fpg  (New England Biolabs)


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    Name:
    Fpg
    Description:
    Fpg 2 500 units
    Catalog Number:
    m0240l
    Price:
    301
    Size:
    2 500 units
    Category:
    Other Enzymes
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    Structured Review

    New England Biolabs fpg
    Fpg
    Fpg 2 500 units
    https://www.bioz.com/result/fpg/product/New England Biolabs
    Average 95 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    fpg - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage"

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage

    Journal: Chembiochem : a European journal of chemical biology

    doi: 10.1002/cbic.201500184

    A) Fluorescence emission of 4 μM probe OGR1 in Fpg reaction buffer at 37 °C without Fpg (gray) and after 25 min with Fpg (black). B) Time course of 4 μM probe OGR1 emission at 465 nm following addition of 658 nM Fpg (black triangles) or hOGG1 (gray circles) at 37 °C. This is a representative curve of n=1.
    Figure Legend Snippet: A) Fluorescence emission of 4 μM probe OGR1 in Fpg reaction buffer at 37 °C without Fpg (gray) and after 25 min with Fpg (black). B) Time course of 4 μM probe OGR1 emission at 465 nm following addition of 658 nM Fpg (black triangles) or hOGG1 (gray circles) at 37 °C. This is a representative curve of n=1.

    Techniques Used: Fluorescence

    A and B) PAGE analysis of 4 μM probe OGR1 radiolabeled at the 5′ end with [ 32 P]-phosphate and reacted with 658 nM hOGG1 (A) or Fpg (B) at 37 °C for various times. Piperidine (200 mM, 95 °C, 5 min), which cleaves the DNA backbone at abasic sites, was added to samples as indicated to identify the 8-oxoguanine excision product and the backbone cleavage product. C and D) MALDI mass spectrometry analysis of probe OGR1 following overnight reaction with hOGG1 (C) or Fpg (D).
    Figure Legend Snippet: A and B) PAGE analysis of 4 μM probe OGR1 radiolabeled at the 5′ end with [ 32 P]-phosphate and reacted with 658 nM hOGG1 (A) or Fpg (B) at 37 °C for various times. Piperidine (200 mM, 95 °C, 5 min), which cleaves the DNA backbone at abasic sites, was added to samples as indicated to identify the 8-oxoguanine excision product and the backbone cleavage product. C and D) MALDI mass spectrometry analysis of probe OGR1 following overnight reaction with hOGG1 (C) or Fpg (D).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Mass Spectrometry

    2) Product Images from "Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair"

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr463

    Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug Fpg or 1.2 µg/plug Nth for 24 h at 20°C before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.
    Figure Legend Snippet: Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug Fpg or 1.2 µg/plug Nth for 24 h at 20°C before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.

    Techniques Used: Incubation, Irradiation

    3) Product Images from "The DNA Glycosylase, Ogg1, Defends Against Oxidant-induced mtDNA Damage and Apoptosis in Pulmonary Artery Endothelial Cells"

    Article Title: The DNA Glycosylase, Ogg1, Defends Against Oxidant-induced mtDNA Damage and Apoptosis in Pulmonary Artery Endothelial Cells

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2010.10.692

    Lack of nuclear DNA fragmentation as detected by the Fpg-FLARE Comet assay in pulmonary artery endothelial cells transfected with either scrambled (A) or Ogg1-specific (B) siRNA and harvested immediately after 1 hour treatment with xanthine oxidase (5 mU/ml). Note lack of Comet “tails”. Negative results also were obtained in cells treated with 2 and 10 mU/ml of XO using either the conventional Comet assay or the Fpg-FLARE. (C) Pulmonary artery endothelial cells without any treatment were used as a negative control. (D) PAECs treated with 1 mM hydrogen peroxide for 15 min at 4°C were used as a positive control for the assay.
    Figure Legend Snippet: Lack of nuclear DNA fragmentation as detected by the Fpg-FLARE Comet assay in pulmonary artery endothelial cells transfected with either scrambled (A) or Ogg1-specific (B) siRNA and harvested immediately after 1 hour treatment with xanthine oxidase (5 mU/ml). Note lack of Comet “tails”. Negative results also were obtained in cells treated with 2 and 10 mU/ml of XO using either the conventional Comet assay or the Fpg-FLARE. (C) Pulmonary artery endothelial cells without any treatment were used as a negative control. (D) PAECs treated with 1 mM hydrogen peroxide for 15 min at 4°C were used as a positive control for the assay.

    Techniques Used: Single Cell Gel Electrophoresis, Transfection, Negative Control, Positive Control

    4) Product Images from "Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations"

    Article Title: Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations

    Journal: eLife

    doi: 10.7554/eLife.51605

    Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).
    Figure Legend Snippet: Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).

    Techniques Used: Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, Staining, Generated, Oligonucleotide Labeling, Ligation

    5) Product Images from "Oxidative DNA damage in lung tissue from patients with COPD is clustered in functionally significant sequences"

    Article Title: Oxidative DNA damage in lung tissue from patients with COPD is clustered in functionally significant sequences

    Journal: International Journal of Chronic Obstructive Pulmonary Disease

    doi: 10.2147/COPD.S15922

    Fpg-sensitive oxidative DNA damage in a VEGF promoter sequence containing the hypoxic response element (HRE; top) but not in a functionally insignificant promoter (middle) or coding (bottom) sequences of the VEGF gene in lung tissue from control subjects and lung tissue from patients with GOLD 4 COPD. See Table 1 for relative positions of sequences examined. Each point represents lung tissue from an individual patient. Note: *Significantly decreased “% intact DNA” in comparison to control at P
    Figure Legend Snippet: Fpg-sensitive oxidative DNA damage in a VEGF promoter sequence containing the hypoxic response element (HRE; top) but not in a functionally insignificant promoter (middle) or coding (bottom) sequences of the VEGF gene in lung tissue from control subjects and lung tissue from patients with GOLD 4 COPD. See Table 1 for relative positions of sequences examined. Each point represents lung tissue from an individual patient. Note: *Significantly decreased “% intact DNA” in comparison to control at P

    Techniques Used: Sequencing

    6) Product Images from "Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells"

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158581

    Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.
    Figure Legend Snippet: Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.

    Techniques Used: Construct, Purification, Negative Control, Positive Control

    7) Product Images from "Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells"

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0158581

    Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.
    Figure Legend Snippet: Optimizations for second strand synthesis. (A) Schematic of the second strand synthesis procedure. Synthetic 5’ phosphorylated ODNs containing the lesion of interest are annealed to phagemid single-stranded DNA, complimentary strands are synthesised by T4 DNA polymerase, and ligated by T4 DNA ligase. (B) Second strand synthesis of HRAS construct using ssDNA purified by silica spin columns or anion-exchange columns. ssDNA purified by anion-exchange column produces high yields of covalently closed product. (C) Schematic of the alkaline gel analysis of the construct nicks positions. Double-digest of pcDNA3.1(+)-HRAS with SmaI and NdeI produces two fragments (labelled 1 and 2). If the synthetic ODN that becomes part of the transcribed strand is not ligated, the transcribed strand fragment 2 produces two smaller fragments (3 and 4). (D) Alkaline gel analysis of HRAS constructs. Negative control HRAS WT T5 exonuclease (T5 exo) treated, covalently closed construct produces only two bands and positive control Fpg nicked HRAS 8-oxoG constructs, treated and not treated with T5 exonuclease, produce the expected four bands. The anion-exchange purified HRAS WT construct produces only two bands, indicating the nicks following second strand synthesis occur at random positions.

    Techniques Used: Construct, Purification, Negative Control, Positive Control

    8) Product Images from "Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site"

    Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site

    Journal: Genes and Environment

    doi: 10.1186/s41021-018-0112-5

    Preparation of DNA template (8-oxoG substrate) with 8-oxoG at a defined position. a Upper strand sequence containing 8-oxoG base and lower strand substrate containing original C base in pBS2/8-oxoG are shown diagrammatically. b Experimental procedure for purification of pBS2/8-oxoG using 8-oxoG oligo. c Identified DNA substrates (100 ng), pBS2/8-oxoG, were incubated with Fpg (2 units) or hOGG1 (0.16 units) at 37 °C for 30 min. Aliquots from the sample were run on 0.8% agarose gel and visualized by staining with EtBr. Lane 1, 1-kbp marker; lane 2, non-treatment; lane 3, Fpg-treatment; lane 4, hOGGI-treatment. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows
    Figure Legend Snippet: Preparation of DNA template (8-oxoG substrate) with 8-oxoG at a defined position. a Upper strand sequence containing 8-oxoG base and lower strand substrate containing original C base in pBS2/8-oxoG are shown diagrammatically. b Experimental procedure for purification of pBS2/8-oxoG using 8-oxoG oligo. c Identified DNA substrates (100 ng), pBS2/8-oxoG, were incubated with Fpg (2 units) or hOGG1 (0.16 units) at 37 °C for 30 min. Aliquots from the sample were run on 0.8% agarose gel and visualized by staining with EtBr. Lane 1, 1-kbp marker; lane 2, non-treatment; lane 3, Fpg-treatment; lane 4, hOGGI-treatment. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows

    Techniques Used: Sequencing, Purification, Incubation, Agarose Gel Electrophoresis, Staining, Marker, Countercurrent Chromatography

    9) Product Images from "Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine"

    Article Title: Widespread transcriptional gene inactivation initiated by a repair intermediate of 8-oxoguanine

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw473

    Construction of plasmid vectors containing the indicated DNA base (8-oxoG) and backbone (phosphorothioate) modifications in the non-transcribed strand of the reporter EGFP gene. ( A ) Out of scale scheme of the pZA expression vector showing the EGFP coding sequence (signed open arrow), transcription start (broken arrow) and tandem Nt.Bpu10I nicking sites used for site-specific insertion of synthetic oligonucleotides. ( B ) Overview of synthetic oligonucleotides and the contained modifications. ( C ) Verification of the incorporation of the indicated synthetic DNA strands into vector DNA. Inhibition of ligation reaction in the absence of polynucleotidekinase (PNK) is an indicator of the successful strand exchange reaction, as described previously ( 31 ). The presence of 8-oxoG is confirmed by DNA strand scission by bacterial Fpg. Positions of covalently closed (cc) and the nicked circular (nc) forms are shown. ( D ) Incision of plasmid vectors containing the specified DNA modifications with 0.5 units human OGG1.
    Figure Legend Snippet: Construction of plasmid vectors containing the indicated DNA base (8-oxoG) and backbone (phosphorothioate) modifications in the non-transcribed strand of the reporter EGFP gene. ( A ) Out of scale scheme of the pZA expression vector showing the EGFP coding sequence (signed open arrow), transcription start (broken arrow) and tandem Nt.Bpu10I nicking sites used for site-specific insertion of synthetic oligonucleotides. ( B ) Overview of synthetic oligonucleotides and the contained modifications. ( C ) Verification of the incorporation of the indicated synthetic DNA strands into vector DNA. Inhibition of ligation reaction in the absence of polynucleotidekinase (PNK) is an indicator of the successful strand exchange reaction, as described previously ( 31 ). The presence of 8-oxoG is confirmed by DNA strand scission by bacterial Fpg. Positions of covalently closed (cc) and the nicked circular (nc) forms are shown. ( D ) Incision of plasmid vectors containing the specified DNA modifications with 0.5 units human OGG1.

    Techniques Used: Plasmid Preparation, Expressing, Sequencing, Inhibition, Ligation

    10) Product Images from "A novel use of random priming-based single-strand library preparation for whole genome sequencing of formalin-fixed paraffin-embedded tissue samples"

    Article Title: A novel use of random priming-based single-strand library preparation for whole genome sequencing of formalin-fixed paraffin-embedded tissue samples

    Journal: Nar Genomics and Bioinformatics

    doi: 10.1093/nargab/lqz017

    Work flow of the standard versus DDAT library preparation method. To generate WGS libraries from low-input, degraded DNA, the complete protocol starts with the addition of enzymes SMUG1 (single-strand-selective monofunctional uracil-DNA glycosylase) and Fpg (formamidopyrimidine [fapy]-DNA glycosylase) to the input DNA ( A and B ) that remove damaged bases such as deoxyuracil and 8-oxoguanine, caused by the FFPE treatment. A short denaturation step (B) is followed by the first strand synthesis; during this step, the genomic DNA, primers and Klenow fragment (3′ → 5′ exo-) are gradually heated from 4 to 37°C with a slow ramping speed of 4°C/min, which is an essential reaction condition (see ‘Discussion’ section), before incubation at 37°C for a further 1.5 h ( C ). The primers contain nine random nucleotides from the 3′-end, in addition to the standard Illumina adaptor sequence, and will anneal to complementary DNA sequences present in the DNA sample. After the first strand synthesis, any remaining primers or short ssDNA fragments are digested with exonuclease I and the dsDNA is purified with AMPure XP beads. Next, the dsDNA is denatured to carry out the second strand synthesis using a second adaptor primer also containing nine random nucleotides, with the same conditions as the first synthesis, followed by bead purification (C). Finally, 10 PCR cycles are carried out using standard Illumina p5 and p7 indexed primers ( D ). The library is purified and assessed using standard quality control methods.
    Figure Legend Snippet: Work flow of the standard versus DDAT library preparation method. To generate WGS libraries from low-input, degraded DNA, the complete protocol starts with the addition of enzymes SMUG1 (single-strand-selective monofunctional uracil-DNA glycosylase) and Fpg (formamidopyrimidine [fapy]-DNA glycosylase) to the input DNA ( A and B ) that remove damaged bases such as deoxyuracil and 8-oxoguanine, caused by the FFPE treatment. A short denaturation step (B) is followed by the first strand synthesis; during this step, the genomic DNA, primers and Klenow fragment (3′ → 5′ exo-) are gradually heated from 4 to 37°C with a slow ramping speed of 4°C/min, which is an essential reaction condition (see ‘Discussion’ section), before incubation at 37°C for a further 1.5 h ( C ). The primers contain nine random nucleotides from the 3′-end, in addition to the standard Illumina adaptor sequence, and will anneal to complementary DNA sequences present in the DNA sample. After the first strand synthesis, any remaining primers or short ssDNA fragments are digested with exonuclease I and the dsDNA is purified with AMPure XP beads. Next, the dsDNA is denatured to carry out the second strand synthesis using a second adaptor primer also containing nine random nucleotides, with the same conditions as the first synthesis, followed by bead purification (C). Finally, 10 PCR cycles are carried out using standard Illumina p5 and p7 indexed primers ( D ). The library is purified and assessed using standard quality control methods.

    Techniques Used: Flow Cytometry, Formalin-fixed Paraffin-Embedded, Incubation, Sequencing, Purification, Polymerase Chain Reaction

    11) Product Images from "Characterization of DNA glycosylase activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry"

    Article Title: Characterization of DNA glycosylase activity by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

    Journal: Analytical biochemistry

    doi: 10.1016/j.ab.2009.07.015

    Gel electrophoretic profiles of substrate specificity of glycosylases. The oligonucleotide containing U was incubated with UNG followed by NaOH (lane 3) or APE1 (lane 4). The oligonucleotide containing FoU was incubated with hSMUG1 followed by APE1 (lane 6), Fpg (lane 8) and endoIII (lane 10). The oligonucleotide containing oxoG was incubated with Fpg (lane 12) and hOGG1 (lane 14).
    Figure Legend Snippet: Gel electrophoretic profiles of substrate specificity of glycosylases. The oligonucleotide containing U was incubated with UNG followed by NaOH (lane 3) or APE1 (lane 4). The oligonucleotide containing FoU was incubated with hSMUG1 followed by APE1 (lane 6), Fpg (lane 8) and endoIII (lane 10). The oligonucleotide containing oxoG was incubated with Fpg (lane 12) and hOGG1 (lane 14).

    Techniques Used: Incubation

    12) Product Images from "Identification of a Chemical That Inhibits the Mycobacterial UvrABC Complex in Nucleotide Excision Repair †"

    Article Title: Identification of a Chemical That Inhibits the Mycobacterial UvrABC Complex in Nucleotide Excision Repair †

    Journal: Biochemistry

    doi: 10.1021/bi101674c

    Nucleotide incision of peroxynitrite-damaged plasmid DNA by Fpg and Mtb UvrA, UvrB, and UvrC proteins. (A) Plasmid DNA (0.5 mg/mL) was incubated with increasing concentrations of peroxynitrite (PN) in buffer containing 150 mM potassium phosphate buffer (pH 7.2) and 25 mM sodium bicarbonate. The DNA (500 ng) was resolved on 1% agarose gels, and percent nicked products was plotted vs concentration of PN. The data show means ± SD from three independent experiments (lower panel). Error bars fall within the symbols. (B) Plasmid (25 nM) treated with PN (open triangles, 50 μM; open circles, 200 μM) and incubated with increasing amounts of Fpg. Means ± SD from three independent experiments (lower panel). (C) Plasmid (25 nM) treated with PN (5.0 × 10 −5 M) was incubated with 100 nM UvrA, increasing concentrations of UvrB (0, 50, 100, 200, or 300 nM), and 150 nM UvrC. The data show means ± SD from three independent experiments (lower panel).
    Figure Legend Snippet: Nucleotide incision of peroxynitrite-damaged plasmid DNA by Fpg and Mtb UvrA, UvrB, and UvrC proteins. (A) Plasmid DNA (0.5 mg/mL) was incubated with increasing concentrations of peroxynitrite (PN) in buffer containing 150 mM potassium phosphate buffer (pH 7.2) and 25 mM sodium bicarbonate. The DNA (500 ng) was resolved on 1% agarose gels, and percent nicked products was plotted vs concentration of PN. The data show means ± SD from three independent experiments (lower panel). Error bars fall within the symbols. (B) Plasmid (25 nM) treated with PN (open triangles, 50 μM; open circles, 200 μM) and incubated with increasing amounts of Fpg. Means ± SD from three independent experiments (lower panel). (C) Plasmid (25 nM) treated with PN (5.0 × 10 −5 M) was incubated with 100 nM UvrA, increasing concentrations of UvrB (0, 50, 100, 200, or 300 nM), and 150 nM UvrC. The data show means ± SD from three independent experiments (lower panel).

    Techniques Used: Plasmid Preparation, Incubation, Concentration Assay

    13) Product Images from "8-oxoguanine DNA glycosylase (OGG1) deficiency elicits coordinated changes in lipid and mitochondrial metabolism in muscle"

    Article Title: 8-oxoguanine DNA glycosylase (OGG1) deficiency elicits coordinated changes in lipid and mitochondrial metabolism in muscle

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181687

    DNA damage estimations: mtDNA long amplicon PCR and total 8-oxoG content. A) Amplification of a long fragment of mtDNA was performed before and after treatment with FPG, a bacterial OGG1 functional analog. Data were normalized to amplification of a short mtDNA fragment. B) Total DNA was isolated by Miniprep and analyzed by ELISA for 8-oxoG content. Data are representative of 6 animals per genotype run in technical duplicates and expressed as mean ± SEM; , p
    Figure Legend Snippet: DNA damage estimations: mtDNA long amplicon PCR and total 8-oxoG content. A) Amplification of a long fragment of mtDNA was performed before and after treatment with FPG, a bacterial OGG1 functional analog. Data were normalized to amplification of a short mtDNA fragment. B) Total DNA was isolated by Miniprep and analyzed by ELISA for 8-oxoG content. Data are representative of 6 animals per genotype run in technical duplicates and expressed as mean ± SEM; , p

    Techniques Used: Amplification, Polymerase Chain Reaction, Functional Assay, Isolation, Enzyme-linked Immunosorbent Assay

    14) Product Images from "The Non-Bulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in vitro"

    Article Title: The Non-Bulky DNA Lesions Spiroiminodihydantoin and 5-Guanidinohydantoin Significantly Block Human RNA Polymerase II Elongation in vitro

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.7b00295

    Verification of template integrity of unmodified (UM), Gh and S -Sp modified linear templates by enzymatic digestion with I-PpoI and Fpg proteins. (A) Representative schematic of enzymatic digests to verify template integrity. I-PpoI digestion is used for verification of complete ligation of the DNA duplex to the CMV promoter fragment. The Fpg assay results in excision of the S- Sp and Gh lesions, generating 67 and 123 nucleotide fragments. (B): Representative denaturing gel showing the products of digestion of the linear templates with I-PpoI. (C) Representative denaturing gel showing the products of digestion first with I-PpoI, followed by treatment with Fpg-protein. Lanes M: oligodeoxynucleotide size markers (5′-[ 32 P]phosphate-labeled 50 bp Ladder (New England Biolabs)).
    Figure Legend Snippet: Verification of template integrity of unmodified (UM), Gh and S -Sp modified linear templates by enzymatic digestion with I-PpoI and Fpg proteins. (A) Representative schematic of enzymatic digests to verify template integrity. I-PpoI digestion is used for verification of complete ligation of the DNA duplex to the CMV promoter fragment. The Fpg assay results in excision of the S- Sp and Gh lesions, generating 67 and 123 nucleotide fragments. (B): Representative denaturing gel showing the products of digestion of the linear templates with I-PpoI. (C) Representative denaturing gel showing the products of digestion first with I-PpoI, followed by treatment with Fpg-protein. Lanes M: oligodeoxynucleotide size markers (5′-[ 32 P]phosphate-labeled 50 bp Ladder (New England Biolabs)).

    Techniques Used: Modification, Ligation, Labeling

    15) Product Images from "HYPOXIA-INDUCED OXIDATIVE BASE MODIFICATIONS IN THE VEGF HYPOXIC RESPONSE ELEMENT ARE ASSOCIATED WITH TRANSCRIPTIONALLY ACTIVE NUCLEOSOMES"

    Article Title: HYPOXIA-INDUCED OXIDATIVE BASE MODIFICATIONS IN THE VEGF HYPOXIC RESPONSE ELEMENT ARE ASSOCIATED WITH TRANSCRIPTIONALLY ACTIVE NUCLEOSOMES

    Journal:

    doi: 10.1016/j.freeradbiomed.2008.09.038

    (A) PCR of treated and untreated with Fpg DNA fragments isolated from monomer, dimer, and trimer nucleosomal repeates and multi-nucleosomal zone with primers specific for the HRE of the VEGF promoter and 28S rRNA. (B) Pooled data for +/−Fpg PCR
    Figure Legend Snippet: (A) PCR of treated and untreated with Fpg DNA fragments isolated from monomer, dimer, and trimer nucleosomal repeates and multi-nucleosomal zone with primers specific for the HRE of the VEGF promoter and 28S rRNA. (B) Pooled data for +/−Fpg PCR

    Techniques Used: Polymerase Chain Reaction, Isolation

    (A) Southern blot analysis of the VEGF hypoxic response element of DNA fragments released from MN-digested (15 min) nuclei of normoxic (N) and hypoxic (H) PAECs with or without myxothiazol treatment. (B) PCR analysis of treated and untreated with Fpg
    Figure Legend Snippet: (A) Southern blot analysis of the VEGF hypoxic response element of DNA fragments released from MN-digested (15 min) nuclei of normoxic (N) and hypoxic (H) PAECs with or without myxothiazol treatment. (B) PCR analysis of treated and untreated with Fpg

    Techniques Used: Southern Blot, Polymerase Chain Reaction

    (A) Southern blot analysis of the VEGF hypoxic response element of DNA fragments released from MN-digested (15 or 30 min) nuclei of normoxic (N) and hypoxic (H) PAECs with or without TSA treatment. (B) PCR analysis of treated and untreated with Fpg DNA
    Figure Legend Snippet: (A) Southern blot analysis of the VEGF hypoxic response element of DNA fragments released from MN-digested (15 or 30 min) nuclei of normoxic (N) and hypoxic (H) PAECs with or without TSA treatment. (B) PCR analysis of treated and untreated with Fpg DNA

    Techniques Used: Southern Blot, Polymerase Chain Reaction

    16) Product Images from "Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site"

    Article Title: Oligo swapping method for in vitro DNA repair substrate containing a single DNA lesion at a specific site

    Journal: Genes and Environment

    doi: 10.1186/s41021-018-0112-5

    Preparation of DNA template (8-oxoG substrate) with 8-oxoG at a defined position. a Upper strand sequence containing 8-oxoG base and lower strand substrate containing original C base in pBS2/8-oxoG are shown diagrammatically. b Experimental procedure for purification of pBS2/8-oxoG using 8-oxoG oligo. c Identified DNA substrates (100 ng), pBS2/8-oxoG, were incubated with Fpg (2 units) or hOGG1 (0.16 units) at 37 °C for 30 min. Aliquots from the sample were run on 0.8% agarose gel and visualized by staining with EtBr. Lane 1, 1-kbp marker; lane 2, non-treatment; lane 3, Fpg-treatment; lane 4, hOGGI-treatment. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows
    Figure Legend Snippet: Preparation of DNA template (8-oxoG substrate) with 8-oxoG at a defined position. a Upper strand sequence containing 8-oxoG base and lower strand substrate containing original C base in pBS2/8-oxoG are shown diagrammatically. b Experimental procedure for purification of pBS2/8-oxoG using 8-oxoG oligo. c Identified DNA substrates (100 ng), pBS2/8-oxoG, were incubated with Fpg (2 units) or hOGG1 (0.16 units) at 37 °C for 30 min. Aliquots from the sample were run on 0.8% agarose gel and visualized by staining with EtBr. Lane 1, 1-kbp marker; lane 2, non-treatment; lane 3, Fpg-treatment; lane 4, hOGGI-treatment. Open circular DNA (OC), liner DNA (Lin), and covalently closed circular DNA (CCC) are indicated by arrows

    Techniques Used: Sequencing, Purification, Incubation, Agarose Gel Electrophoresis, Staining, Marker, Countercurrent Chromatography

    17) Product Images from "Oxidative Stress Induces Persistent Telomeric DNA Damage Responsible for Nuclear Morphology Change in Mammalian Cells"

    Article Title: Oxidative Stress Induces Persistent Telomeric DNA Damage Responsible for Nuclear Morphology Change in Mammalian Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0110963

    FPG-sensitive sites within telomeric DNA. Quantitative PCR was used to evaluate the amount of FPG-sensitive sites within telomeric sequences after 1 hr treatment with hydrogen peroxide. The columns denote the results obtained from two different doses of H 2 O 2 (100 and 200 µM). We observed a significant increase (p
    Figure Legend Snippet: FPG-sensitive sites within telomeric DNA. Quantitative PCR was used to evaluate the amount of FPG-sensitive sites within telomeric sequences after 1 hr treatment with hydrogen peroxide. The columns denote the results obtained from two different doses of H 2 O 2 (100 and 200 µM). We observed a significant increase (p

    Techniques Used: Real-time Polymerase Chain Reaction

    Standard and FPG-modified alkaline comet assay. Standard and FPG-modified versions of the comet assay have been used to evaluate the fold increase in genomic damage induced by hydrogen peroxide with respect to the control value. The net cleavage sites generated by FPG activity were calculated subtracting the value of total DNA damage yielded by the samples not treated with the enzyme from the DNA damage value obtained from samples treated with the enzyme. The value of this subtraction is shown in the graph as a light grey portion labelled ‘‘FPG sites.’’ To assess any changes in Tail DNA values, we analysed control cells immediately (time 0) and after 15 hrs treatment. All values are normalised to the control values. A significant increase (p
    Figure Legend Snippet: Standard and FPG-modified alkaline comet assay. Standard and FPG-modified versions of the comet assay have been used to evaluate the fold increase in genomic damage induced by hydrogen peroxide with respect to the control value. The net cleavage sites generated by FPG activity were calculated subtracting the value of total DNA damage yielded by the samples not treated with the enzyme from the DNA damage value obtained from samples treated with the enzyme. The value of this subtraction is shown in the graph as a light grey portion labelled ‘‘FPG sites.’’ To assess any changes in Tail DNA values, we analysed control cells immediately (time 0) and after 15 hrs treatment. All values are normalised to the control values. A significant increase (p

    Techniques Used: Modification, Alkaline Single Cell Gel Electrophoresis, Single Cell Gel Electrophoresis, Generated, Activity Assay

    Related Articles

    Amplification:

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: .. The total 10 μl mixture was denatured at 95 °C for 3 min and left on ice immediately for another 3 min before incubation with 9 μl Reaction buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl of Enzyme Mix (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) at 30 °C for 35 ~ 65 min. .. The reaction was stopped by incubation at 65 °C for 10 min when the amplification product reached to 0.5 ~ 1 μg (monitored by Qubit).

    Article Title: DNA damage in normally and prematurely aged mice
    Article Snippet: .. Additionally, in parallel with FPG treatment, samples intended for analysis of the mitochondrial genome were cleaved with XhoI restriction endonuclease (NEB), which has a unique recognition side outside of the amplified region, which helps to relax supercoiled mtDNA, making it more suitable for PCR amplification. .. Electrophoresis was performed in 0.8% agarose (Bio-Rad, Hercules, CA, USA) gels prepared and run with standard 1x TBE buffer (Fisher Scientific, Pittsburgh, PA, USA).

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq
    Article Snippet: .. The total 10 μl mixture was denatured at 95 °C for 3 min and left on ice immediately for another 3 min before incubation with 9 μl Reaction Buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl of Enzyme Mix (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl UDG (Uracil-DNA Glycosylase, NEB, M0280S), 1 μl Fpg (NEB, M0240S) at 30 °C for 35–65 min. .. The total 10 μl mixture was denatured at 95 °C for 3 min and left on ice immediately for another 3 min before incubation with 9 μl Reaction Buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl of Enzyme Mix (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl UDG (Uracil-DNA Glycosylase, NEB, M0280S), 1 μl Fpg (NEB, M0240S) at 30 °C for 35–65 min.

    Article Title: Ultra-precise detection of mutations by droplet-based amplification of circularized DNA
    Article Snippet: A column of 2.5 μl circularized DNA was placed into 0.2-ml tubes containing 5 μl 10X phi29 DNA Polymerase Reaction Buffer (NEB, M0269S), 2.5 μl exonuclease-resistant hexamer (500 μM), 2.5 μl 10 mM dNTP, 1 μl 100XBSA, 27 μl ddH2 O, which was denatured at 95 °C for 3 min, then put on ice immediately for another 3 min, and further added by a mixture of 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) and 2.5 μl phi29 DNA Polymerase (NEB, M0269S). .. The emulsion was broken using PFO (1H, 1H, 2H, 2H, - Perflurooctanol) to recover the amplified DNA as follows: adding 2 ~ 3 volume of PFO (100 ~ 150 μl) to the reaction mixture and admixing thoroughly, incubating at room temperature for 5 min, then centrifuging at 3000 rpm for 5 min.

    Synthesized:

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq
    Article Snippet: The adapter was synthesized from two oligonucleotides, /5phos/5′-GATCAGTCGTACGTGCTTACTCTCAATAGCAGCTT-3′ and /5phos/5′-GTGGGCAGTCGGTGAACGACTGAUCT-3′ (Invitrogen, Life Technologies). .. Subsequently, 1 μl Exonuclease I (NEB, M0293S), 1 μl Exonuclease III (NEB, M0206S) and 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) were added into the reaction and jointly incubated at 37 °C for 1 h. Then the mixture was purified with MinElute Reaction Cleanup Kit (3 × ERC) (QIAGEN) and its final concentration was calibrated using Qubit ssDNA Assay Kit.

    Construct:

    Article Title: Efficient and Reliable Production of Vectors for the Study of the Repair, Mutagenesis, and Phenotypic Consequences of Defined DNA Damage Lesions in Mammalian Cells
    Article Snippet: .. Fpg and Nth Nicking Assays 250 ng of each construct were digested with Formamidopyrimidine DNA glycosylase (Fpg, New England Biolabs, Cat. #M0240S) using 1 μL of Fpg (8 units) in the presence of BSA, according to the manufacturer’s instructions, in 1X NEBuffer 1 for 1 hr at 37°C. ..

    Incubation:

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: .. The total 10 μl mixture was denatured at 95 °C for 3 min and left on ice immediately for another 3 min before incubation with 9 μl Reaction buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl of Enzyme Mix (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) at 30 °C for 35 ~ 65 min. .. The reaction was stopped by incubation at 65 °C for 10 min when the amplification product reached to 0.5 ~ 1 μg (monitored by Qubit).

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq
    Article Snippet: .. Subsequently, 1 μl Exonuclease I (NEB, M0293S), 1 μl Exonuclease III (NEB, M0206S) and 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) were added into the reaction and jointly incubated at 37 °C for 1 h. Then the mixture was purified with MinElute Reaction Cleanup Kit (3 × ERC) (QIAGEN) and its final concentration was calibrated using Qubit ssDNA Assay Kit. .. A 15.8 μl sample of circularized DNA, 1 μl of primers and 2 μl of NEBuffer 4 were mixed well and incubated at 95 °C for 3 min, followed by 45 °C for 5 min and immediately placed on ice for another 3 min. Then, 0.5 μl of 2.5 mM dNTPs, 0.2 μl of 100 × BSA and 0.5 μl of T4 DNA Polymerase were added to the mixture, followed by incubation at 25 °C for 30 min and 75 °C for 20 min. Then, 1 μl of USER Enzyme (NEB, M5505S) was added, followed by incubation at 37 °C for 30 min and 50 °C for 4 min, and the sample was immediately placed on ice thereafter.

    Article Title: DNA damage in normally and prematurely aged mice
    Article Snippet: For detection of oxidative damage 100 ng of sample DNA (2 µl of stock solution) were incubated in 50 µl of reaction mixture containing 4 units of FPG enzyme (NEB, Ipswich, MA, USA) and 5 µl of NEBuffer 4 (NEB) at 37°C for 1 hour followed by 20 min incubation at 65°C. .. Additionally, in parallel with FPG treatment, samples intended for analysis of the mitochondrial genome were cleaved with XhoI restriction endonuclease (NEB), which has a unique recognition side outside of the amplified region, which helps to relax supercoiled mtDNA, making it more suitable for PCR amplification.

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair
    Article Snippet: Irradiated and non-irradiated DNA obtained by LTL of cells embedded in agarose (~1.2 µg DNA per plug) was treated for 24 h at 20°C with Fpg (400 ng, New England Biolabs, M0240 L) in the buffer provided by the manufacturer, or Nth (1.2 µg, NEB, M0268 L) in a buffer [70 mM HEPES/KOH pH 7.6, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT) and 50 µg/ml bovine serum albumin] reducing non-specific nuclease activity ( ). .. After enzyme treatment agarose blocks were incubated at 20°C for 2 h with 1 mg/ml protease in TEN-buffer and washed once in TEN-buffer before PFGE.

    Article Title: Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer
    Article Snippet: .. After permeabilization, cells were incubated with uracil DNA glycosylase (UDG) to remove uracil (NEB #M0304S), formamidopyrimidine [Fapy]-DNA glycosylase (Fapy-DNA glycosylase NEB #M0240S) to remove Fapy lesions, T4 Pyrimidine dimer glycosylase (T4PDG NEB #M-308S) to remove pyrimidine dimer lesions, endonuclease IV (Endo IV NEB #M0304S) to process oxidative damage, AP sites and modifies 3’ phosphates to 3’ OH, and endonuclease VIII (Endo VIII NEB #M0299S) to remove damaged pyrimidines diluted in 1X Thermpol buffer and incubated at 37°C for 1 h. Damage sites are labeled by DNA polymerase I Klenow large fragment (lacking 5’ to 3’ exonuclease activity) incubated with Digoxigenin-11-dUTP, alkali-labile (Dig) (Sigma-Aldrich #DIUTP-RO) at 37°C for 1 h. The Dig-dUTP is covalently incorporated into the DNA for detection of damage sites. .. Cells were then washed in PBS, blocked using 2% BSA in PBS and Dig was then detected using an anti-Dig antibody (abcam #ab420 clone 21H8) at a dilution of 1:250 in 2% BSA in PBS for 1 h at RT.

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq
    Article Snippet: .. The total 10 μl mixture was denatured at 95 °C for 3 min and left on ice immediately for another 3 min before incubation with 9 μl Reaction Buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl of Enzyme Mix (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl UDG (Uracil-DNA Glycosylase, NEB, M0280S), 1 μl Fpg (NEB, M0240S) at 30 °C for 35–65 min. .. The total 10 μl mixture was denatured at 95 °C for 3 min and left on ice immediately for another 3 min before incubation with 9 μl Reaction Buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl of Enzyme Mix (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl UDG (Uracil-DNA Glycosylase, NEB, M0280S), 1 μl Fpg (NEB, M0240S) at 30 °C for 35–65 min.

    Article Title: Ultra-precise detection of mutations by droplet-based amplification of circularized DNA
    Article Snippet: Subsequently, 0.5 μl Exonuclease I (NEB, M0293S) and 0.5 μl Exonuclease III (NEB, M0206S) were added into the reaction and jointly incubated at 37 °C for 1 h. The inner enzymes were inactivated at 80 °C for another 20 min. .. A column of 2.5 μl circularized DNA was placed into 0.2-ml tubes containing 5 μl 10X phi29 DNA Polymerase Reaction Buffer (NEB, M0269S), 2.5 μl exonuclease-resistant hexamer (500 μM), 2.5 μl 10 mM dNTP, 1 μl 100XBSA, 27 μl ddH2 O, which was denatured at 95 °C for 3 min, then put on ice immediately for another 3 min, and further added by a mixture of 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) and 2.5 μl phi29 DNA Polymerase (NEB, M0269S).

    Activity Assay:

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair
    Article Snippet: .. Irradiated and non-irradiated DNA obtained by LTL of cells embedded in agarose (~1.2 µg DNA per plug) was treated for 24 h at 20°C with Fpg (400 ng, New England Biolabs, M0240 L) in the buffer provided by the manufacturer, or Nth (1.2 µg, NEB, M0268 L) in a buffer [70 mM HEPES/KOH pH 7.6, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT) and 50 µg/ml bovine serum albumin] reducing non-specific nuclease activity ( ). .. After enzyme treatment agarose blocks were incubated at 20°C for 2 h with 1 mg/ml protease in TEN-buffer and washed once in TEN-buffer before PFGE.

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage
    Article Snippet: Paragraph title: Fluorescence analysis of in vitro enzymatic activity ... Fpg, hOGG1, Fpg reaction buffer (NEB buffer 1: 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.0 at 25 °C), hOGG1 reaction buffer (NEB buffer 4: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9 at 25 °C), and BSA (10 mg/mL) were purchased from New England Biolabs.

    Article Title: Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer
    Article Snippet: .. After permeabilization, cells were incubated with uracil DNA glycosylase (UDG) to remove uracil (NEB #M0304S), formamidopyrimidine [Fapy]-DNA glycosylase (Fapy-DNA glycosylase NEB #M0240S) to remove Fapy lesions, T4 Pyrimidine dimer glycosylase (T4PDG NEB #M-308S) to remove pyrimidine dimer lesions, endonuclease IV (Endo IV NEB #M0304S) to process oxidative damage, AP sites and modifies 3’ phosphates to 3’ OH, and endonuclease VIII (Endo VIII NEB #M0299S) to remove damaged pyrimidines diluted in 1X Thermpol buffer and incubated at 37°C for 1 h. Damage sites are labeled by DNA polymerase I Klenow large fragment (lacking 5’ to 3’ exonuclease activity) incubated with Digoxigenin-11-dUTP, alkali-labile (Dig) (Sigma-Aldrich #DIUTP-RO) at 37°C for 1 h. The Dig-dUTP is covalently incorporated into the DNA for detection of damage sites. .. Cells were then washed in PBS, blocked using 2% BSA in PBS and Dig was then detected using an anti-Dig antibody (abcam #ab420 clone 21H8) at a dilution of 1:250 in 2% BSA in PBS for 1 h at RT.

    Modification:

    Article Title: Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer
    Article Snippet: The Repair Assisted Damage Detection (RADD) assay detects DNA base lesions and strand breaks using an enzymatic cocktail specific for these lesions that removes the lesion and tags the resulting gap or strand break with a modified base for fluorescent detection [ ]. .. After permeabilization, cells were incubated with uracil DNA glycosylase (UDG) to remove uracil (NEB #M0304S), formamidopyrimidine [Fapy]-DNA glycosylase (Fapy-DNA glycosylase NEB #M0240S) to remove Fapy lesions, T4 Pyrimidine dimer glycosylase (T4PDG NEB #M-308S) to remove pyrimidine dimer lesions, endonuclease IV (Endo IV NEB #M0304S) to process oxidative damage, AP sites and modifies 3’ phosphates to 3’ OH, and endonuclease VIII (Endo VIII NEB #M0299S) to remove damaged pyrimidines diluted in 1X Thermpol buffer and incubated at 37°C for 1 h. Damage sites are labeled by DNA polymerase I Klenow large fragment (lacking 5’ to 3’ exonuclease activity) incubated with Digoxigenin-11-dUTP, alkali-labile (Dig) (Sigma-Aldrich #DIUTP-RO) at 37°C for 1 h. The Dig-dUTP is covalently incorporated into the DNA for detection of damage sites.

    Western Blot:

    Article Title: MacroH2A1 Regulation of Poly(ADP-Ribose) Synthesis and Stability Prevents Necrosis and Promotes DNA Repair
    Article Snippet: Total genomic DNA was extracted from Luc KD and mH2A KD IMR90 cells using a DNA Extractor WB kit (Wako; code no. 291-50502), and genomic DNA was kept on ice during the process. .. For Fpg enrichment, 5 μg of gDNA was digested with recombinant Fpg (New England Biolabs [NEB]; M0240S) and purified by ethanol precipitation; 0.1 μg Fpg enzyme was used for 1 μg of genomic DNA in NEB buffer 1 and BSA for 1 h at 37°C.

    Digital PCR:

    Article Title: Ultra-precise detection of mutations by droplet-based amplification of circularized DNA
    Article Snippet: A column of 2.5 μl circularized DNA was placed into 0.2-ml tubes containing 5 μl 10X phi29 DNA Polymerase Reaction Buffer (NEB, M0269S), 2.5 μl exonuclease-resistant hexamer (500 μM), 2.5 μl 10 mM dNTP, 1 μl 100XBSA, 27 μl ddH2 O, which was denatured at 95 °C for 3 min, then put on ice immediately for another 3 min, and further added by a mixture of 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) and 2.5 μl phi29 DNA Polymerase (NEB, M0269S). .. The thoroughly mixed 45 μl RCA reaction and 5 μl stabilizer were put into RainDrop Source chip (RainDance Technologies , RainDrop Digital PCR System) together to produce water-in-oil emulsion droplets as the manufacturer’s recommendations.

    Polymerase Chain Reaction:

    Article Title: Integrated digital error suppression for improved detection of circulating tumor DNA
    Article Snippet: .. DNA repair enzymes Related to , we tested enzymatic removal of damaged DNA bases in a representative 32ng cfDNA sample from a healthy adult donor using the following products: (i) uracil DNA-glycosylase (UDG; NEB catalog number M0372S), which leaves an abasic site in place of uracil (a cytosine oxidation product), thereby preventing PCR from continuing through the site of oxidation, eliminating C > T errors due to cytosine oxidation; (ii) 8-oxoguanine DNA glycosylate (FPG; NEB catalog number M0240S), which removes damaged purines and cleaves at the site of the damaged bases, eliminating G > T errors due to guanine oxidation, and (iii) PreCR repair mix (NEB catalog number M0309S), which is designed to remove a variety of damaged bases, including oxidized guanines and cytosines. ..

    Article Title: DNA damage in normally and prematurely aged mice
    Article Snippet: .. Additionally, in parallel with FPG treatment, samples intended for analysis of the mitochondrial genome were cleaved with XhoI restriction endonuclease (NEB), which has a unique recognition side outside of the amplified region, which helps to relax supercoiled mtDNA, making it more suitable for PCR amplification. .. Electrophoresis was performed in 0.8% agarose (Bio-Rad, Hercules, CA, USA) gels prepared and run with standard 1x TBE buffer (Fisher Scientific, Pittsburgh, PA, USA).

    Recombinant:

    Article Title: MacroH2A1 Regulation of Poly(ADP-Ribose) Synthesis and Stability Prevents Necrosis and Promotes DNA Repair
    Article Snippet: .. For Fpg enrichment, 5 μg of gDNA was digested with recombinant Fpg (New England Biolabs [NEB]; M0240S) and purified by ethanol precipitation; 0.1 μg Fpg enzyme was used for 1 μg of genomic DNA in NEB buffer 1 and BSA for 1 h at 37°C. .. C olorimetric measurement of AP sites was performed using a commercial kit (Abcam; ab211154) following the manufacturer’s protocol.

    Immunofluorescence:

    Article Title: Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer
    Article Snippet: DNA damage analysis utilizing RADD Cells were plated in 8-well chambered coverglass, fixed, and permeabilized using the same procedure as for immunofluorescence. .. After permeabilization, cells were incubated with uracil DNA glycosylase (UDG) to remove uracil (NEB #M0304S), formamidopyrimidine [Fapy]-DNA glycosylase (Fapy-DNA glycosylase NEB #M0240S) to remove Fapy lesions, T4 Pyrimidine dimer glycosylase (T4PDG NEB #M-308S) to remove pyrimidine dimer lesions, endonuclease IV (Endo IV NEB #M0304S) to process oxidative damage, AP sites and modifies 3’ phosphates to 3’ OH, and endonuclease VIII (Endo VIII NEB #M0299S) to remove damaged pyrimidines diluted in 1X Thermpol buffer and incubated at 37°C for 1 h. Damage sites are labeled by DNA polymerase I Klenow large fragment (lacking 5’ to 3’ exonuclease activity) incubated with Digoxigenin-11-dUTP, alkali-labile (Dig) (Sigma-Aldrich #DIUTP-RO) at 37°C for 1 h. The Dig-dUTP is covalently incorporated into the DNA for detection of damage sites.

    Fluorescence:

    Article Title: MacroH2A1 Regulation of Poly(ADP-Ribose) Synthesis and Stability Prevents Necrosis and Promotes DNA Repair
    Article Snippet: For Fpg enrichment, 5 μg of gDNA was digested with recombinant Fpg (New England Biolabs [NEB]; M0240S) and purified by ethanol precipitation; 0.1 μg Fpg enzyme was used for 1 μg of genomic DNA in NEB buffer 1 and BSA for 1 h at 37°C. .. The genomic-DNA concentration was determined using a Denovix QFX and a DeNovix double-stranded DAN (dsDNA) high-sensitivity fluorescence assay kit to ensure equal loading of genomic DNA.

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage
    Article Snippet: Paragraph title: Fluorescence analysis of in vitro enzymatic activity ... Fpg, hOGG1, Fpg reaction buffer (NEB buffer 1: 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.0 at 25 °C), hOGG1 reaction buffer (NEB buffer 4: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9 at 25 °C), and BSA (10 mg/mL) were purchased from New England Biolabs.

    Labeling:

    Article Title: Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer
    Article Snippet: .. After permeabilization, cells were incubated with uracil DNA glycosylase (UDG) to remove uracil (NEB #M0304S), formamidopyrimidine [Fapy]-DNA glycosylase (Fapy-DNA glycosylase NEB #M0240S) to remove Fapy lesions, T4 Pyrimidine dimer glycosylase (T4PDG NEB #M-308S) to remove pyrimidine dimer lesions, endonuclease IV (Endo IV NEB #M0304S) to process oxidative damage, AP sites and modifies 3’ phosphates to 3’ OH, and endonuclease VIII (Endo VIII NEB #M0299S) to remove damaged pyrimidines diluted in 1X Thermpol buffer and incubated at 37°C for 1 h. Damage sites are labeled by DNA polymerase I Klenow large fragment (lacking 5’ to 3’ exonuclease activity) incubated with Digoxigenin-11-dUTP, alkali-labile (Dig) (Sigma-Aldrich #DIUTP-RO) at 37°C for 1 h. The Dig-dUTP is covalently incorporated into the DNA for detection of damage sites. .. Cells were then washed in PBS, blocked using 2% BSA in PBS and Dig was then detected using an anti-Dig antibody (abcam #ab420 clone 21H8) at a dilution of 1:250 in 2% BSA in PBS for 1 h at RT.

    Purification:

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: The successfully circularized DNA was purified using the Oligo Clean & concentrator kit (Zymo, D4060). .. The total 10 μl mixture was denatured at 95 °C for 3 min and left on ice immediately for another 3 min before incubation with 9 μl Reaction buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl of Enzyme Mix (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) at 30 °C for 35 ~ 65 min.

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq
    Article Snippet: .. Subsequently, 1 μl Exonuclease I (NEB, M0293S), 1 μl Exonuclease III (NEB, M0206S) and 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) were added into the reaction and jointly incubated at 37 °C for 1 h. Then the mixture was purified with MinElute Reaction Cleanup Kit (3 × ERC) (QIAGEN) and its final concentration was calibrated using Qubit ssDNA Assay Kit. .. A 15.8 μl sample of circularized DNA, 1 μl of primers and 2 μl of NEBuffer 4 were mixed well and incubated at 95 °C for 3 min, followed by 45 °C for 5 min and immediately placed on ice for another 3 min. Then, 0.5 μl of 2.5 mM dNTPs, 0.2 μl of 100 × BSA and 0.5 μl of T4 DNA Polymerase were added to the mixture, followed by incubation at 25 °C for 30 min and 75 °C for 20 min. Then, 1 μl of USER Enzyme (NEB, M5505S) was added, followed by incubation at 37 °C for 30 min and 50 °C for 4 min, and the sample was immediately placed on ice thereafter.

    Article Title: MacroH2A1 Regulation of Poly(ADP-Ribose) Synthesis and Stability Prevents Necrosis and Promotes DNA Repair
    Article Snippet: .. For Fpg enrichment, 5 μg of gDNA was digested with recombinant Fpg (New England Biolabs [NEB]; M0240S) and purified by ethanol precipitation; 0.1 μg Fpg enzyme was used for 1 μg of genomic DNA in NEB buffer 1 and BSA for 1 h at 37°C. .. C olorimetric measurement of AP sites was performed using a commercial kit (Abcam; ab211154) following the manufacturer’s protocol.

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq
    Article Snippet: The total 10 μl mixture was denatured at 95 °C for 3 min and left on ice immediately for another 3 min before incubation with 9 μl Reaction Buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl of Enzyme Mix (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl UDG (Uracil-DNA Glycosylase, NEB, M0280S), 1 μl Fpg (NEB, M0240S) at 30 °C for 35–65 min. .. The total 10 μl mixture was denatured at 95 °C for 3 min and left on ice immediately for another 3 min before incubation with 9 μl Reaction Buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl of Enzyme Mix (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl UDG (Uracil-DNA Glycosylase, NEB, M0280S), 1 μl Fpg (NEB, M0240S) at 30 °C for 35–65 min.

    Article Title: Ultra-precise detection of mutations by droplet-based amplification of circularized DNA
    Article Snippet: The successfully circularized DNA was purified using the QIAquick Nucleotide Removal Kit (10X PN1) and its final concentration was calibrated using Qubit® ssDNA Assay Kit. .. A column of 2.5 μl circularized DNA was placed into 0.2-ml tubes containing 5 μl 10X phi29 DNA Polymerase Reaction Buffer (NEB, M0269S), 2.5 μl exonuclease-resistant hexamer (500 μM), 2.5 μl 10 mM dNTP, 1 μl 100XBSA, 27 μl ddH2 O, which was denatured at 95 °C for 3 min, then put on ice immediately for another 3 min, and further added by a mixture of 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) and 2.5 μl phi29 DNA Polymerase (NEB, M0269S).

    Gel Extraction:

    Article Title: Ultra-precise detection of mutations by droplet-based amplification of circularized DNA
    Article Snippet: Subsequently, the gels with DNAs in length of 80 ~ 140 bp marked with 20 bp DNA ladder (Takara) were particularly cut off, and further extracted using QIAGEN MinElute Gel Extraction Kit (6X buffer QG). .. A column of 2.5 μl circularized DNA was placed into 0.2-ml tubes containing 5 μl 10X phi29 DNA Polymerase Reaction Buffer (NEB, M0269S), 2.5 μl exonuclease-resistant hexamer (500 μM), 2.5 μl 10 mM dNTP, 1 μl 100XBSA, 27 μl ddH2 O, which was denatured at 95 °C for 3 min, then put on ice immediately for another 3 min, and further added by a mixture of 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) and 2.5 μl phi29 DNA Polymerase (NEB, M0269S).

    Chromatin Immunoprecipitation:

    Article Title: Ultra-precise detection of mutations by droplet-based amplification of circularized DNA
    Article Snippet: A column of 2.5 μl circularized DNA was placed into 0.2-ml tubes containing 5 μl 10X phi29 DNA Polymerase Reaction Buffer (NEB, M0269S), 2.5 μl exonuclease-resistant hexamer (500 μM), 2.5 μl 10 mM dNTP, 1 μl 100XBSA, 27 μl ddH2 O, which was denatured at 95 °C for 3 min, then put on ice immediately for another 3 min, and further added by a mixture of 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) and 2.5 μl phi29 DNA Polymerase (NEB, M0269S). .. The thoroughly mixed 45 μl RCA reaction and 5 μl stabilizer were put into RainDrop Source chip (RainDance Technologies , RainDrop Digital PCR System) together to produce water-in-oil emulsion droplets as the manufacturer’s recommendations.

    Irradiation:

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair
    Article Snippet: .. Irradiated and non-irradiated DNA obtained by LTL of cells embedded in agarose (~1.2 µg DNA per plug) was treated for 24 h at 20°C with Fpg (400 ng, New England Biolabs, M0240 L) in the buffer provided by the manufacturer, or Nth (1.2 µg, NEB, M0268 L) in a buffer [70 mM HEPES/KOH pH 7.6, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT) and 50 µg/ml bovine serum albumin] reducing non-specific nuclease activity ( ). .. After enzyme treatment agarose blocks were incubated at 20°C for 2 h with 1 mg/ml protease in TEN-buffer and washed once in TEN-buffer before PFGE.

    In Vitro:

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage
    Article Snippet: Paragraph title: Fluorescence analysis of in vitro enzymatic activity ... Fpg, hOGG1, Fpg reaction buffer (NEB buffer 1: 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.0 at 25 °C), hOGG1 reaction buffer (NEB buffer 4: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9 at 25 °C), and BSA (10 mg/mL) were purchased from New England Biolabs.

    Ethanol Precipitation:

    Article Title: MacroH2A1 Regulation of Poly(ADP-Ribose) Synthesis and Stability Prevents Necrosis and Promotes DNA Repair
    Article Snippet: .. For Fpg enrichment, 5 μg of gDNA was digested with recombinant Fpg (New England Biolabs [NEB]; M0240S) and purified by ethanol precipitation; 0.1 μg Fpg enzyme was used for 1 μg of genomic DNA in NEB buffer 1 and BSA for 1 h at 37°C. .. C olorimetric measurement of AP sites was performed using a commercial kit (Abcam; ab211154) following the manufacturer’s protocol.

    Next-Generation Sequencing:

    Article Title: Using ultra-sensitive next generation sequencing to dissect DNA damage-induced mutagenesis
    Article Snippet: The total 10 μl mixture was denatured at 95 °C for 3 min and left on ice immediately for another 3 min before incubation with 9 μl Reaction buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl of Enzyme Mix (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) at 30 °C for 35 ~ 65 min. .. The recovered DNA can be used for preparing standard NGS libraries.

    Spectrophotometry:

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage
    Article Snippet: Fpg, hOGG1, Fpg reaction buffer (NEB buffer 1: 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.0 at 25 °C), hOGG1 reaction buffer (NEB buffer 4: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9 at 25 °C), and BSA (10 mg/mL) were purchased from New England Biolabs. .. The emission spectra were obtained with a Jobin Yvon-Spex Fluorolog 3 spectrophotometer by exciting the samples at the fluorophore's excitation maximum (pyrene 342 nm, perylene 440 nm, 2-aminopurine 303 nm, ethenoadenine 276 nm, pyrrolo-dC 347 nm, Dss 380 nm, tCo 360 nm, phenylethynylpyrene 363 nm, and QB 400 nm) and measuring the emission starting at 10 nm above the excitation wavelength with a step size of 1 nm.

    Concentration Assay:

    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. .. The total 10 μl mixture was denatured at 95 °C for 3 min and left on ice immediately for another 3 min before incubation with 9 μl Reaction buffer (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl of Enzyme Mix (illustra GenomiPhi V2 DNA Amplification Kit, GE Healthcare, 25-6600-30), 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) at 30 °C for 35 ~ 65 min.

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq
    Article Snippet: .. Subsequently, 1 μl Exonuclease I (NEB, M0293S), 1 μl Exonuclease III (NEB, M0206S) and 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) were added into the reaction and jointly incubated at 37 °C for 1 h. Then the mixture was purified with MinElute Reaction Cleanup Kit (3 × ERC) (QIAGEN) and its final concentration was calibrated using Qubit ssDNA Assay Kit. .. A 15.8 μl sample of circularized DNA, 1 μl of primers and 2 μl of NEBuffer 4 were mixed well and incubated at 95 °C for 3 min, followed by 45 °C for 5 min and immediately placed on ice for another 3 min. Then, 0.5 μl of 2.5 mM dNTPs, 0.2 μl of 100 × BSA and 0.5 μl of T4 DNA Polymerase were added to the mixture, followed by incubation at 25 °C for 30 min and 75 °C for 20 min. Then, 1 μl of USER Enzyme (NEB, M5505S) was added, followed by incubation at 37 °C for 30 min and 50 °C for 4 min, and the sample was immediately placed on ice thereafter.

    Article Title: MacroH2A1 Regulation of Poly(ADP-Ribose) Synthesis and Stability Prevents Necrosis and Promotes DNA Repair
    Article Snippet: For Fpg enrichment, 5 μg of gDNA was digested with recombinant Fpg (New England Biolabs [NEB]; M0240S) and purified by ethanol precipitation; 0.1 μg Fpg enzyme was used for 1 μg of genomic DNA in NEB buffer 1 and BSA for 1 h at 37°C. .. The genomic-DNA concentration was determined using a Denovix QFX and a DeNovix double-stranded DAN (dsDNA) high-sensitivity fluorescence assay kit to ensure equal loading of genomic DNA.

    Article Title: Ultra-precise detection of mutations by droplet-based amplification of circularized DNA
    Article Snippet: The successfully circularized DNA was purified using the QIAquick Nucleotide Removal Kit (10X PN1) and its final concentration was calibrated using Qubit® ssDNA Assay Kit. .. A column of 2.5 μl circularized DNA was placed into 0.2-ml tubes containing 5 μl 10X phi29 DNA Polymerase Reaction Buffer (NEB, M0269S), 2.5 μl exonuclease-resistant hexamer (500 μM), 2.5 μl 10 mM dNTP, 1 μl 100XBSA, 27 μl ddH2 O, which was denatured at 95 °C for 3 min, then put on ice immediately for another 3 min, and further added by a mixture of 1 μl UDG (uracil-DNA glycosylase, NEB, M0280S), 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) and 2.5 μl phi29 DNA Polymerase (NEB, M0269S).

    Staining:

    Article Title: Defective base excision repair in the response to DNA damaging agents in triple negative breast cancer
    Article Snippet: After permeabilization, cells were incubated with uracil DNA glycosylase (UDG) to remove uracil (NEB #M0304S), formamidopyrimidine [Fapy]-DNA glycosylase (Fapy-DNA glycosylase NEB #M0240S) to remove Fapy lesions, T4 Pyrimidine dimer glycosylase (T4PDG NEB #M-308S) to remove pyrimidine dimer lesions, endonuclease IV (Endo IV NEB #M0304S) to process oxidative damage, AP sites and modifies 3’ phosphates to 3’ OH, and endonuclease VIII (Endo VIII NEB #M0299S) to remove damaged pyrimidines diluted in 1X Thermpol buffer and incubated at 37°C for 1 h. Damage sites are labeled by DNA polymerase I Klenow large fragment (lacking 5’ to 3’ exonuclease activity) incubated with Digoxigenin-11-dUTP, alkali-labile (Dig) (Sigma-Aldrich #DIUTP-RO) at 37°C for 1 h. The Dig-dUTP is covalently incorporated into the DNA for detection of damage sites. .. Samples were then stained with goat anti-Mouse Alexa Fluor 546 at 1:400 in 2% BSA in PBS for 45 min at RT, and nuclei were stained as for immunofluorescence.

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    New England Biolabs fpg
    A) Fluorescence emission of 4 μM probe OGR1 in <t>Fpg</t> reaction buffer at 37 °C without Fpg (gray) and after 25 min with Fpg (black). B) Time course of 4 μM probe OGR1 emission at 465 nm following addition of 658 nM Fpg (black triangles) or <t>hOGG1</t> (gray circles) at 37 °C. This is a representative curve of n=1.
    Fpg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A) Fluorescence emission of 4 μM probe OGR1 in Fpg reaction buffer at 37 °C without Fpg (gray) and after 25 min with Fpg (black). B) Time course of 4 μM probe OGR1 emission at 465 nm following addition of 658 nM Fpg (black triangles) or hOGG1 (gray circles) at 37 °C. This is a representative curve of n=1.

    Journal: Chembiochem : a European journal of chemical biology

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage

    doi: 10.1002/cbic.201500184

    Figure Lengend Snippet: A) Fluorescence emission of 4 μM probe OGR1 in Fpg reaction buffer at 37 °C without Fpg (gray) and after 25 min with Fpg (black). B) Time course of 4 μM probe OGR1 emission at 465 nm following addition of 658 nM Fpg (black triangles) or hOGG1 (gray circles) at 37 °C. This is a representative curve of n=1.

    Article Snippet: Fpg, hOGG1, Fpg reaction buffer (NEB buffer 1: 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.0 at 25 °C), hOGG1 reaction buffer (NEB buffer 4: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9 at 25 °C), and BSA (10 mg/mL) were purchased from New England Biolabs.

    Techniques: Fluorescence

    A and B) PAGE analysis of 4 μM probe OGR1 radiolabeled at the 5′ end with [ 32 P]-phosphate and reacted with 658 nM hOGG1 (A) or Fpg (B) at 37 °C for various times. Piperidine (200 mM, 95 °C, 5 min), which cleaves the DNA backbone at abasic sites, was added to samples as indicated to identify the 8-oxoguanine excision product and the backbone cleavage product. C and D) MALDI mass spectrometry analysis of probe OGR1 following overnight reaction with hOGG1 (C) or Fpg (D).

    Journal: Chembiochem : a European journal of chemical biology

    Article Title: In Vitro Fluorogenic Real-time Assay of the Repair of Oxidative DNA Damage

    doi: 10.1002/cbic.201500184

    Figure Lengend Snippet: A and B) PAGE analysis of 4 μM probe OGR1 radiolabeled at the 5′ end with [ 32 P]-phosphate and reacted with 658 nM hOGG1 (A) or Fpg (B) at 37 °C for various times. Piperidine (200 mM, 95 °C, 5 min), which cleaves the DNA backbone at abasic sites, was added to samples as indicated to identify the 8-oxoguanine excision product and the backbone cleavage product. C and D) MALDI mass spectrometry analysis of probe OGR1 following overnight reaction with hOGG1 (C) or Fpg (D).

    Article Snippet: Fpg, hOGG1, Fpg reaction buffer (NEB buffer 1: 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.0 at 25 °C), hOGG1 reaction buffer (NEB buffer 4: 50 mM potassium acetate, 20 mM Tris-acetate, 10 mM magnesium acetate, 1 mM dithiothreitol, pH 7.9 at 25 °C), and BSA (10 mg/mL) were purchased from New England Biolabs.

    Techniques: Polyacrylamide Gel Electrophoresis, Mass Spectrometry

    Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug Fpg or 1.2 µg/plug Nth for 24 h at 20°C before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.

    Journal: Nucleic Acids Research

    Article Title: Post-irradiation chemical processing of DNA damage generates double-strand breaks in cells already engaged in repair

    doi: 10.1093/nar/gkr463

    Figure Lengend Snippet: Apurinic or apyrimidinic sites are unlikely to represent radiation-induced labile lesions. LIG4 − / − MEFs embedded in agarose were exposed to 20 Gy X-rays and subjected to LTL. Subsequently, agarose blocks were incubated at 4, 37 and 50°C for 48 h and then treated with 400 ng/plug Fpg or 1.2 µg/plug Nth for 24 h at 20°C before analysing by PFGE. ( A ) FDR measured at the different treatment conditions as indicated. Control: samples maintained in TEN-buffer throughout. Buffer: samples maintained in enzyme buffer during the enzyme treatment step. Buffer + enzyme: samples maintained in enzyme buffer with the indicated amount of Fpg during the enzyme treatment step. ( B ) As is A but for Nth. ( C ) Net increase in FDR as a result of Fpg treatment in irradiated samples pre-treated as indicated. Net increase was calculated by subtracting the FDR of buffer-only samples from that obtained in the presence of the enzyme. ( D ) Same as in C but for Nth.

    Article Snippet: Irradiated and non-irradiated DNA obtained by LTL of cells embedded in agarose (~1.2 µg DNA per plug) was treated for 24 h at 20°C with Fpg (400 ng, New England Biolabs, M0240 L) in the buffer provided by the manufacturer, or Nth (1.2 µg, NEB, M0268 L) in a buffer [70 mM HEPES/KOH pH 7.6, 100 mM KCl, 1 mM EDTA, 1 mM dithiothreitol (DTT) and 50 µg/ml bovine serum albumin] reducing non-specific nuclease activity ( ).

    Techniques: Incubation, Irradiation

    Lack of nuclear DNA fragmentation as detected by the Fpg-FLARE Comet assay in pulmonary artery endothelial cells transfected with either scrambled (A) or Ogg1-specific (B) siRNA and harvested immediately after 1 hour treatment with xanthine oxidase (5 mU/ml). Note lack of Comet “tails”. Negative results also were obtained in cells treated with 2 and 10 mU/ml of XO using either the conventional Comet assay or the Fpg-FLARE. (C) Pulmonary artery endothelial cells without any treatment were used as a negative control. (D) PAECs treated with 1 mM hydrogen peroxide for 15 min at 4°C were used as a positive control for the assay.

    Journal: Free radical biology & medicine

    Article Title: The DNA Glycosylase, Ogg1, Defends Against Oxidant-induced mtDNA Damage and Apoptosis in Pulmonary Artery Endothelial Cells

    doi: 10.1016/j.freeradbiomed.2010.10.692

    Figure Lengend Snippet: Lack of nuclear DNA fragmentation as detected by the Fpg-FLARE Comet assay in pulmonary artery endothelial cells transfected with either scrambled (A) or Ogg1-specific (B) siRNA and harvested immediately after 1 hour treatment with xanthine oxidase (5 mU/ml). Note lack of Comet “tails”. Negative results also were obtained in cells treated with 2 and 10 mU/ml of XO using either the conventional Comet assay or the Fpg-FLARE. (C) Pulmonary artery endothelial cells without any treatment were used as a negative control. (D) PAECs treated with 1 mM hydrogen peroxide for 15 min at 4°C were used as a positive control for the assay.

    Article Snippet: To reveal oxidative base modifications, DNA was treated with formamidopyrimidine glycosylase, Fpg (New England Biolabs, Beverly, MA), a bacterial DNA repair enzyme that cleaves DNA at sites of oxidized purines, thereby creating single-strand breaks detectable on alkaline Southern blot.

    Techniques: Single Cell Gel Electrophoresis, Transfection, Negative Control, Positive Control

    Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).

    Journal: eLife

    Article Title: Perturbation of base excision repair sensitizes breast cancer cells to APOBEC3 deaminase-mediated mutations

    doi: 10.7554/eLife.51605

    Figure Lengend Snippet: Purification and activity assays of PNKP and Polβ. ( A ) Purified Polβ-His 6 (17 ng) and PNKP-His 6 (127 ng) from E. coli were subjected to PAGE and stained with Coomassie blue. ( B ) Incorporation of [α- 32 P]-dCTP by Polβ using APE1-generated product. ddC, di-deoxynucleotide; P, product. ( C ) Schematic of the preparation of S (substrate) and subsequent enzymatic reactions for testing PNKP activity. ( D ) Efficiency of oligonucleotide labeling, annealing, and ligation leading to S indicated in ( C ). ( E ) Fpg (NEB, 1 U) completely digested S and the 3’ phosphate was completely removed by PNKP (12.7 ng and 127 ng, lanes 2 and 3), or by T4 PNK (NEB, 0.1 U and 1 U, lanes 7 and 8). NEIL2 (272 ng) only partially digested S and its 3’P was resistant to the PNKP phosphatase (lanes 4 and 5).

    Article Snippet: The oligonucleotide (2 pmol) was treated with UDG (1 U, NEB) and Fpg (1U, NEB) or NEIL2 (272 ng) in a 10 μl reaction at 37°C for 30 min.

    Techniques: Purification, Activity Assay, Polyacrylamide Gel Electrophoresis, Staining, Generated, Oligonucleotide Labeling, Ligation