exonuclease iii e coli  (New England Biolabs)


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    Name:
    Exonuclease III (E.coli) - 2
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    Catalog Number:
    M0206L
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    Score:
    85
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    Structured Review

    New England Biolabs exonuclease iii e coli
    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq <t>(three</t> experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

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    Images

    1) Product Images from "Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq"

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq

    Journal: Nature Communications

    doi: 10.1038/ncomms15335

    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.
    Figure Legend Snippet: Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Techniques Used: Mutagenesis, Two Tailed Test

    2) Product Images from "Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq"

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq

    Journal: Nature Communications

    doi: 10.1038/ncomms15335

    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.
    Figure Legend Snippet: Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Techniques Used: Mutagenesis, Two Tailed Test

    Related Articles

    Clone Assay:

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: These subclones were tested for phenotypic rescue of lf3-2 , lf3-5 , and lf3-6 by cotransformation. .. Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. The sequence of the LF3 gene was obtained by sequencing plasmid clones with universal sequencing primers or gene-specific primers (DNA Sequencing and Synthesis Facility, Iowa State University, or Advanced Genetics Analysis Center, University of Minnesota).

    Centrifugation:

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: For denaturing polyacrylamide gel electrophoresis (PAGE) analysis, samples were extracted with 150 μl of CHCl3 containing 1/25 volume of isoamyl alcohol, the aqueous phase was precipitated with 2.5 volumes of 95% ethanol, and the DNA was recovered by centrifugation. .. Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above.

    Amplification:

    Article Title: A paired-end sequencing strategy to map the complex landscape of transcription initiation
    Article Snippet: Paragraph title: Circularization and rolling circle amplification ... Ligation was performed with 5 cycles of 95°C for 30 sec, 68°C for 2 min, 55°C for 1 min and 60°C for 5 min, followed by 5 cycles of 95°C for 30 sec, 65°C for 2 min, 55°C for 1 min and 60°C for 5 min. To remove excess oligonucleotides and unligated DNA fragments, we added 3 μl Exonuclease I (NEB) and 0.6 μl Exonuclease III (NEB) to the ligation reaction and the mixture was incubated at 37°C for 45 min followed by 80°C for 20 min.

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: HIS4 (−96/+112) was amplified by PCR in a 1–2-ml reaction using the 32 P-labeled upstream primer and downstream primer (5′-TATTCCATGAGGCCAGATC-3′) and purified by electrophoresis on a 2% agarose gel. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer. .. In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer.

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: When comparing nucleosome positions in different strains or conditions it is critical to match the digestion levels for each sample analyzed ( ). .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification .. 1 Dilute overnight culture to OD600 = 0.1 in 250 ml fresh YPD media.

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. The sequence of the LF3 gene was obtained by sequencing plasmid clones with universal sequencing primers or gene-specific primers (DNA Sequencing and Synthesis Facility, Iowa State University, or Advanced Genetics Analysis Center, University of Minnesota).

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Second-strand cDNA was then bulk amplified with LongAmp HotStart Taq using Pac_for_dU and Pac_rev_dU primers, which contain an internal 5′-dUTP. .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min. .. The successfully circularized DNA was purified using the Oligo Clean & concentrator kit (Zymo, D4060).

    Article Title: Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence
    Article Snippet: Amplified DNA was quantified using a standard curve from a p8TR-gB plasmid dilution series. .. To detect replicated p8TR-gB, 6 μg of Hirt DNA was digested with 60 U DpnI in NEB buffer 2 in a 50-μl reaction mixture overnight at 37°C, followed by exonuclease III (ExoIII) (NEB) treatment (120 U) for 30 min at 37°C to reduce the background due to incompletely DpnI digested DNA ( , ).

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each). .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Autoradiography:

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above. .. To recover intact nucleosomes, 32 μg of salmon testes DNA (Sigma) was added to each 12-μl sample after the DNase I digestion, and then native electrophoresis was performed with gels containing 3.8% polyacrylamide (0.1% bisacrylamide) and 25% Tris-borate-EDTA ( ).

    Electrophoresis:

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: HIS4 (−96/+112) was amplified by PCR in a 1–2-ml reaction using the 32 P-labeled upstream primer and downstream primer (5′-TATTCCATGAGGCCAGATC-3′) and purified by electrophoresis on a 2% agarose gel. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Samples were incubated for several minutes at 65°C and then subjected to electrophoresis through 0.4-mm gels consisting of 8% polyacrylamide (0.4% bisacrylamide), 7 M urea, and 70% Tris-borate-EDTA ( ). .. Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above.

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each). .. Illumina paired-end sequencing adaptors were ligated using Rapid T4 DNA ligase (Enzymatics, MA).

    Microarray:

    Article Title: Structural and Regulatory Characterization of the Placental Epigenome at Its Maternal Interface
    Article Snippet: Paragraph title: Target DNA Preparation for Agilent Microarray Analysis ... Reactions were then incubated further with 75U (0.75 µl) Exonuclease III (NEB) and incubated at 30°C for 1 hour.

    Random Hexamer Labeling:

    Article Title: A paired-end sequencing strategy to map the complex landscape of transcription initiation
    Article Snippet: Ligation was performed with 5 cycles of 95°C for 30 sec, 68°C for 2 min, 55°C for 1 min and 60°C for 5 min, followed by 5 cycles of 95°C for 30 sec, 65°C for 2 min, 55°C for 1 min and 60°C for 5 min. To remove excess oligonucleotides and unligated DNA fragments, we added 3 μl Exonuclease I (NEB) and 0.6 μl Exonuclease III (NEB) to the ligation reaction and the mixture was incubated at 37°C for 45 min followed by 80°C for 20 min. .. This removes all linear DNA fragments (ssDNAs and dsDNAs) and the remaining circular DNAs were further amplified by rolling circle amplification (RCA).

    Activity Assay:

    Article Title: Dissection of the sequence-specific DNA binding and exonuclease activities reveals a superactive yet apoptotically impaired mutant p53 protein
    Article Snippet: Typically, 2–4 times molar excess of tetrameric p53 was incubated with DNA substrates in an exonuclease activity assay buffer containing 50 mM TrisHCl (pH 8.0), and 10 mM MgCl2 at 30°C for 30 min. .. For exonuclease III (New England BioLabs, Beverly, MA), 1–1 ×10−7 pmol of protein was incubated with 0.25 pmol of substrate as described above.

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Subsequently, 50 U (5 μl) of XmaI endonuclease (NEB) was added, and digestion continued for an additional 16 h. The digested DNA was purified using a QIAquick PCR purification kit (Qiagen, CA). .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each). .. Illumina paired-end sequencing adaptors were ligated using Rapid T4 DNA ligase (Enzymatics, MA).

    Expressing:

    Article Title: A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions
    Article Snippet: Plasmid midi-preps from E. coli cells expressing STEC mod methyltransferase and the negative control expressing a non-methyltransferase (SiaB), were prepared using the Qiagen plasmid midi kit according to the manufacturer's instructions. .. Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (USB; Cleveland, OH, USA).

    Genome Wide:

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Genome-wide DNA methylation analysis using next-generation sequencing ( , ) was performed for 20 samples for which a sufficient amount of DNA was available. .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Modification:

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta
    Article Snippet: An Exonuclease III deletion procedure, modified from , was used to produce progressive deletions from the telomere repeat end of the putative AVR-Pita clone. .. Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs).

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer. .. Circular DNA was then purified on Macherey-Nagel columns following a 25-min heating step at 85°C to inactivate the enzymes.

    Article Title: A highly selective, label-free, homogenous luminescent switch-on probe for the detection of nanomolar transcription factor NF-kappaB
    Article Snippet: The p50 protein was expressed and purified based on a modified procedure from Leung et al. ( ). .. Exonuclease III (ExoIII) was purchased from New England Biolabs.

    Hybridization:

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: A 4-kb PstI–SstI fragment from the cloned region was used as a hybridization probe to screen a λ phage library of Chlamydomonas WT genomic DNA and positive clones were tested for phenotypic rescue of lf3-5 or lf3-6 mutants by cotransformation using the pARG7.8 plasmid ( ) as described previously ( ). .. Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to .

    High Performance Liquid Chromatography:

    Article Title: Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes
    Article Snippet: The synthetic oligodeoxyribonucleotides and biotinylated oligodeoxyribonucleotides purchased from VBC Biotech (Vienna, Austria) were purified by HPLC as described previously . .. T4 DNA ligase and Exonuclease III were from New England Biolabs (Beverly, MA, USA).

    Sequencing:

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta
    Article Snippet: Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs). .. Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs).

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: These subclones were tested for phenotypic rescue of lf3-2 , lf3-5 , and lf3-6 by cotransformation. .. Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. The sequence of the LF3 gene was obtained by sequencing plasmid clones with universal sequencing primers or gene-specific primers (DNA Sequencing and Synthesis Facility, Iowa State University, or Advanced Genetics Analysis Center, University of Minnesota).

    Article Title: A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions
    Article Snippet: Paragraph title: Single-molecule, real-time (SMRT) sequencing and methylome analysis ... Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (USB; Cleveland, OH, USA).

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Paragraph title: SMRTbell library preparation and SMRT Sequencing ... After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Ligation:

    Article Title: A paired-end sequencing strategy to map the complex landscape of transcription initiation
    Article Snippet: 9 pmol “bridge” oligonucleotide (5′-GCC GTC TTC AGC CGC CTCA AGC TTC TAA CGA TGT ACG-3′) and 7.5 units of Ampligase (Epicentre) were then added. .. Ligation was performed with 5 cycles of 95°C for 30 sec, 68°C for 2 min, 55°C for 1 min and 60°C for 5 min, followed by 5 cycles of 95°C for 30 sec, 65°C for 2 min, 55°C for 1 min and 60°C for 5 min. To remove excess oligonucleotides and unligated DNA fragments, we added 3 μl Exonuclease I (NEB) and 0.6 μl Exonuclease III (NEB) to the ligation reaction and the mixture was incubated at 37°C for 45 min followed by 80°C for 20 min. .. This removes all linear DNA fragments (ssDNAs and dsDNAs) and the remaining circular DNAs were further amplified by rolling circle amplification (RCA).

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: The cross-linking of either 3C or 4C DNA products was then reversed overnight at 65°C following the addition of EDTA (final concentration, 1 mM), NaCl (final concentration, 0.2 M), and 10 or 20 μl of 10 μg/μl proteinase K. The samples were then subjected to three successive phenol-chloroform-isoamyl alcohol (25:24:1) extractions, followed by a chloroform washing step, diluted 4 times in water, and precipitated at −20°C for 2 h by 2 volumes of isopropanol. .. In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer. .. Circular DNA was then purified on Macherey-Nagel columns following a 25-min heating step at 85°C to inactivate the enzymes.

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: The the sticky ended cDNA for each size-bin was then ligated to 1 μM hairpin adapters that contain complementary sticky ends to create SMRTbell templates using 2000 Units T4 DNA ligase (New England Biolabs). .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates. .. SMRTbell libraries for the 2–3 kb and 3–6 kb size-bins were subjected to an additional round of size selection on the Sage BluePippin to remove trace amounts of small inserts.

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: Following ligation, the beads were washed three times with Dynabead wash buffer: twice at 58°C and once at room temperature. .. 1 μl Exo III (NEB catalog no. M0206S)) was added to the MIP reaction and samples were incubated at 37°C for 45 minutes.

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each). .. Illumina paired-end sequencing adaptors were ligated using Rapid T4 DNA ligase (Enzymatics, MA).

    SYBR Green Assay:

    Article Title: Introducing structure-switching functionality into small-molecule-binding aptamers via nuclease-directed truncation
    Article Snippet: Exonuclease III and exonuclease I were purchased from New England Biolabs. .. Exonuclease III and exonuclease I were purchased from New England Biolabs.

    Footprinting:

    Article Title: Multiple sequence-directed possibilities provide a pool of nucleosome position choices in different states of activity of a gene
    Article Snippet: Paragraph title: Exonuclease III footprinting ... The reconstituted mononucleosomes were digested with 20 U/ml Exonuclease III (NEB), for 0, 3, 6, and 9 minutes as compared with 0, .5, 1.5, and 2 minutes for the naked DNA samples.

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: Paragraph title: Exonuclease Footprinting ... Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Cell Culture:

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: For chromosome orientation FISH (CO-FISH), cells were cultured in 7.5 mM of 5-bromo-2′-deoxyuridine (BrdU) and 2.5 mM of 5-bromo-2′-deoxycytosine (BrdC) for 20 h prior to colcemid treatment and chromosome spread. .. Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described.

    Generated:

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: The cross-linking of either 3C or 4C DNA products was then reversed overnight at 65°C following the addition of EDTA (final concentration, 1 mM), NaCl (final concentration, 0.2 M), and 10 or 20 μl of 10 μg/μl proteinase K. The samples were then subjected to three successive phenol-chloroform-isoamyl alcohol (25:24:1) extractions, followed by a chloroform washing step, diluted 4 times in water, and precipitated at −20°C for 2 h by 2 volumes of isopropanol. .. In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer. .. Circular DNA was then purified on Macherey-Nagel columns following a 25-min heating step at 85°C to inactivate the enzymes.

    Imaging:

    Article Title: Multiple sequence-directed possibilities provide a pool of nucleosome position choices in different states of activity of a gene
    Article Snippet: The reconstituted mononucleosomes were digested with 20 U/ml Exonuclease III (NEB), for 0, 3, 6, and 9 minutes as compared with 0, .5, 1.5, and 2 minutes for the naked DNA samples. .. The reconstituted mononucleosomes were digested with 20 U/ml Exonuclease III (NEB), for 0, 3, 6, and 9 minutes as compared with 0, .5, 1.5, and 2 minutes for the naked DNA samples.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. The sequence of the LF3 gene was obtained by sequencing plasmid clones with universal sequencing primers or gene-specific primers (DNA Sequencing and Synthesis Facility, Iowa State University, or Advanced Genetics Analysis Center, University of Minnesota).

    Binding Assay:

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Paragraph title: Binding and DNase I assays. ... Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above.

    Fluorescence:

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: Paragraph title: Fluorescence in situ hybridization and immuno-fluorescence in situ hybridization ... Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described.

    Methylation:

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Paragraph title: Digital restriction enzyme analysis of methylation (DREAM) ... The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Mutagenesis:

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Paragraph title: Cloning and sequencing of the LF3 gene and the mutant lf3-2 allele ... Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to .

    Size-exclusion Chromatography:

    Article Title: A paired-end sequencing strategy to map the complex landscape of transcription initiation
    Article Snippet: 9 pmol “bridge” oligonucleotide (5′-GCC GTC TTC AGC CGC CTCA AGC TTC TAA CGA TGT ACG-3′) and 7.5 units of Ampligase (Epicentre) were then added. .. Ligation was performed with 5 cycles of 95°C for 30 sec, 68°C for 2 min, 55°C for 1 min and 60°C for 5 min, followed by 5 cycles of 95°C for 30 sec, 65°C for 2 min, 55°C for 1 min and 60°C for 5 min. To remove excess oligonucleotides and unligated DNA fragments, we added 3 μl Exonuclease I (NEB) and 0.6 μl Exonuclease III (NEB) to the ligation reaction and the mixture was incubated at 37°C for 45 min followed by 80°C for 20 min. .. This removes all linear DNA fragments (ssDNAs and dsDNAs) and the remaining circular DNAs were further amplified by rolling circle amplification (RCA).

    Labeling:

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: The labeled DNA (1 pmol) was incubated for 1 h at room temperature with 2 pmol of TFIIB, 1.6 pmol of TFIIA, 1.1 pmol of TBP, 2.4 pmol of TFIIE, 1.5 pmol of TFIIH*, and 1.2 pmol of pol II-TFIIF complex in 8 μl of buffer A; combined with 12 μl of buffer B; and incubated for 1 h at 4 °C. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Article Title: Dissection of the sequence-specific DNA binding and exonuclease activities reveals a superactive yet apoptotically impaired mutant p53 protein
    Article Snippet: Duplex substrates were prepared by purifying annealed DNA from 10% (w/v) polyacrylamide gels and quantitated as described in Ahn et al. DNA substrates were labeled at the 5'-end with 32 P by incubating with T4 polynucleotide kinase and [γ-32 P] ATP (4,500 Ci/mmol). .. For exonuclease III (New England BioLabs, Beverly, MA), 1–1 ×10−7 pmol of protein was incubated with 0.25 pmol of substrate as described above.

    Purification:

    Article Title: Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes
    Article Snippet: The synthetic oligodeoxyribonucleotides and biotinylated oligodeoxyribonucleotides purchased from VBC Biotech (Vienna, Austria) were purified by HPLC as described previously . .. T4 DNA ligase and Exonuclease III were from New England Biolabs (Beverly, MA, USA).

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: HIS4 (−96/+112) was amplified by PCR in a 1–2-ml reaction using the 32 P-labeled upstream primer and downstream primer (5′-TATTCCATGAGGCCAGATC-3′) and purified by electrophoresis on a 2% agarose gel. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: The cross-linking of either 3C or 4C DNA products was then reversed overnight at 65°C following the addition of EDTA (final concentration, 1 mM), NaCl (final concentration, 0.2 M), and 10 or 20 μl of 10 μg/μl proteinase K. The samples were then subjected to three successive phenol-chloroform-isoamyl alcohol (25:24:1) extractions, followed by a chloroform washing step, diluted 4 times in water, and precipitated at −20°C for 2 h by 2 volumes of isopropanol. .. In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer. .. Circular DNA was then purified on Macherey-Nagel columns following a 25-min heating step at 85°C to inactivate the enzymes.

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: When comparing nucleosome positions in different strains or conditions it is critical to match the digestion levels for each sample analyzed ( ). .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification .. 1 Dilute overnight culture to OD600 = 0.1 in 250 ml fresh YPD media.

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. The gene prediction programs GeneMark ( http://opal.biology.gatech.edu/GeneMark/eukhmm.cgi ) or Genscan ( http://genes.mit.edu/GENSCAN.html ) were used to predict the exon/intron structure of LF3 . cDNA sequences were obtained by RT-PCR using primers designed for putative exons under conditions described previously , except that RNA was treated with amplification-grade DNaseI (GIBCO BRL) for 15 min at RT to remove DNA contamination before reverse transcription.

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Purified, size-selected cDNA was eluted in 20 μl Low TE and then digested using 1 unit USER (New England Biolabs) to create sticky ends. .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: The size-selected and purified DNA was concentrated to 12 μl, then denatured at 95 °C for 3 min followed by incubation on ice for 3 min. Then the sample was supplemented with a mixture of 10X Cirligase buffer (1.5 μl), 50 mM MnCl2 (0.75 μl), Cirligase (0.75 μl) (Epicentre CL9025K) and further incubated at 60°Cfor 14 h before the reaction was stopped by heating at 80 °C for 10 min. .. Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: The ligated MIPs were further purified and fully eluted from the immobilized cDNA by digesting all linear DNA with exonucleases; 4 μl of exonuclease mix (3.5 μl 10× Exonuclease Buffer, 0.5 μl Exo I (NEB catalog no. M0293S. .. 1 μl Exo III (NEB catalog no. M0206S)) was added to the MIP reaction and samples were incubated at 37°C for 45 minutes.

    Article Title: A highly selective, label-free, homogenous luminescent switch-on probe for the detection of nanomolar transcription factor NF-kappaB
    Article Snippet: The p50 protein was expressed and purified based on a modified procedure from Leung et al. ( ). .. Exonuclease III (ExoIII) was purchased from New England Biolabs.

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Subsequently, 50 U (5 μl) of XmaI endonuclease (NEB) was added, and digestion continued for an additional 16 h. The digested DNA was purified using a QIAquick PCR purification kit (Qiagen, CA). .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Polymerase Chain Reaction:

    Article Title: A paired-end sequencing strategy to map the complex landscape of transcription initiation
    Article Snippet: First-strand cDNAs were amplified by 2-5 cycles of PCR with high-fidelity DNA polymerase (Finnzymes) to generate blunt-end dsDNAs. .. Ligation was performed with 5 cycles of 95°C for 30 sec, 68°C for 2 min, 55°C for 1 min and 60°C for 5 min, followed by 5 cycles of 95°C for 30 sec, 65°C for 2 min, 55°C for 1 min and 60°C for 5 min. To remove excess oligonucleotides and unligated DNA fragments, we added 3 μl Exonuclease I (NEB) and 0.6 μl Exonuclease III (NEB) to the ligation reaction and the mixture was incubated at 37°C for 45 min followed by 80°C for 20 min.

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: HIS4 (−96/+112) was amplified by PCR in a 1–2-ml reaction using the 32 P-labeled upstream primer and downstream primer (5′-TATTCCATGAGGCCAGATC-3′) and purified by electrophoresis on a 2% agarose gel. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: When comparing nucleosome positions in different strains or conditions it is critical to match the digestion levels for each sample analyzed ( ). .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification .. 1 Dilute overnight culture to OD600 = 0.1 in 250 ml fresh YPD media.

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to . .. Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to .

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Large-scale PCR products were purified with AMPure PB beads (Pacific Biosciences) and quality control was performed on a BioAnalyzer (Agilent). cDNA was then subjected to size fractionation using the Sage BluePippin system (Sage Science), collecting three size-bins: 1–2 kb, 2–3 kb, and 3–6 kb. .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Article Title: Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence
    Article Snippet: To detect total p8TR-gB, 2 μg of Hirt DNA was digested with 30 U of EcoRV (New England Biolabs [NEB]) in a 50-μl reaction mixture overnight at 37°C, followed by heat inactivation of EcoRV at 80°C for 30 min. PCR amplification was performed in triplicate using 3.75 μl of the EcoRV-digested product as the template. .. To detect replicated p8TR-gB, 6 μg of Hirt DNA was digested with 60 U DpnI in NEB buffer 2 in a 50-μl reaction mixture overnight at 37°C, followed by exonuclease III (ExoIII) (NEB) treatment (120 U) for 30 min at 37°C to reduce the background due to incompletely DpnI digested DNA ( , ).

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Subsequently, 50 U (5 μl) of XmaI endonuclease (NEB) was added, and digestion continued for an additional 16 h. The digested DNA was purified using a QIAquick PCR purification kit (Qiagen, CA). .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)). .. The DNA pellet was recovered by centrifugation at maximum speed for 1 h, dried at 37 °C, and resuspended in 10 μl of gel loading buffer (95% formamide, 0.02% bromphenol blue, 5 m m EDTA, and 0.025% xylene cyanol).

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Gels were fixed in 12% methanol and 10% acetic acid, dried, and then autoradiographed. .. Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above. .. DraI, HhaI, and StyI (New England Biolabs) were used in the same way except the NaCl was omitted from the binding reaction and replaced with 1× reaction buffer supplied with the enzyme, and 20 U of each enzyme was added per 100 fmol of DNA.

    Staining:

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: For chromosome orientation FISH (CO-FISH), cells were cultured in 7.5 mM of 5-bromo-2′-deoxyuridine (BrdU) and 2.5 mM of 5-bromo-2′-deoxycytosine (BrdC) for 20 h prior to colcemid treatment and chromosome spread. .. Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described. .. For immuno-FISH, the slides were immunostained with anti-γH2AX antibody and fluorescein-labeled second antibody.

    Agarose Gel Electrophoresis:

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: HIS4 (−96/+112) was amplified by PCR in a 1–2-ml reaction using the 32 P-labeled upstream primer and downstream primer (5′-TATTCCATGAGGCCAGATC-3′) and purified by electrophoresis on a 2% agarose gel. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    In Situ Hybridization:

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: Paragraph title: Fluorescence in situ hybridization and immuno-fluorescence in situ hybridization ... Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described.

    Plasmid Preparation:

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta
    Article Snippet: An Exonuclease III deletion procedure, modified from , was used to produce progressive deletions from the telomere repeat end of the putative AVR-Pita clone. .. Plasmid pCB783, containing the 791-bp telomeric SacI fragment, was digested first with KpnI and XhoI and then with Exonuclease III (New England Biolabs). .. The ExoIII-digested DNAs were treated with S1 nuclease (Sigma) followed by a fill-in reaction with the Klenow fragment of DNA polymerase I, ligation, and transformation into DH5αMCR cells (Gibco BRL).

    Article Title: Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas
    Article Snippet: Fragments of a lambda clone λ12D9, which was able to rescue the mutant phenotype, were subcloned into the vector pBluescript KS+ (Stratagene) using restriction sites indicated in A. .. Nested-deletion clones for sequencing were made using Exonuclease III (New England Biolabs, Inc.) and S1 nuclease (GIBCO BRL) according to .

    Article Title: A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions
    Article Snippet: Plasmid midi-preps from E. coli cells expressing STEC mod methyltransferase and the negative control expressing a non-methyltransferase (SiaB), were prepared using the Qiagen plasmid midi kit according to the manufacturer's instructions. .. Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (USB; Cleveland, OH, USA).

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: Alternatively, if the plasmid DNA less than 100 ng, the fragmentation step should be performed in the following shearing conditions: duty cycle: 10%, intensity: 5, cycles per burst: 100, time: 900 s, temperature < 4 °C, and purified with MinElute Reaction Cleanup Kit (3X buffer ERC) to remove small fragments (less than 70 bp). .. Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Article Title: Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence
    Article Snippet: Amplified DNA was quantified using a standard curve from a p8TR-gB plasmid dilution series. .. To detect replicated p8TR-gB, 6 μg of Hirt DNA was digested with 60 U DpnI in NEB buffer 2 in a 50-μl reaction mixture overnight at 37°C, followed by exonuclease III (ExoIII) (NEB) treatment (120 U) for 30 min at 37°C to reduce the background due to incompletely DpnI digested DNA ( , ).

    Software:

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above. .. DraI, HhaI, and StyI (New England Biolabs) were used in the same way except the NaCl was omitted from the binding reaction and replaced with 1× reaction buffer supplied with the enzyme, and 20 U of each enzyme was added per 100 fmol of DNA.

    Real-time Polymerase Chain Reaction:

    Article Title: Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence
    Article Snippet: PCRs were performed using an ABI 7300 real-time PCR system (Applied Biosystems) (1 cycle at 50°C for 2 min; 1 cycle at 95°C for 10 min; 40 cycles of 95°C for 30 s and 60°C for 1 min). .. To detect replicated p8TR-gB, 6 μg of Hirt DNA was digested with 60 U DpnI in NEB buffer 2 in a 50-μl reaction mixture overnight at 37°C, followed by exonuclease III (ExoIII) (NEB) treatment (120 U) for 30 min at 37°C to reduce the background due to incompletely DpnI digested DNA ( , ).

    Negative Control:

    Article Title: A survey of Type III restriction-modification systems reveals numerous, novel epigenetic regulators controlling phase-variable regulons; phasevarions
    Article Snippet: Plasmid midi-preps from E. coli cells expressing STEC mod methyltransferase and the negative control expressing a non-methyltransferase (SiaB), were prepared using the Qiagen plasmid midi kit according to the manufacturer's instructions. .. Incompletely formed SMRTbell templates were degraded with a combination of Exonuclease III (New England Biolabs; Ipswich, MA, USA) and Exonuclease VII (USB; Cleveland, OH, USA).

    Selection:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Following size selection, size-selected cDNA was re-amplified using LongAmp HotStart Taq. .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Sample Prep:

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: When comparing nucleosome positions in different strains or conditions it is critical to match the digestion levels for each sample analyzed ( ). .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification .. 1 Dilute overnight culture to OD600 = 0.1 in 250 ml fresh YPD media.

    Next-Generation Sequencing:

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Genome-wide DNA methylation analysis using next-generation sequencing ( , ) was performed for 20 samples for which a sufficient amount of DNA was available. .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Incubation:

    Article Title: A paired-end sequencing strategy to map the complex landscape of transcription initiation
    Article Snippet: 9 pmol “bridge” oligonucleotide (5′-GCC GTC TTC AGC CGC CTCA AGC TTC TAA CGA TGT ACG-3′) and 7.5 units of Ampligase (Epicentre) were then added. .. Ligation was performed with 5 cycles of 95°C for 30 sec, 68°C for 2 min, 55°C for 1 min and 60°C for 5 min, followed by 5 cycles of 95°C for 30 sec, 65°C for 2 min, 55°C for 1 min and 60°C for 5 min. To remove excess oligonucleotides and unligated DNA fragments, we added 3 μl Exonuclease I (NEB) and 0.6 μl Exonuclease III (NEB) to the ligation reaction and the mixture was incubated at 37°C for 45 min followed by 80°C for 20 min. .. This removes all linear DNA fragments (ssDNAs and dsDNAs) and the remaining circular DNAs were further amplified by rolling circle amplification (RCA).

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: For chromosome orientation FISH (CO-FISH), cells were cultured in 7.5 mM of 5-bromo-2′-deoxyuridine (BrdU) and 2.5 mM of 5-bromo-2′-deoxycytosine (BrdC) for 20 h prior to colcemid treatment and chromosome spread. .. Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described. .. For immuno-FISH, the slides were immunostained with anti-γH2AX antibody and fluorescein-labeled second antibody.

    Article Title: Formation and Fate of a Complete 31-Protein RNA Polymerase II Transcription Preinitiation Complex
    Article Snippet: The reconstituted PIC was combined with an equal volume of 2× NTP buffer (1.6 m m NTP(s) or 0.5 m m non-hydrolyzable analog ATPγS, 10 m m magnesium acetate, and 5 units of RNaseOUT) and incubated for 30 min at 30 °C. .. Exonuclease III digestion was performed with 200 units of exonuclease III (New England Biolabs) for 9 min at 30 °C and stopped by adding 185 μl of stop buffer II (10 m m Tris (pH 7.5), 300 m m sodium acetate (pH 5.5), 5 m m EDTA, 0.7% SDS, 0.1 mg/ml glycogen, 0.013 mg/ml proteinase K, and 0.5 mg/ml salmon sperm DNA (Invitrogen)).

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: Following these 5 days of ligation, 4C samples were further incubated for 1.5 h with 1 μl of T4 DNA ligase in order to ensure that the ligase filled any nicks in the circularized 3C products. .. In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer.

    Article Title: Dissection of the sequence-specific DNA binding and exonuclease activities reveals a superactive yet apoptotically impaired mutant p53 protein
    Article Snippet: Product formation was analyzed by PhosphoImager (Molecular Dynamics). .. For exonuclease III (New England BioLabs, Beverly, MA), 1–1 ×10−7 pmol of protein was incubated with 0.25 pmol of substrate as described above. .. For immunoprecipitation-exonuclease assays, each p53 protein (200 ng) purified from Ni-NTA column was immunoprecipitated with 20 µL of mAb1801 conjugated to Protein A-Agarose beads (Pharmacia LKB, Piscataway, NJ) in an exonuclease buffer.

    Article Title: Structural Features of Nucleosomes Reorganized by Yeast FACT and Its HMG Box Component, Nhp6
    Article Snippet: Gels were fixed in 12% methanol and 10% acetic acid, dried, and then autoradiographed. .. Exonuclease III (New England Biolabs) digestions were performed as for DNase I except that various amounts of exonuclease III were added and incubated at 30°C for 10 min before the reaction was terminated and samples were prepared for denaturing PAGE as above. .. DraI, HhaI, and StyI (New England Biolabs) were used in the same way except the NaCl was omitted from the binding reaction and replaced with 1× reaction buffer supplied with the enzyme, and 20 U of each enzyme was added per 100 fmol of DNA.

    Article Title: Structural and Regulatory Characterization of the Placental Epigenome at Its Maternal Interface
    Article Snippet: Following overnight digestion, reactions were digested further with 5 uL (50U) of TspRI (NEB) at 65°C for three hours. .. Reactions were then incubated further with 75U (0.75 µl) Exonuclease III (NEB) and incubated at 30°C for 1 hour. .. Enzymatic activity was then nullified by heating at 70°C for 20 min after which 50U of RecJF (NEB) were added to remove single stranded DNA.

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: The the sticky ended cDNA for each size-bin was then ligated to 1 μM hairpin adapters that contain complementary sticky ends to create SMRTbell templates using 2000 Units T4 DNA ligase (New England Biolabs). .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates. .. SMRTbell libraries for the 2–3 kb and 3–6 kb size-bins were subjected to an additional round of size selection on the Sage BluePippin to remove trace amounts of small inserts.

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: The size-selected and purified DNA was concentrated to 12 μl, then denatured at 95 °C for 3 min followed by incubation on ice for 3 min. Then the sample was supplemented with a mixture of 10X Cirligase buffer (1.5 μl), 50 mM MnCl2 (0.75 μl), Cirligase (0.75 μl) (Epicentre CL9025K) and further incubated at 60°Cfor 14 h before the reaction was stopped by heating at 80 °C for 10 min. .. Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min. .. The successfully circularized DNA was purified using the Oligo Clean & concentrator kit (Zymo, D4060).

    Article Title: A molecular inversion probe assay for detecting alternative splicing
    Article Snippet: The ligated MIPs were further purified and fully eluted from the immobilized cDNA by digesting all linear DNA with exonucleases; 4 μl of exonuclease mix (3.5 μl 10× Exonuclease Buffer, 0.5 μl Exo I (NEB catalog no. M0293S. .. 1 μl Exo III (NEB catalog no. M0206S)) was added to the MIP reaction and samples were incubated at 37°C for 45 minutes. .. The specific process used for amplifying circularized asMIPs was tailored to the method of quantitation; for arrays, MIPs were amplified with biotinylated PCR primers before being hybridized to microarrays.

    Spectrophotometry:

    Article Title: Structural and Regulatory Characterization of the Placental Epigenome at Its Maternal Interface
    Article Snippet: Reactions were then incubated further with 75U (0.75 µl) Exonuclease III (NEB) and incubated at 30°C for 1 hour. .. Reactions were then incubated further with 75U (0.75 µl) Exonuclease III (NEB) and incubated at 30°C for 1 hour.

    DNA Methylation Assay:

    Article Title: TET2 mutations affect non-CpG island DNA methylation at enhancers and transcription factor binding sites in chronic myelomonocytic leukemia
    Article Snippet: Genome-wide DNA methylation analysis using next-generation sequencing ( , ) was performed for 20 samples for which a sufficient amount of DNA was available. .. The 3′ recessed ends of the DNA created by XmaI digestion were filled in with 3′-dA tails added by Klenow DNA polymerase lacking 3′-to-5′ exonuclease activity (New England Biolabs, MA) and a dCTP, dGTP, and dATP mix (0.4 mM of each).

    Concentration Assay:

    Article Title: Dynamic Estrogen Receptor Interactomes Control Estrogen-Responsive Trefoil Factor (TFF) Locus Cell-Specific Activities
    Article Snippet: The cross-linking of either 3C or 4C DNA products was then reversed overnight at 65°C following the addition of EDTA (final concentration, 1 mM), NaCl (final concentration, 0.2 M), and 10 or 20 μl of 10 μg/μl proteinase K. The samples were then subjected to three successive phenol-chloroform-isoamyl alcohol (25:24:1) extractions, followed by a chloroform washing step, diluted 4 times in water, and precipitated at −20°C for 2 h by 2 volumes of isopropanol. .. In contrast, the 4C products generated by the 5-day ligation were then purified from linear DNA by a combined digestion with 7 μl of exonuclease I and 2 μl of exonuclease III (New England BioLabs) in a total volume of 100 μl of 1× exonuclease I buffer.

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: The ultimate DNA concentration was calibrated using Qubit 2.0 dsDNA HS Assay kit. .. Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Fractionation:

    Article Title: SMRT-Cappable-seq reveals complex operon variants in bacteria
    Article Snippet: Large-scale PCR products were purified with AMPure PB beads (Pacific Biosciences) and quality control was performed on a BioAnalyzer (Agilent). cDNA was then subjected to size fractionation using the Sage BluePippin system (Sage Science), collecting three size-bins: 1–2 kb, 2–3 kb, and 3–6 kb. .. After incubation at room temperature for 1 h for ligation, 100 Units Exonuclease III (New England Biolabs) and 10 Units Exonuclease VII (USB) were added into the ligation reaction to remove the incompletely formed SMRTbell templates.

    Gel Extraction:

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: When comparing nucleosome positions in different strains or conditions it is critical to match the digestion levels for each sample analyzed ( ). .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification .. 1 Dilute overnight culture to OD600 = 0.1 in 250 ml fresh YPD media.

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: Subsequently, the gel regions with DNAs in length of 60 ~ 100 bp marked by the 20 bp DNA ladder (Takara) were specifically cut out, and further extracted using QIAGEN MinElute Gel Extraction Kit (6X buffer QG). .. Subsequently, 0.75 μl Exonuclease I (NEB, M0293S) and 0.75 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Fluorescence In Situ Hybridization:

    Article Title: DNA-PKcs phosphorylates hnRNP-A1 to facilitate the RPA-to-POT1 switch and telomere capping after replication
    Article Snippet: For chromosome orientation FISH (CO-FISH), cells were cultured in 7.5 mM of 5-bromo-2′-deoxyuridine (BrdU) and 2.5 mM of 5-bromo-2′-deoxycytosine (BrdC) for 20 h prior to colcemid treatment and chromosome spread. .. Slides were stained with Hoechst 33258 (10 μg/ml), exposed to ultraviolet light (365-nm UV-A, 30 min), incubated with exonuclease III (1.6% v/v, New England Biolabs) for 10 min at RT, hybridized with Cy3-labeled C-rich and fluorescein isothiocyanate-labeled G-rich telomeric PNA probes (Panagene), followed by PBS washes and dehydrated as described.

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    New England Biolabs exonuclease iii e coli
    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq <t>(three</t> experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.
    Exonuclease Iii E Coli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Journal: Nature Communications

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq

    doi: 10.1038/ncomms15335

    Figure Lengend Snippet: Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Article Snippet: Subsequently, 1 μl Exonuclease I (NEB, M0293S) and 1 μl Exonuclease III (NEB, M0206S) were added into the reaction and incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Techniques: Mutagenesis, Two Tailed Test

    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: As positive control, the oligo AP was incubated with 1U Endo III (E . coli NTH1, New England Biolabs), or with 2U Exonuclease III (Exo III, E . coli AP endonuclease, New England Biolabs) or with 1 μg of purified recombinant TcAP1 T . cruzi AP endonuclease.

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

    DNA repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and (iii) the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.

    Journal: Nucleic Acids Research

    Article Title: Linking uracil base excision repair and 5-fluorouracil toxicity in yeast

    doi: 10.1093/nar/gkj430

    Figure Lengend Snippet: DNA repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and (iii) the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.

    Article Snippet: Briefly, 4 µg of each DNA sample was digested with E.coli exonuclease III (145 U; New England Biolabs) for 1 min at 37°C, 100 mM putrescine at 37°C for 30 min (Acros Organics), both exonuclease III and putrescine, or left undigested.

    Techniques: Derivative Assay, Knock-Out