy-guanine [ , ]. ..

    Hybridization:

    Article Title: Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets
    Article Snippet: Paragraph title: Hybridization of oligonucleotides and site-specific cleavage of mismatch with MutY glycosylase and AP-cleaving enzyme ... For comparison of the AP-cleaving enzymes, 0.05 U/μl UDG (Fermentas) was combined pair wise with 0.05 U/μl of the following AP-cleaving enzymes: Ape 1 (New England Biolabs), Endonuclease III (New England Biolabs), Endonuclease IV (Fermentas), Endonuclease V (Fermentas), Exonuclease III (Fermentas) and Fpg (New England Biolabs).

    Southern Blot:

    Article Title:
    Article Snippet: Paragraph title: DNA Isolation and Quantitative Southern Blot Analysis ... The quantitated total cellular DNA was heated to 70 °C for 15 min, cooled to room temperature for 10 min, and treated for 30 min with 5 units/μg endonuclease III and 5 units/μg formamidopyrimidine glycosylase (New England Biolabs).

    Generated:

    Article Title: Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Repairs DNA Damage Induced by Topoisomerases I and II and Base Alkylation in Vertebrate Cells
    Article Snippet: Double-stranded OH40/36 and P40/36 were generated in the same manner. .. Annealed DNA was incubated with uracil-DNA glycosylase for 1 h at 37 °C, and then Endonuclease III (New England BioLabs) was added for 1 h at 37 °C to generate the 3′-dRP at a nicked DNA site.

    Article Title: HMGA2 exhibits dRP/AP site cleavage activity and protects cancer cells from DNA-damage-induced cytotoxicity during chemotherapy
    Article Snippet: The nature of the 5′ and 3′ termini generated by HMGA2 on DNA containing an AP site was determined by comparing the electrophoretic mobility of products generated by HMGA2 with those created by E. coli enzymes Endo III, Endo IV, or Fpg. .. For control reactions, eight units of Endonuclease III and FPG (NEB) was used.

    Article Title: Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields
    Article Snippet: .. Endonuclease III and VIII (New England Biolabs, Ipswich, MA) digestion creates a strand break (nicked plasmid) at Apurinic/apyrimidinic (AP) abasic sites (AP sites are generated by various intracellular free radicals); .. RNase H (New England Biolabs, Ipswich, MA) digestion of plasmid-encoded RNA ( e.g. RNAII-derived primer) resulted in a nicked plasmid; and

    other:

    Article Title: Chemical repair activity of free radical scavenger edaravone: reduction reactions with dGMP hydroxyl radical adducts and suppression of base lesions and AP sites on irradiated plasmid DNA
    Article Snippet: Formamidopyrimidine-DNA glycosylase (Fpg), endonuclease III (Nth), endonuclease IV (Nfo), and reaction buffer solutions used for the treatment of these enzymes were purchased from New England BioLabs Inc. (Ipswich, MA).

    Article Title: Genotoxicity, cytotoxicity and gene expression in patients undergoing elective surgery under isoflurane anaesthesia
    Article Snippet: Ethidium bromide, HEPES and bovine serum albumin were purchased from Sigma (St Louis, MO, USA); normal and low melting point agaroses, EDTA and Tris from Invitrogen (Carlsbad, CA, USA); hydrogen peroxide (H2 O2 ), H3 BO3 , NaCl, NaOH, KCl, HCl, NaHCO3 , KH2 PO4 and Na2 HPO4 from Merck (Germany); Ficoll–Paque® from GE (Sweden); dimethylsulfoxide from Mallinckrodt (Mexico); Triton X-100 from J. T. Baker (Phillipsburg, NJ, USA); annexin labelled with fluorescein isothiocyanate (FITC) and annexin V buffer, 7-amino-actinomycin D (7-AAD), phycoerythrin (PE)-labelled monoclonal antibodies anti-hCD4+ and anti-hCD8+ were purchased from Becton Dickinson (San Jose, CA, USA) and endonuclease III (endo III) and formamidopyrimidine DNA glycosylase (Fpg) from New England Biolabs (Ipswich, MA, USA).

    Sequencing:

    Article Title: AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis
    Article Snippet: The double-stranded 71AP substrate contained an abasic furanose analogue at position 36, the sequence was 5′-(HEX)-GAG CAG AGT CAG TGC TGC TAC GAG CGG ATC ACT AT F ATA GGT AGT TTA TCC TAC GAA CTC CGT CCG TAC CG-3′, where F is the abasic furanose analogue. .. Nicked DNA was produced by reacting 200 nM of 50AP DNA with 25 U of endonuclease III (New England Biolabs) in standard reaction buffer (20 mM Tris pH 7.5, 1 mM EDTA, 20 mM NaCl, and 0.1 mg/ml BSA) at 37°C for 90 min. Nuclease assays were performed in a standard reaction buffer supplemented with 15 mM MgCl2 at 25°C.

    Recombinant:

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. Recombinant core histones from yeast H2A, H2B, H2A.Z, and fly H3, H4 were purified following methods described earlier ( ; ).

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase
    Article Snippet: .. Afterwards, 1μg of TcNTH1 native recombinant protein was incubated with 2 pg of the oligo substrate in Endonuclease III (Endo III) buffer solution (New England Biolabs, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM DTT) for 1 h at 37°C. ..

    DNA Extraction:

    Article Title: Antimicrobial photodynamic therapy with fulleropyrrolidine: photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies
    Article Snippet: Paragraph title: Genomic DNA extraction and polymerase chain reaction ... Post irradiation, the genomic DNA (10 ng) was extracted from the S. aureus cells using Extract ME DNA Bacteria purification kit (Blirt S.A.) and treated with endonuclease III (New England, BioLabs).

    Article Title:
    Article Snippet: Paragraph title: DNA Isolation and Quantitative Southern Blot Analysis ... The quantitated total cellular DNA was heated to 70 °C for 15 min, cooled to room temperature for 10 min, and treated for 30 min with 5 units/μg endonuclease III and 5 units/μg formamidopyrimidine glycosylase (New England Biolabs).

    Isolation:

    Article Title: Defective Repair of Oxidative Base Lesions by the DNA Glycosylase Nth1 Associates with Multiple Telomere Defects
    Article Snippet: In brief, DNA was isolated from mouse kidney and primary MEFs by salting out , . .. 400 ng of duplex oligomer or genomic DNA were incubated overnight with 12 units of Endonuclease III (New England Biolabs) in 1× NEB Endonuclease III buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM Dithiothreitol, pH 8.0).

    Microscopy:

    Article Title: DNA damage and repair in Fuchs endothelial corneal dystrophy
    Article Snippet: To assess the level of oxidative DNA damage, the modified comet assay with human 8-oxoguanine DNA glycosylase (hOGG1; New England Biolabs, Ipswich, MA, USA) or Endonuclease III (EndoIII; New England Biolabs, Ipswich, MA, USA) was conducted according to the procedure described earlier [ ]. hOGG1 recognizes and removes mainly 7,8-dihydro-8-oxoguanine (8-oxoguanine) when paired with cytosine, 8-oxoadenine when paired with cytosine, foramidopyrimidine (fapy)-guanine and methy-fapy-guanine [ , ]. .. The cell pellets were gently mixed with 50 μl of 0.75 % low melting point agarose in PBS cooled to 37 °C and spread onto microscope slides precoated with 0.5 % normal melting point agarose (two gels per slide).

    Purification:

    Article Title: Fine-tuning recA expression in Staphylococcus aureus for antimicrobial photoinactivation: importance of photo-induced DNA damage in the photoinactivation mechanism
    Article Snippet: .. Post-irradiation, the genomic DNA (10 ng) was extracted from the S . aureus cells using the ExtractME DNA Bacteria purification kit (Blirt S.A., Poland) and treated with Endonuclease III (BioLabs, New England). ..

    Article Title: Antimicrobial photodynamic therapy with fulleropyrrolidine: photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies
    Article Snippet: .. Post irradiation, the genomic DNA (10 ng) was extracted from the S. aureus cells using Extract ME DNA Bacteria purification kit (Blirt S.A.) and treated with endonuclease III (New England, BioLabs). ..

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. Recombinant core histones from yeast H2A, H2B, H2A.Z, and fly H3, H4 were purified following methods described earlier ( ; ).

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase
    Article Snippet: 2.7 DNA glycosylase activity assay The bifunctional DNA glycosylase activity of the purified native recombinant TcNTH1 from E . coli and from T . cruzi epimastigotes as well as from epimastigote and trypomastigote homogenates was determined using a 32 mer synthetic DNA oligonucleotide containing a thymine glycol residue at position 18 (5’-CCGGTGCATGACACTGT(Tg)ACCTATCCTCAGCG-3’; where Tg indicates thymine glycol). .. Afterwards, 1μg of TcNTH1 native recombinant protein was incubated with 2 pg of the oligo substrate in Endonuclease III (Endo III) buffer solution (New England Biolabs, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM DTT) for 1 h at 37°C.

    Polymerase Chain Reaction:

    Article Title: Antimicrobial photodynamic therapy with fulleropyrrolidine: photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies
    Article Snippet: Paragraph title: Genomic DNA extraction and polymerase chain reaction ... Post irradiation, the genomic DNA (10 ng) was extracted from the S. aureus cells using Extract ME DNA Bacteria purification kit (Blirt S.A.) and treated with endonuclease III (New England, BioLabs).

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: .. For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. DNA fragments with a gap have slower mobility on 6% native PAGE; and this was used to monitor completion of digestion.

    Article Title: NEIL1 and NEIL2 DNA glycosylases protect neural crest development against mitochondrial oxidative stress
    Article Snippet: PCR mixture contained 1 ng of DNA and gDNA- and mtDNA-specific primers (mmActB and mmCytB, see for sequences). .. After phenol/chloroform extraction, DNA was further incubated with 20 units Endonuclease III (Nth, NEB, M0268) in a 50 µl reaction volume for 2 hr at 37°C, phenol/chloroform extracted, ethanol precipitated and resuspended in H2 O supplemented with 40 µM BHT and DFOM.

    Labeling:

    Article Title: Glycosylases and AP-cleaving enzymes as a general tool for probe-directed cleavage of ssDNA targets
    Article Snippet: Hybridization of oligonucleotides and site-specific cleavage of mismatch with MutY glycosylase and AP-cleaving enzyme Two nanomolar of labeled target (target_MutY for the MutY glycosylase test and target_AP for the AP test) and 4 nM of probe (probe_MutY for MutY glycosylase test and probe_AP for AP test) were hybridized in 1 × G/AP buffer (20 mM Tris–HCl, 30 mM NaCl, 1 mM EDTA, 100 mM KCl and 1 mM DTT) for 10 min at 65°C and then slowly cooled to room temperature. .. For comparison of the AP-cleaving enzymes, 0.05 U/μl UDG (Fermentas) was combined pair wise with 0.05 U/μl of the following AP-cleaving enzymes: Ape 1 (New England Biolabs), Endonuclease III (New England Biolabs), Endonuclease IV (Fermentas), Endonuclease V (Fermentas), Exonuclease III (Fermentas) and Fpg (New England Biolabs).

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: Primers labeled with Cy5, Cy3, or 6-FAM (6-carboxyfluorescein) were used for PCR. .. For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ).

    Article Title: Tyrosyl-DNA Phosphodiesterase 1 (TDP1) Repairs DNA Damage Induced by Topoisomerases I and II and Base Alkylation in Vertebrate Cells
    Article Snippet: The resulting internally labeled 40-nt product harboring 5′-phosphotyrosine, -hydroxyl, or -phosphate was then gel-purified and eluted for use (named Y40, OH40, and P40, respectively). .. Annealed DNA was incubated with uracil-DNA glycosylase for 1 h at 37 °C, and then Endonuclease III (New England BioLabs) was added for 1 h at 37 °C to generate the 3′-dRP at a nicked DNA site.

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase
    Article Snippet: This oligo was labeled at the 5’ end with [γ-32 P]ATP (3x103 Ci/mmol, 20 μCi per 40 pmol of substrate) using the DNA 5’ End Labeling System kit (Promega) and then hybridized with unlabeled complementary oligonucleotide (5’-CGCTGAGGATAGGT(A/G)ACAGTGTCATGCACCGG-3’). .. Afterwards, 1μg of TcNTH1 native recombinant protein was incubated with 2 pg of the oligo substrate in Endonuclease III (Endo III) buffer solution (New England Biolabs, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM DTT) for 1 h at 37°C.

    Polyacrylamide Gel Electrophoresis:

    Article Title: AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis
    Article Snippet: Nicked DNA was produced by reacting 200 nM of 50AP DNA with 25 U of endonuclease III (New England Biolabs) in standard reaction buffer (20 mM Tris pH 7.5, 1 mM EDTA, 20 mM NaCl, and 0.1 mg/ml BSA) at 37°C for 90 min. Nuclease assays were performed in a standard reaction buffer supplemented with 15 mM MgCl2 at 25°C. .. Aliquots were removed at specified time points as detailed in the figure legends, and quenched in formamide loading buffer (0.01% xylene cyanol, 0.01% bromophenol blue, 30 mM EDTA in formamide) before separation via denaturing polyacrylamide gel electrophoresis.

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. All DNAs, with and without gap, were PAGE-purified using a Mini Prep Cell (Bio-Rad, Hercules, CA).

    Salting Out:

    Article Title: Defective Repair of Oxidative Base Lesions by the DNA Glycosylase Nth1 Associates with Multiple Telomere Defects
    Article Snippet: In brief, DNA was isolated from mouse kidney and primary MEFs by salting out , . .. 400 ng of duplex oligomer or genomic DNA were incubated overnight with 12 units of Endonuclease III (New England Biolabs) in 1× NEB Endonuclease III buffer (20 mM Tris-HCl, 1 mM EDTA, 1 mM Dithiothreitol, pH 8.0).

    Activated Clotting Time Assay:

    Article Title: AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis
    Article Snippet: The double-stranded 71AP substrate contained an abasic furanose analogue at position 36, the sequence was 5′-(HEX)-GAG CAG AGT CAG TGC TGC TAC GAG CGG ATC ACT AT F ATA GGT AGT TTA TCC TAC GAA CTC CGT CCG TAC CG-3′, where F is the abasic furanose analogue. .. Nicked DNA was produced by reacting 200 nM of 50AP DNA with 25 U of endonuclease III (New England Biolabs) in standard reaction buffer (20 mM Tris pH 7.5, 1 mM EDTA, 20 mM NaCl, and 0.1 mg/ml BSA) at 37°C for 90 min. Nuclease assays were performed in a standard reaction buffer supplemented with 15 mM MgCl2 at 25°C.

    Plasmid Preparation:

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: Nucleosome reconstitution DNA for nucleosome preparations was PCR amplified from a plasmid containing the Widom's 601 DNA ( ). .. For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ).

    Article Title: Critical design criteria for minimal antibiotic-free plasmid vectors necessary to combine robust RNA Pol II and Pol III-mediated eukaryotic expression with high bacterial production yields
    Article Snippet: .. Endonuclease III and VIII (New England Biolabs, Ipswich, MA) digestion creates a strand break (nicked plasmid) at Apurinic/apyrimidinic (AP) abasic sites (AP sites are generated by various intracellular free radicals); .. RNase H (New England Biolabs, Ipswich, MA) digestion of plasmid-encoded RNA ( e.g. RNAII-derived primer) resulted in a nicked plasmid; and

    Irradiation:

    Article Title: Antimicrobial photodynamic therapy with fulleropyrrolidine: photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies
    Article Snippet: .. Post irradiation, the genomic DNA (10 ng) was extracted from the S. aureus cells using Extract ME DNA Bacteria purification kit (Blirt S.A.) and treated with endonuclease III (New England, BioLabs). ..

    In Vitro:

    Article Title: HMGA2 exhibits dRP/AP site cleavage activity and protects cancer cells from DNA-damage-induced cytotoxicity during chemotherapy
    Article Snippet: Paragraph title: In vitro lyase assays ... For control reactions, eight units of Endonuclease III and FPG (NEB) was used.

    Produced:

    Article Title: AP endonuclease paralogues with distinct activities in DNA repair and bacterial pathogenesis
    Article Snippet: .. Nicked DNA was produced by reacting 200 nM of 50AP DNA with 25 U of endonuclease III (New England Biolabs) in standard reaction buffer (20 mM Tris pH 7.5, 1 mM EDTA, 20 mM NaCl, and 0.1 mg/ml BSA) at 37°C for 90 min. Nuclease assays were performed in a standard reaction buffer supplemented with 15 mM MgCl2 at 25°C. .. Aliquots were removed at specified time points as detailed in the figure legends, and quenched in formamide loading buffer (0.01% xylene cyanol, 0.01% bromophenol blue, 30 mM EDTA in formamide) before separation via denaturing polyacrylamide gel electrophoresis.

    Concentration Assay:

    Article Title:
    Article Snippet: The precipitated samples then were pelleted and resuspended in distilled H2 O, and the sample was treated with RNase (final concentration, 1.25 μg/ml; Roche Applied Science) and BamHI restriction enzyme (Roche Applied Science). .. The quantitated total cellular DNA was heated to 70 °C for 15 min, cooled to room temperature for 10 min, and treated for 30 min with 5 units/μg endonuclease III and 5 units/μg formamidopyrimidine glycosylase (New England Biolabs).

    End Labeling:

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase
    Article Snippet: This oligo was labeled at the 5’ end with [γ-32 P]ATP (3x103 Ci/mmol, 20 μCi per 40 pmol of substrate) using the DNA 5’ End Labeling System kit (Promega) and then hybridized with unlabeled complementary oligonucleotide (5’-CGCTGAGGATAGGT(A/G)ACAGTGTCATGCACCGG-3’). .. Afterwards, 1μg of TcNTH1 native recombinant protein was incubated with 2 pg of the oligo substrate in Endonuclease III (Endo III) buffer solution (New England Biolabs, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM DTT) for 1 h at 37°C.

    Staining:

    Article Title:
    Article Snippet: The quantitated total cellular DNA was heated to 70 °C for 15 min, cooled to room temperature for 10 min, and treated for 30 min with 5 units/μg endonuclease III and 5 units/μg formamidopyrimidine glycosylase (New England Biolabs). .. The resulting gel was stained with ethidium bromide and imaged using UV light to obtain a gel picture.

    Clear Native PAGE:

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast
    Article Snippet: For nucleosomes with DNA gaps: primers containing deoxyuridine residues at gap sites were used for PCR amplification, and the PCR product was treated with a mix of Uracil-DNA glycosylase and endonuclease III (USER Enzyme from NEB, Ipswich, MA) ( ). .. DNA fragments with a gap have slower mobility on 6% native PAGE; and this was used to monitor completion of digestion.

    Activity Assay:

    Article Title: Antimicrobial photodynamic therapy with fulleropyrrolidine: photoinactivation mechanism of Staphylococcus aureus, in vitro and in vivo studies
    Article Snippet: Post irradiation, the genomic DNA (10 ng) was extracted from the S. aureus cells using Extract ME DNA Bacteria purification kit (Blirt S.A.) and treated with endonuclease III (New England, BioLabs). .. The N -glycosylase activity releases damaged pyrimidines from double-stranded DNA, generating a basic (AP site).

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase
    Article Snippet: Paragraph title: 2.7 DNA glycosylase activity assay ... Afterwards, 1μg of TcNTH1 native recombinant protein was incubated with 2 pg of the oligo substrate in Endonuclease III (Endo III) buffer solution (New England Biolabs, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM DTT) for 1 h at 37°C.

    Article Title: HMGA2 exhibits dRP/AP site cleavage activity and protects cancer cells from DNA-damage-induced cytotoxicity during chemotherapy
    Article Snippet: The AP lyase activity was assayed in a 10 μl reaction containing 1× reaction buffer (10 mM Tris–HCl, pH 7.5; 1 mM EDTA; 50 mM KCl) and 1.5 μM of 5′-32 P-labeled 31-mer-AP substrate plus 15 μM of HMGA proteins or hook 3. .. For control reactions, eight units of Endonuclease III and FPG (NEB) was used.

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  • 90
    New England Biolabs endonuclease iii
    TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo <t>III</t> (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same <t>oligo</t> incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.
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    TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.

    Journal: PLoS ONE

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

    doi: 10.1371/journal.pone.0157270

    Figure Lengend Snippet: TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.

    Article Snippet: Afterwards, 1μg of TcNTH1 native recombinant protein was incubated with 2 pg of the oligo substrate in Endonuclease III (Endo III) buffer solution (New England Biolabs, 20 mM Tris-HCl pH 8.0, 1 mM EDTA, 1 mM DTT) for 1 h at 37°C.

    Techniques: Activity Assay, Labeling, Incubation, Negative Control, Positive Control, Purification, Transformation Assay, Transfection