residual endonuclease activity  (New England Biolabs)


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    New England Biolabs residual endonuclease activity
    Residual Endonuclease Activity, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/residual endonuclease activity/product/New England Biolabs
    Average 90 stars, based on 1 article reviews
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    residual endonuclease activity - by Bioz Stars, 2020-04
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    Related Products / Commonly Used Together

    mgcl2
    radiation-treated bam hi
    tris-hcl
    nacl
    dithiothreitol

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    Related Articles

    Clone Assay:

    Article Title: Using shotgun sequence data to find active restriction enzyme genes
    Article Snippet: Cells (0.9 g) were suspended in 10 ml sonication buffer (20 mM Tris–HCL, 1 mM DTT, 0.1 mM EDTA, pH7.5 at 25°C), lysed by sonication and assayed for endonuclease activity using λDNA (40 sites for GRCGYC) and pBR322 DNA linearized with PstI (six sites for GRCGYC) in NEBuffer 4. .. No endonuclease activity was observed for any of the clones, even though the plasmids had the correct HindV putative gene sequence.

    Amplification:

    Article Title: Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)
    Article Snippet: 16S-23S rDNA spacer amplicons were digested with 4 U of each endonuclease (New England BioLabs, Saint-Quentin-en-Yvelines, France) and fractionated by electrophoresis in 2.5% high-resolution agarose gels (MetaPhor; TEBU, Le Perray en Yvelines, France) at 100 V for 3 h. With PCR-ribotyping, all of the strains of R. paucula exhibited a single fragment of between 831 and 947 bp in size, based on the use of DNA Molecular Weight Marker III. .. 16S-23S rDNA spacer amplicons were digested with 4 U of each endonuclease (New England BioLabs, Saint-Quentin-en-Yvelines, France) and fractionated by electrophoresis in 2.5% high-resolution agarose gels (MetaPhor; TEBU, Le Perray en Yvelines, France) at 100 V for 3 h. With PCR-ribotyping, all of the strains of R. paucula exhibited a single fragment of between 831 and 947 bp in size, based on the use of DNA Molecular Weight Marker III.

    Article Title: Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae
    Article Snippet: Amplification conditions, using GoTaq reagents (Promega, Madison, Wisconsin), were as described in section 4.2, with the exception of extension steps of 3 min at 72°C. .. Genomic DNA from the strains with the highest (WVU1853T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with endonuclease Vsp I (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% agarose gel.

    Article Title: Digital MDA for enumeration of total nucleic acid contamination
    Article Snippet: The crude products were also analyzed by agarose gel electrophoresis to assess the presence of high molecular weight amplified DNA. .. Endonuclease activity was tested by incubation of enzyme samples with 1 μg ϕ × 174 virion (ss, closed circular) DNA (New England Biolabs) at 37°C for 4 h in DNAse I reaction buffer (New England Biolabs).

    Article Title: Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures?
    Article Snippet: To study the epigenetic changes in specific DNA sequences it was used MS-RAPD, a modification of the original RAPD technique which is closer to MSAP (methylation sensitive amplified polymorphisms) analysis. .. In the first reaction, 250 ηg of each extracted DNA was added to a restriction mixture containing: 1× restriction buffer and 10/20 U restriction endonuclease to a final volume of 30 µL, one mixture per endonuclease (Hpa II/Msp I; all endonucleases and buffers were supplied by New England Biolabs, USA).

    Chromatography:

    Article Title: Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans
    Article Snippet: The synthetic decapeptide (H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH) was obtained from Elim Biopharmaceuticals, Inc. Hayward, California, USA, and was authenticated at NHLBI by high performance liquid chromatography-mass spectrometry (HPLC-MS). .. Following irradiation, 20 µl of each ionizing radiation-treated Bam HI sample were assayed for residual endonuclease activity in separate reaction mixtures (final volume, 30 µl) containing 125 ng μ-phage DNA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 , and 1 mM dithiothreitol (New England Biolabs).

    Lambda DNA Preparation:

    Article Title: A Sequence-Specific Nicking Endonuclease from Streptomyces: Purification, Physical and Catalytic Properties
    Article Snippet: DNA Cleavage Assay Enzyme activity was measured by incubating the endonuclease with supercoiled plasmid or lambda DNA in buffer R (10 mM Tris-HCl (pH 8.0), 10 mM MgCl2 , 50 mM NaCl, and 5 mM β -mercaptoethanol). .. The unit of endonuclease activity was quantified by titrating against known units of commercial Eco RI (New England Biolabs).

    Construct:

    Article Title: Using shotgun sequence data to find active restriction enzyme genes
    Article Snippet: Cells (0.9 g) were suspended in 10 ml sonication buffer (20 mM Tris–HCL, 1 mM DTT, 0.1 mM EDTA, pH7.5 at 25°C), lysed by sonication and assayed for endonuclease activity using λDNA (40 sites for GRCGYC) and pBR322 DNA linearized with PstI (six sites for GRCGYC) in NEBuffer 4. .. A vector carrying the HindV putative endonuclease gene was transformed into ER2566 cells lacking the methyltransferase construct, but even when induced there was no deleterious effect on cell growth, nor was any endonuclease activity detected in cell extracts.

    SYBR Green Assay:

    Article Title: Digital MDA for enumeration of total nucleic acid contamination
    Article Snippet: Reaction master mixes were made up according to the manufacturers’ instructions, omitting any chemical or thermal template denaturation steps and supplementing 0.3% Tween-20 and 1× SYBR GREEN I (Invitrogen), used to follow the reactions in real time during a 16 h, 30°C incubation on the MX3005-P thermocycler (Stratagene). .. Endonuclease activity was tested by incubation of enzyme samples with 1 μg ϕ × 174 virion (ss, closed circular) DNA (New England Biolabs) at 37°C for 4 h in DNAse I reaction buffer (New England Biolabs).

    Incubation:

    Article Title: A Sequence-Specific Nicking Endonuclease from Streptomyces: Purification, Physical and Catalytic Properties
    Article Snippet: After 30 min of incubation, the reactions were quenched by addition of 1/3 volume of loading dye solution containing 0.5% SDS and heating at 80°C for 5 min. Cleavage products were analyzed on 1 or 1.5% agarose gels. .. The unit of endonuclease activity was quantified by titrating against known units of commercial Eco RI (New England Biolabs).

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases
    Article Snippet: After adding serially diluted samples (1:5) to the reaction mixture (1 μg pBR322 plasmid DNA, 2 mM CaCl2 , 5 mM MgCl2 , 10 mM Tris-HCl, pH 7.4, and 0.5 mM dithiothreitol) the reaction was incubated for 1 h at 37°C, after which the reaction was terminated by adding Stop-solution (10 mM Tris-HCl, pH 7.4, 1% SDS, 25 mM Na2 EDTA, 7.5 mM bromophenol blue). .. Endonuclease activity was measured the same way as above in samples containing serially diluted protein (1:5), 1 μg plasmid pBR322 DNA (New England Biolabs), 2 mM CaCl2 , 5 mM MgCl2 , 10 mM Tris-HCl, pH 7.4, and 0.5 mM dithiothreitol to determine the Ca/Mg-dependent (primarily DNase I) endonuclease activity or 5 mM MnCl2 , 10 mM Tris-HCl (pH 7.4), and 0.5 mM dithiothreitol for the Mn-dependent (mainly EndoG) activity.

    Article Title: Digital MDA for enumeration of total nucleic acid contamination
    Article Snippet: .. Endonuclease activity was tested by incubation of enzyme samples with 1 μg ϕ × 174 virion (ss, closed circular) DNA (New England Biolabs) at 37°C for 4 h in DNAse I reaction buffer (New England Biolabs). .. DNA shearing Twenty nanograms E. coli K12 genomic DNA [again prepared by the DNeasy method (Qiagen) for Gram-negative cells] was diluted in 200 μl 20 mM Tris, pH 7.5, containing 0.05% Tween-20.

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases
    Article Snippet: Endonuclease activity in total protein extracts from kidney cells and in culture medium was determined using the plasmid incision assay (PIA) with pBR322 plasmid (New England Biolabs, Beverly, MA) as the substrate as described previously (Basnakian et al ., ). .. Plasmid and Lipofectamine were diluted separately in serum-free DMEM/Ham's F-12 medium (Sigma-Aldrich), mixed together, and incubated for 20 min at room temperature.

    Article Title: Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans
    Article Snippet: Following irradiation, 20 µl of each ionizing radiation-treated Bam HI sample were assayed for residual endonuclease activity in separate reaction mixtures (final volume, 30 µl) containing 125 ng μ-phage DNA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 , and 1 mM dithiothreitol (New England Biolabs). .. Bam HI/μ DNA mixtures were incubated for 1.25 h at 37°C, followed by agarose (0.8%) gel electrophoresis (AGE).

    Activity Assay:

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: .. Endonuclease activity was assayed by incubating various amounts of MmeI at 37°C in MmeI reaction buffer (NEBuffer 4: 20 mM Tris–acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT, supplemented with 100 µg/ml BSA and 80 µM AdoMet) containing 1 µg substrate DNA per 50 µl. .. Reactions were terminated by addition of stop solution (50 mM EDTA, pH 8.0, 50% glycerol, 0.02% bromophenol blue), and reaction products were analyzed by electrophoresis in 1% LE agarose or 3% NuSieve GTG agarose gels alongside DNA size standards lambda-HindIII with PhiX174-HaeIII, or lambda-BstEII with pBR322-MspI.

    Article Title: A Sequence-Specific Nicking Endonuclease from Streptomyces: Purification, Physical and Catalytic Properties
    Article Snippet: .. The unit of endonuclease activity was quantified by titrating against known units of commercial Eco RI (New England Biolabs). .. Protein concentration was determined by the Bradford method using BioRad protein assay reagent (BioRad, USA) and BSA as the standard.

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases
    Article Snippet: .. Endonuclease activity was measured the same way as above in samples containing serially diluted protein (1:5), 1 μg plasmid pBR322 DNA (New England Biolabs), 2 mM CaCl2 , 5 mM MgCl2 , 10 mM Tris-HCl, pH 7.4, and 0.5 mM dithiothreitol to determine the Ca/Mg-dependent (primarily DNase I) endonuclease activity or 5 mM MnCl2 , 10 mM Tris-HCl (pH 7.4), and 0.5 mM dithiothreitol for the Mn-dependent (mainly EndoG) activity. .. As opposed to PIA, zymogram gel electrophoresis, previously used by us to assess DNase I activity (Basnakian et al ., ), was not applicable for EndoG due to low specific activity of the enzyme in the used tubular epithelial cells.

    Article Title: Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae
    Article Snippet: .. Genomic DNA from the strains with the highest (WVU1853T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with endonuclease Vsp I (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% agarose gel. ..

    Article Title: Digital MDA for enumeration of total nucleic acid contamination
    Article Snippet: .. Endonuclease activity was tested by incubation of enzyme samples with 1 μg ϕ × 174 virion (ss, closed circular) DNA (New England Biolabs) at 37°C for 4 h in DNAse I reaction buffer (New England Biolabs). .. DNA shearing Twenty nanograms E. coli K12 genomic DNA [again prepared by the DNeasy method (Qiagen) for Gram-negative cells] was diluted in 200 μl 20 mM Tris, pH 7.5, containing 0.05% Tween-20.

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases
    Article Snippet: .. Endonuclease activity in total protein extracts from kidney cells and in culture medium was determined using the plasmid incision assay (PIA) with pBR322 plasmid (New England Biolabs, Beverly, MA) as the substrate as described previously (Basnakian et al ., ). .. To determine whether Lipofectamine is protecting plasmid DNA from degradation by endonucleases, pBR322 plasmid was pretreated with Lipofectamine before it was exposed to the culture medium.

    Article Title: Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans
    Article Snippet: .. Following irradiation, 20 µl of each ionizing radiation-treated Bam HI sample were assayed for residual endonuclease activity in separate reaction mixtures (final volume, 30 µl) containing 125 ng μ-phage DNA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 , and 1 mM dithiothreitol (New England Biolabs). .. Bam HI/μ DNA mixtures were incubated for 1.25 h at 37°C, followed by agarose (0.8%) gel electrophoresis (AGE).

    Article Title: Tear lipocalin is the major endonuclease in tears
    Article Snippet: .. Endonuclease activity assay In general, DNA-hydrolyzing activity was determined in 20 mM Tris-HCl, pH 7.5, 1 mM MgCl2 , 1mM CaCl2, 50 mM NaCl, and 0.1 μg sc pUC19 plasmid DNA (New England Biolabs, Beverly, MA) in a volume of 20 μl. .. For experiments investigating activity with varying ions, the other components of the assay buffer are specifically indicated.

    Article Title: Using shotgun sequence data to find active restriction enzyme genes
    Article Snippet: .. Cells (0.9 g) were suspended in 10 ml sonication buffer (20 mM Tris–HCL, 1 mM DTT, 0.1 mM EDTA, pH7.5 at 25°C), lysed by sonication and assayed for endonuclease activity using λDNA (40 sites for GRCGYC) and pBR322 DNA linearized with PstI (six sites for GRCGYC) in NEBuffer 4. .. No endonuclease activity was observed for any of the clones, even though the plasmids had the correct HindV putative gene sequence.

    Mass Spectrometry:

    Article Title: Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures?
    Article Snippet: Paragraph title: Methylation-sensitive random-amplified polymorphic DNA (MS-RAPD) ... In the first reaction, 250 ηg of each extracted DNA was added to a restriction mixture containing: 1× restriction buffer and 10/20 U restriction endonuclease to a final volume of 30 µL, one mixture per endonuclease (Hpa II/Msp I; all endonucleases and buffers were supplied by New England Biolabs, USA).

    Modification:

    Article Title: Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures?
    Article Snippet: To study the epigenetic changes in specific DNA sequences it was used MS-RAPD, a modification of the original RAPD technique which is closer to MSAP (methylation sensitive amplified polymorphisms) analysis. .. In the first reaction, 250 ηg of each extracted DNA was added to a restriction mixture containing: 1× restriction buffer and 10/20 U restriction endonuclease to a final volume of 30 µL, one mixture per endonuclease (Hpa II/Msp I; all endonucleases and buffers were supplied by New England Biolabs, USA).

    Transformation Assay:

    Article Title: Using shotgun sequence data to find active restriction enzyme genes
    Article Snippet: Cells (0.9 g) were suspended in 10 ml sonication buffer (20 mM Tris–HCL, 1 mM DTT, 0.1 mM EDTA, pH7.5 at 25°C), lysed by sonication and assayed for endonuclease activity using λDNA (40 sites for GRCGYC) and pBR322 DNA linearized with PstI (six sites for GRCGYC) in NEBuffer 4. .. A vector carrying the HindV putative endonuclease gene was transformed into ER2566 cells lacking the methyltransferase construct, but even when induced there was no deleterious effect on cell growth, nor was any endonuclease activity detected in cell extracts.

    Hybridization:

    Article Title: Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae
    Article Snippet: Genomic DNA from the strains with the highest (WVU1853T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with endonuclease Vsp I (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% agarose gel. .. Hybridization of the nanI probe and detection of the digoxygenin label were carried out using the DIG EasyHyb system (Roche Applied Sciences) according to the manufacturer’s instructions.

    High Performance Liquid Chromatography:

    Article Title: Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans
    Article Snippet: The synthetic decapeptide (H-Asp-Glu-His-Gly-Thr-Ala-Val-Met-Leu-Lys-OH) was obtained from Elim Biopharmaceuticals, Inc. Hayward, California, USA, and was authenticated at NHLBI by high performance liquid chromatography-mass spectrometry (HPLC-MS). .. Following irradiation, 20 µl of each ionizing radiation-treated Bam HI sample were assayed for residual endonuclease activity in separate reaction mixtures (final volume, 30 µl) containing 125 ng μ-phage DNA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 , and 1 mM dithiothreitol (New England Biolabs).

    Southern Blot:

    Article Title: Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae
    Article Snippet: Paragraph title: 4.4. Southern blotting to determine nanI copy number ... Genomic DNA from the strains with the highest (WVU1853T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with endonuclease Vsp I (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% agarose gel.

    DNA Sequencing:

    Article Title: Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)
    Article Snippet: 16S-23S rDNA spacer amplicons were digested with 4 U of each endonuclease (New England BioLabs, Saint-Quentin-en-Yvelines, France) and fractionated by electrophoresis in 2.5% high-resolution agarose gels (MetaPhor; TEBU, Le Perray en Yvelines, France) at 100 V for 3 h. With PCR-ribotyping, all of the strains of R. paucula exhibited a single fragment of between 831 and 947 bp in size, based on the use of DNA Molecular Weight Marker III. .. 16S-23S rDNA spacer amplicons were digested with 4 U of each endonuclease (New England BioLabs, Saint-Quentin-en-Yvelines, France) and fractionated by electrophoresis in 2.5% high-resolution agarose gels (MetaPhor; TEBU, Le Perray en Yvelines, France) at 100 V for 3 h. With PCR-ribotyping, all of the strains of R. paucula exhibited a single fragment of between 831 and 947 bp in size, based on the use of DNA Molecular Weight Marker III.

    Protein Concentration:

    Article Title: A Sequence-Specific Nicking Endonuclease from Streptomyces: Purification, Physical and Catalytic Properties
    Article Snippet: The unit of endonuclease activity was quantified by titrating against known units of commercial Eco RI (New England Biolabs). .. Protein concentration was determined by the Bradford method using BioRad protein assay reagent (BioRad, USA) and BSA as the standard.

    Sequencing:

    Article Title: Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)
    Article Snippet: 16S-23S rDNA spacer amplicons were digested with 4 U of each endonuclease (New England BioLabs, Saint-Quentin-en-Yvelines, France) and fractionated by electrophoresis in 2.5% high-resolution agarose gels (MetaPhor; TEBU, Le Perray en Yvelines, France) at 100 V for 3 h. With PCR-ribotyping, all of the strains of R. paucula exhibited a single fragment of between 831 and 947 bp in size, based on the use of DNA Molecular Weight Marker III. .. 16S-23S rDNA spacer amplicons were digested with 4 U of each endonuclease (New England BioLabs, Saint-Quentin-en-Yvelines, France) and fractionated by electrophoresis in 2.5% high-resolution agarose gels (MetaPhor; TEBU, Le Perray en Yvelines, France) at 100 V for 3 h. With PCR-ribotyping, all of the strains of R. paucula exhibited a single fragment of between 831 and 947 bp in size, based on the use of DNA Molecular Weight Marker III.

    Article Title: Using shotgun sequence data to find active restriction enzyme genes
    Article Snippet: Cells (0.9 g) were suspended in 10 ml sonication buffer (20 mM Tris–HCL, 1 mM DTT, 0.1 mM EDTA, pH7.5 at 25°C), lysed by sonication and assayed for endonuclease activity using λDNA (40 sites for GRCGYC) and pBR322 DNA linearized with PstI (six sites for GRCGYC) in NEBuffer 4. .. No endonuclease activity was observed for any of the clones, even though the plasmids had the correct HindV putative gene sequence.

    Sonication:

    Article Title: Using shotgun sequence data to find active restriction enzyme genes
    Article Snippet: .. Cells (0.9 g) were suspended in 10 ml sonication buffer (20 mM Tris–HCL, 1 mM DTT, 0.1 mM EDTA, pH7.5 at 25°C), lysed by sonication and assayed for endonuclease activity using λDNA (40 sites for GRCGYC) and pBR322 DNA linearized with PstI (six sites for GRCGYC) in NEBuffer 4. .. No endonuclease activity was observed for any of the clones, even though the plasmids had the correct HindV putative gene sequence.

    Molecular Weight:

    Article Title: Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)
    Article Snippet: .. 16S-23S rDNA spacer amplicons were digested with 4 U of each endonuclease (New England BioLabs, Saint-Quentin-en-Yvelines, France) and fractionated by electrophoresis in 2.5% high-resolution agarose gels (MetaPhor; TEBU, Le Perray en Yvelines, France) at 100 V for 3 h. With PCR-ribotyping, all of the strains of R. paucula exhibited a single fragment of between 831 and 947 bp in size, based on the use of DNA Molecular Weight Marker III. ..

    Article Title: Digital MDA for enumeration of total nucleic acid contamination
    Article Snippet: The crude products were also analyzed by agarose gel electrophoresis to assess the presence of high molecular weight amplified DNA. .. Endonuclease activity was tested by incubation of enzyme samples with 1 μg ϕ × 174 virion (ss, closed circular) DNA (New England Biolabs) at 37°C for 4 h in DNAse I reaction buffer (New England Biolabs).

    Imaging:

    Article Title: Tear lipocalin is the major endonuclease in tears
    Article Snippet: Endonuclease activity assay In general, DNA-hydrolyzing activity was determined in 20 mM Tris-HCl, pH 7.5, 1 mM MgCl2 , 1mM CaCl2, 50 mM NaCl, and 0.1 μg sc pUC19 plasmid DNA (New England Biolabs, Beverly, MA) in a volume of 20 μl. .. Ethidium-bromide stained gels were photographed and scanned with a densitometer (IS-100 Digital Imaging System; Alpha Innotech Corporation, San Leandro, CA).

    DNA Extraction:

    Article Title: Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures?
    Article Snippet: Methylation-sensitive random-amplified polymorphic DNA (MS-RAPD) Genomic DNA was extracted from 75 mg of frozen leaves with a plant genomic DNA extraction kit (DNeasy Plant Mini Kit, Qiagen, Germany) according to the manufacturer's instructions. .. In the first reaction, 250 ηg of each extracted DNA was added to a restriction mixture containing: 1× restriction buffer and 10/20 U restriction endonuclease to a final volume of 30 µL, one mixture per endonuclease (Hpa II/Msp I; all endonucleases and buffers were supplied by New England Biolabs, USA).

    Nucleic Acid Electrophoresis:

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases
    Article Snippet: Endonuclease activity was measured the same way as above in samples containing serially diluted protein (1:5), 1 μg plasmid pBR322 DNA (New England Biolabs), 2 mM CaCl2 , 5 mM MgCl2 , 10 mM Tris-HCl, pH 7.4, and 0.5 mM dithiothreitol to determine the Ca/Mg-dependent (primarily DNase I) endonuclease activity or 5 mM MnCl2 , 10 mM Tris-HCl (pH 7.4), and 0.5 mM dithiothreitol for the Mn-dependent (mainly EndoG) activity. .. As opposed to PIA, zymogram gel electrophoresis, previously used by us to assess DNase I activity (Basnakian et al ., ), was not applicable for EndoG due to low specific activity of the enzyme in the used tubular epithelial cells.

    Article Title: Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans
    Article Snippet: Following irradiation, 20 µl of each ionizing radiation-treated Bam HI sample were assayed for residual endonuclease activity in separate reaction mixtures (final volume, 30 µl) containing 125 ng μ-phage DNA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 , and 1 mM dithiothreitol (New England Biolabs). .. Bam HI/μ DNA mixtures were incubated for 1.25 h at 37°C, followed by agarose (0.8%) gel electrophoresis (AGE).

    Article Title: Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures?
    Article Snippet: DNA yield and purity were assessed by spectrophotometry (as described in Valledor et al. ) and gel electrophoresis on agarose gel by direct comparison with phage λ DNA. .. In the first reaction, 250 ηg of each extracted DNA was added to a restriction mixture containing: 1× restriction buffer and 10/20 U restriction endonuclease to a final volume of 30 µL, one mixture per endonuclease (Hpa II/Msp I; all endonucleases and buffers were supplied by New England Biolabs, USA).

    DNA Cleavage Assay:

    Article Title: A Sequence-Specific Nicking Endonuclease from Streptomyces: Purification, Physical and Catalytic Properties
    Article Snippet: Paragraph title: 2.5. DNA Cleavage Assay ... The unit of endonuclease activity was quantified by titrating against known units of commercial Eco RI (New England Biolabs).

    Methylation:

    Article Title: Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures?
    Article Snippet: Paragraph title: Methylation-sensitive random-amplified polymorphic DNA (MS-RAPD) ... In the first reaction, 250 ηg of each extracted DNA was added to a restriction mixture containing: 1× restriction buffer and 10/20 U restriction endonuclease to a final volume of 30 µL, one mixture per endonuclease (Hpa II/Msp I; all endonucleases and buffers were supplied by New England Biolabs, USA).

    Multiple Displacement Amplification:

    Article Title: Digital MDA for enumeration of total nucleic acid contamination
    Article Snippet: Activity assays The commercial and high-purity ϕ29 DNAP reagents were tested for activity in 50 μl MDA reactions. .. Endonuclease activity was tested by incubation of enzyme samples with 1 μg ϕ × 174 virion (ss, closed circular) DNA (New England Biolabs) at 37°C for 4 h in DNAse I reaction buffer (New England Biolabs).

    Labeling:

    Article Title: Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae
    Article Snippet: The amplicons were labeled with digoxygenin (DIG Hi prime, Roche Applied Sciences) according to the manufacturer’s instructions. .. Genomic DNA from the strains with the highest (WVU1853T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with endonuclease Vsp I (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% agarose gel.

    Polymerase Chain Reaction:

    Article Title: Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)
    Article Snippet: .. 16S-23S rDNA spacer amplicons were digested with 4 U of each endonuclease (New England BioLabs, Saint-Quentin-en-Yvelines, France) and fractionated by electrophoresis in 2.5% high-resolution agarose gels (MetaPhor; TEBU, Le Perray en Yvelines, France) at 100 V for 3 h. With PCR-ribotyping, all of the strains of R. paucula exhibited a single fragment of between 831 and 947 bp in size, based on the use of DNA Molecular Weight Marker III. ..

    Article Title: Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae
    Article Snippet: To compare the nanI copy number among strains by Southern blotting, a probe consisting of the 3′ 2.8 kb of nanI was amplified from WVU1853T genomic DNA using PCR primers 5′-TCT CTT CCT TTT TGA GGG CTA-3′ and 5′-GCA AAT CAT CTT AAG AAA AGT CAT T-3′. .. Genomic DNA from the strains with the highest (WVU1853T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with endonuclease Vsp I (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% agarose gel.

    Article Title: Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures?
    Article Snippet: In the first reaction, 250 ηg of each extracted DNA was added to a restriction mixture containing: 1× restriction buffer and 10/20 U restriction endonuclease to a final volume of 30 µL, one mixture per endonuclease (Hpa II/Msp I; all endonucleases and buffers were supplied by New England Biolabs, USA). .. PCR amplification was performed according to Cocconcelli et al. : 20 µL reaction mixtures containing: 12–14 ng DNA, 1× PCR buffer (Invitrogen, USA), 3.5 mM MgCl2 , 75 pM dNTP, 0.25 mM each primer, 1 U Taq polymerase (Invitrogen, USA).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae
    Article Snippet: To compare the nanI copy number among strains by Southern blotting, a probe consisting of the 3′ 2.8 kb of nanI was amplified from WVU1853T genomic DNA using PCR primers 5′-TCT CTT CCT TTT TGA GGG CTA-3′ and 5′-GCA AAT CAT CTT AAG AAA AGT CAT T-3′. .. Genomic DNA from the strains with the highest (WVU1853T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with endonuclease Vsp I (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% agarose gel.

    Plasmid Preparation:

    Article Title: A Sequence-Specific Nicking Endonuclease from Streptomyces: Purification, Physical and Catalytic Properties
    Article Snippet: DNA Cleavage Assay Enzyme activity was measured by incubating the endonuclease with supercoiled plasmid or lambda DNA in buffer R (10 mM Tris-HCl (pH 8.0), 10 mM MgCl2 , 50 mM NaCl, and 5 mM β -mercaptoethanol). .. The unit of endonuclease activity was quantified by titrating against known units of commercial Eco RI (New England Biolabs).

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases
    Article Snippet: .. Endonuclease activity was measured the same way as above in samples containing serially diluted protein (1:5), 1 μg plasmid pBR322 DNA (New England Biolabs), 2 mM CaCl2 , 5 mM MgCl2 , 10 mM Tris-HCl, pH 7.4, and 0.5 mM dithiothreitol to determine the Ca/Mg-dependent (primarily DNase I) endonuclease activity or 5 mM MnCl2 , 10 mM Tris-HCl (pH 7.4), and 0.5 mM dithiothreitol for the Mn-dependent (mainly EndoG) activity. .. As opposed to PIA, zymogram gel electrophoresis, previously used by us to assess DNase I activity (Basnakian et al ., ), was not applicable for EndoG due to low specific activity of the enzyme in the used tubular epithelial cells.

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases
    Article Snippet: .. Endonuclease activity in total protein extracts from kidney cells and in culture medium was determined using the plasmid incision assay (PIA) with pBR322 plasmid (New England Biolabs, Beverly, MA) as the substrate as described previously (Basnakian et al ., ). .. To determine whether Lipofectamine is protecting plasmid DNA from degradation by endonucleases, pBR322 plasmid was pretreated with Lipofectamine before it was exposed to the culture medium.

    Article Title: Tear lipocalin is the major endonuclease in tears
    Article Snippet: .. Endonuclease activity assay In general, DNA-hydrolyzing activity was determined in 20 mM Tris-HCl, pH 7.5, 1 mM MgCl2 , 1mM CaCl2, 50 mM NaCl, and 0.1 μg sc pUC19 plasmid DNA (New England Biolabs, Beverly, MA) in a volume of 20 μl. .. For experiments investigating activity with varying ions, the other components of the assay buffer are specifically indicated.

    Article Title: Using shotgun sequence data to find active restriction enzyme genes
    Article Snippet: Cells (0.9 g) were suspended in 10 ml sonication buffer (20 mM Tris–HCL, 1 mM DTT, 0.1 mM EDTA, pH7.5 at 25°C), lysed by sonication and assayed for endonuclease activity using λDNA (40 sites for GRCGYC) and pBR322 DNA linearized with PstI (six sites for GRCGYC) in NEBuffer 4. .. A vector carrying the HindV putative endonuclease gene was transformed into ER2566 cells lacking the methyltransferase construct, but even when induced there was no deleterious effect on cell growth, nor was any endonuclease activity detected in cell extracts.

    Irradiation:

    Article Title: DNA lesions induced by UV A1 and B radiation in human cells: Comparative analyses in the overall genome and in the p53 tumor suppressor gene
    Article Snippet: The standard controls were made from the genomic DNAs of normal human skin fibroblasts irradiated with increasing doses of UVC (i.e., 1, 10, 25, 50, 100, 150, 200, and 250 J/m2 ) emitted from a germicidal lamp and UVB (i.e., 200, 600, 1,200, and 2,400 J/m2 ). .. The standard controls for CPD determination were subjected to T4 endonuclease V digestion, and then run on a 1.5% alkaline/agarose electrophoresis gel ( ) with specific ladder markers in the range of 100 to 10,000 bp (New England Biolabs).

    Article Title: Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans
    Article Snippet: .. Following irradiation, 20 µl of each ionizing radiation-treated Bam HI sample were assayed for residual endonuclease activity in separate reaction mixtures (final volume, 30 µl) containing 125 ng μ-phage DNA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 , and 1 mM dithiothreitol (New England Biolabs). .. Bam HI/μ DNA mixtures were incubated for 1.25 h at 37°C, followed by agarose (0.8%) gel electrophoresis (AGE).

    Agarose Gel Electrophoresis:

    Article Title: Uptake of Foreign Nucleic Acids in Kidney Tubular Epithelial Cells Deficient in Proapoptotic Endonucleases
    Article Snippet: The samples were run on a 1% agarose gel in Tris-acetate-EDTA buffer, pH 8 (7V/cm, 35 min), and the DNA was viewed with ethidium bromide. .. Endonuclease activity was measured the same way as above in samples containing serially diluted protein (1:5), 1 μg plasmid pBR322 DNA (New England Biolabs), 2 mM CaCl2 , 5 mM MgCl2 , 10 mM Tris-HCl, pH 7.4, and 0.5 mM dithiothreitol to determine the Ca/Mg-dependent (primarily DNase I) endonuclease activity or 5 mM MnCl2 , 10 mM Tris-HCl (pH 7.4), and 0.5 mM dithiothreitol for the Mn-dependent (mainly EndoG) activity.

    Article Title: Genetic Variation in Sialidase and Linkage to N-acetylneuraminate Catabolism in Mycoplasma synoviae
    Article Snippet: .. Genomic DNA from the strains with the highest (WVU1853T ) and lowest (K4907A and K5395B) levels of sialidase activity was digested with endonuclease Vsp I (New England Biolabs, Ipswich, Massachusetts), then separated on a 0.6% agarose gel. ..

    Article Title: Digital MDA for enumeration of total nucleic acid contamination
    Article Snippet: The crude products were also analyzed by agarose gel electrophoresis to assess the presence of high molecular weight amplified DNA. .. Endonuclease activity was tested by incubation of enzyme samples with 1 μg ϕ × 174 virion (ss, closed circular) DNA (New England Biolabs) at 37°C for 4 h in DNAse I reaction buffer (New England Biolabs).

    Article Title: Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures?
    Article Snippet: DNA yield and purity were assessed by spectrophotometry (as described in Valledor et al. ) and gel electrophoresis on agarose gel by direct comparison with phage λ DNA. .. In the first reaction, 250 ηg of each extracted DNA was added to a restriction mixture containing: 1× restriction buffer and 10/20 U restriction endonuclease to a final volume of 30 µL, one mixture per endonuclease (Hpa II/Msp I; all endonucleases and buffers were supplied by New England Biolabs, USA).

    In Vitro:

    Article Title: Small-Molecule Antioxidant Proteome-Shields in Deinococcus radiodurans
    Article Snippet: Assays for enzyme activity and DNA damage The chemical agents identified in the DR-ultrafiltrate ( , , and ; and ) were reconstituted in vitro using reagents from Sigma Chemical Company (St. Louis, Missouri, USA). .. Following irradiation, 20 µl of each ionizing radiation-treated Bam HI sample were assayed for residual endonuclease activity in separate reaction mixtures (final volume, 30 µl) containing 125 ng μ-phage DNA, 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl2 , and 1 mM dithiothreitol (New England Biolabs).

    Electrophoresis:

    Article Title: Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)
    Article Snippet: .. 16S-23S rDNA spacer amplicons were digested with 4 U of each endonuclease (New England BioLabs, Saint-Quentin-en-Yvelines, France) and fractionated by electrophoresis in 2.5% high-resolution agarose gels (MetaPhor; TEBU, Le Perray en Yvelines, France) at 100 V for 3 h. With PCR-ribotyping, all of the strains of R. paucula exhibited a single fragment of between 831 and 947 bp in size, based on the use of DNA Molecular Weight Marker III. ..

    Article Title: MmeI: a minimal Type II restriction-modification system that only modifies one DNA strand for host protection
    Article Snippet: Endonuclease activity was assayed by incubating various amounts of MmeI at 37°C in MmeI reaction buffer (NEBuffer 4: 20 mM Tris–acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT, supplemented with 100 µg/ml BSA and 80 µM AdoMet) containing 1 µg substrate DNA per 50 µl. .. Reactions were terminated by addition of stop solution (50 mM EDTA, pH 8.0, 50% glycerol, 0.02% bromophenol blue), and reaction products were analyzed by electrophoresis in 1% LE agarose or 3% NuSieve GTG agarose gels alongside DNA size standards lambda-HindIII with PhiX174-HaeIII, or lambda-BstEII with pBR322-MspI.

    Article Title: DNA lesions induced by UV A1 and B radiation in human cells: Comparative analyses in the overall genome and in the p53 tumor suppressor gene
    Article Snippet: .. The standard controls for CPD determination were subjected to T4 endonuclease V digestion, and then run on a 1.5% alkaline/agarose electrophoresis gel ( ) with specific ladder markers in the range of 100 to 10,000 bp (New England Biolabs). .. The standard controls for (6-4)PPs determination were subjected to two consecutive treatments with ( i ) CPD photolyase and ( ii ) UV damage endonuclease (Trevigen, Gaithersburg, MD) , and subsequently run on an electrophoresis gel as described above.

    Article Title: Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures?
    Article Snippet: In the first reaction, 250 ηg of each extracted DNA was added to a restriction mixture containing: 1× restriction buffer and 10/20 U restriction endonuclease to a final volume of 30 µL, one mixture per endonuclease (Hpa II/Msp I; all endonucleases and buffers were supplied by New England Biolabs, USA). .. The reaction was stopped by placing the tubes on ice and restriction was checked by agarose electrophoresis.

    Spectrophotometry:

    Article Title: Is the Interplay between Epigenetic Markers Related to the Acclimation of Cork Oak Plants to High Temperatures?
    Article Snippet: DNA yield and purity were assessed by spectrophotometry (as described in Valledor et al. ) and gel electrophoresis on agarose gel by direct comparison with phage λ DNA. .. In the first reaction, 250 ηg of each extracted DNA was added to a restriction mixture containing: 1× restriction buffer and 10/20 U restriction endonuclease to a final volume of 30 µL, one mixture per endonuclease (Hpa II/Msp I; all endonucleases and buffers were supplied by New England Biolabs, USA).

    Marker:

    Article Title: Ralstonia paucula (Formerly CDC Group IV c-2): Unsuccessful Strain Differentiation with PCR-Based Methods, Study of the 16S-23S Spacer of the rRNA Operon, and Comparison with Other Ralstonia Species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)
    Article Snippet: .. 16S-23S rDNA spacer amplicons were digested with 4 U of each endonuclease (New England BioLabs, Saint-Quentin-en-Yvelines, France) and fractionated by electrophoresis in 2.5% high-resolution agarose gels (MetaPhor; TEBU, Le Perray en Yvelines, France) at 100 V for 3 h. With PCR-ribotyping, all of the strains of R. paucula exhibited a single fragment of between 831 and 947 bp in size, based on the use of DNA Molecular Weight Marker III. ..

    Staining:

    Article Title: Tear lipocalin is the major endonuclease in tears
    Article Snippet: Endonuclease activity assay In general, DNA-hydrolyzing activity was determined in 20 mM Tris-HCl, pH 7.5, 1 mM MgCl2 , 1mM CaCl2, 50 mM NaCl, and 0.1 μg sc pUC19 plasmid DNA (New England Biolabs, Beverly, MA) in a volume of 20 μl. .. Ethidium-bromide stained gels were photographed and scanned with a densitometer (IS-100 Digital Imaging System; Alpha Innotech Corporation, San Leandro, CA).

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    New England Biolabs t4 endonuclease v digestion
    Mapping of CPDs and Fpg-sensitive sites in exon 6 of the  p53  gene (nontranscribed strand) by LM-PCR. Genomic DNAs of UVB-irradiated (0.26 and 0.78 J/cm 2 ) or UVA1-irradiated (216 and 648 J/cm 2 ) human fibroblasts were subjected to T4 endonuclease V cleavage and CPD-photolyase reactivation (to create ligatable ends) ( a ), or treatment with Fpg (+) or digestion buffer only (-), and subsequently assayed by LM-PCR ( b ), as described in  Materials and Methods . Hotspots of lesion formation are indicated by arrows, and the corresponding nucleotide positions, e.g., respective codons, are specified. Sequence contexts of the lesions in introns and exons are written in lowercase and uppercase, respectively. Underlined bases are the exact positions where the lesions are formed. Major Fpg-sensitive sites are identified by  * ) was included in all runs. All samples were processed in parallel and run on the same gel but were separated for better illustration. M, sizing standard.
    T4 Endonuclease V Digestion, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Mapping of CPDs and Fpg-sensitive sites in exon 6 of the  p53  gene (nontranscribed strand) by LM-PCR. Genomic DNAs of UVB-irradiated (0.26 and 0.78 J/cm 2 ) or UVA1-irradiated (216 and 648 J/cm 2 ) human fibroblasts were subjected to T4 endonuclease V cleavage and CPD-photolyase reactivation (to create ligatable ends) ( a ), or treatment with Fpg (+) or digestion buffer only (-), and subsequently assayed by LM-PCR ( b ), as described in  Materials and Methods . Hotspots of lesion formation are indicated by arrows, and the corresponding nucleotide positions, e.g., respective codons, are specified. Sequence contexts of the lesions in introns and exons are written in lowercase and uppercase, respectively. Underlined bases are the exact positions where the lesions are formed. Major Fpg-sensitive sites are identified by  * ) was included in all runs. All samples were processed in parallel and run on the same gel but were separated for better illustration. M, sizing standard.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: DNA lesions induced by UV A1 and B radiation in human cells: Comparative analyses in the overall genome and in the p53 tumor suppressor gene

    doi: 10.1073/pnas.0502311102

    Figure Lengend Snippet: Mapping of CPDs and Fpg-sensitive sites in exon 6 of the p53 gene (nontranscribed strand) by LM-PCR. Genomic DNAs of UVB-irradiated (0.26 and 0.78 J/cm 2 ) or UVA1-irradiated (216 and 648 J/cm 2 ) human fibroblasts were subjected to T4 endonuclease V cleavage and CPD-photolyase reactivation (to create ligatable ends) ( a ), or treatment with Fpg (+) or digestion buffer only (-), and subsequently assayed by LM-PCR ( b ), as described in Materials and Methods . Hotspots of lesion formation are indicated by arrows, and the corresponding nucleotide positions, e.g., respective codons, are specified. Sequence contexts of the lesions in introns and exons are written in lowercase and uppercase, respectively. Underlined bases are the exact positions where the lesions are formed. Major Fpg-sensitive sites are identified by * ) was included in all runs. All samples were processed in parallel and run on the same gel but were separated for better illustration. M, sizing standard.

    Article Snippet: The standard controls for CPD determination were subjected to T4 endonuclease V digestion, and then run on a 1.5% alkaline/agarose electrophoresis gel ( ) with specific ladder markers in the range of 100 to 10,000 bp (New England Biolabs).

    Techniques: Polymerase Chain Reaction, Irradiation, Sequencing