nt phix174 virion dna  (New England Biolabs)


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    Name:
    PhiX174 Virion DNA
    Description:
    PhiX174 Virion DNA 250 ug
    Catalog Number:
    N3023L
    Price:
    320
    Category:
    Genomic DNA
    Size:
    250 ug
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    Structured Review

    New England Biolabs nt phix174 virion dna
    PhiX174 Virion DNA
    PhiX174 Virion DNA 250 ug
    https://www.bioz.com/result/nt phix174 virion dna/product/New England Biolabs
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nt phix174 virion dna - by Bioz Stars, 2021-04
    96/100 stars

    Images

    1) Product Images from "A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors"

    Article Title: A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors

    Journal: eLife

    doi: 10.7554/eLife.64131

    Rad51-EED binds both ssDNA and dsDNA like wild-type Rad51. ( A ) Purified Rad51-EED was examined by SDS-PAGE and coomassie staining. ( B, C ) The indicated concentrations of Rad51 (WT) or Rad51-EED (EED) were incubated with 30 µM nt PhiX174 virion DNA (ssDNA; B ) or 20 µM nt of linearized PhiX174 RF I DNA (dsDNA; C ) in the presence or absence of ATP. Protein-DNA complexes were crosslinked with glutaraldehyde and then resolved by agarose gel electrophoresis. ( D ) Purified Rad51 (WT or E206K) was incubated with purified Swi5-Sfr1 (or the equivalent volume of protein storage buffer) and subjected to immunoprecipitation with an anti-Sfr1 antibody. For quantification, Rad51 signal was normalized to Sfr1 signal and expressed relative to wild type. Data in ( D ) are the means of two independent experiments with individual values shown. Relative co-IP of Rad51 (%) for Figure 6—figure supplement 1D .
    Figure Legend Snippet: Rad51-EED binds both ssDNA and dsDNA like wild-type Rad51. ( A ) Purified Rad51-EED was examined by SDS-PAGE and coomassie staining. ( B, C ) The indicated concentrations of Rad51 (WT) or Rad51-EED (EED) were incubated with 30 µM nt PhiX174 virion DNA (ssDNA; B ) or 20 µM nt of linearized PhiX174 RF I DNA (dsDNA; C ) in the presence or absence of ATP. Protein-DNA complexes were crosslinked with glutaraldehyde and then resolved by agarose gel electrophoresis. ( D ) Purified Rad51 (WT or E206K) was incubated with purified Swi5-Sfr1 (or the equivalent volume of protein storage buffer) and subjected to immunoprecipitation with an anti-Sfr1 antibody. For quantification, Rad51 signal was normalized to Sfr1 signal and expressed relative to wild type. Data in ( D ) are the means of two independent experiments with individual values shown. Relative co-IP of Rad51 (%) for Figure 6—figure supplement 1D .

    Techniques Used: Purification, SDS Page, Staining, Incubation, Agarose Gel Electrophoresis, Immunoprecipitation, Co-Immunoprecipitation Assay

    2) Product Images from "Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation"

    Article Title: Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1358

    Bcl2 directly inhibits MRN-mediated DNA resection. ( A ) DNA resection activity of purified Mre11, Rad50, NBS1 and MRN complex were measured using PhiX174 circular ssDNA as substrate (left panel: endonuclease activity) and dsDNA as substrate (right panel: exonuclease activity). ( B ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified Bcl2. BSA was used as control. ( C ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified WT Bcl2 or Bcl2 BH deletion mutant protein(s).
    Figure Legend Snippet: Bcl2 directly inhibits MRN-mediated DNA resection. ( A ) DNA resection activity of purified Mre11, Rad50, NBS1 and MRN complex were measured using PhiX174 circular ssDNA as substrate (left panel: endonuclease activity) and dsDNA as substrate (right panel: exonuclease activity). ( B ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified Bcl2. BSA was used as control. ( C ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified WT Bcl2 or Bcl2 BH deletion mutant protein(s).

    Techniques Used: Activity Assay, Purification, Mutagenesis

    3) Product Images from "Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System *"

    Article Title: Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.542787

    EXO1-dependent ATR Activation. A , a model system for excision repair-checkpoint coupling. ATR kinase reactions were carried out with ATR-ATRIP, TopBP1, RPA, and EXO1 as indicated. 0.6 ng (27 p m ) single-stranded φX174 DNA ( ssDNA ), plasmid DNA (
    Figure Legend Snippet: EXO1-dependent ATR Activation. A , a model system for excision repair-checkpoint coupling. ATR kinase reactions were carried out with ATR-ATRIP, TopBP1, RPA, and EXO1 as indicated. 0.6 ng (27 p m ) single-stranded φX174 DNA ( ssDNA ), plasmid DNA (

    Techniques Used: Activation Assay, Recombinase Polymerase Amplification, Plasmid Preparation

    4) Product Images from "A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors"

    Article Title: A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors

    Journal: eLife

    doi: 10.7554/eLife.64131

    Rad51-EED binds both ssDNA and dsDNA like wild-type Rad51. ( A ) Purified Rad51-EED was examined by SDS-PAGE and coomassie staining. ( B, C ) The indicated concentrations of Rad51 (WT) or Rad51-EED (EED) were incubated with 30 µM nt PhiX174 virion DNA (ssDNA; B ) or 20 µM nt of linearized PhiX174 RF I DNA (dsDNA; C ) in the presence or absence of ATP. Protein-DNA complexes were crosslinked with glutaraldehyde and then resolved by agarose gel electrophoresis. ( D ) Purified Rad51 (WT or E206K) was incubated with purified Swi5-Sfr1 (or the equivalent volume of protein storage buffer) and subjected to immunoprecipitation with an anti-Sfr1 antibody. For quantification, Rad51 signal was normalized to Sfr1 signal and expressed relative to wild type. Data in ( D ) are the means of two independent experiments with individual values shown. Relative co-IP of Rad51 (%) for Figure 6—figure supplement 1D .
    Figure Legend Snippet: Rad51-EED binds both ssDNA and dsDNA like wild-type Rad51. ( A ) Purified Rad51-EED was examined by SDS-PAGE and coomassie staining. ( B, C ) The indicated concentrations of Rad51 (WT) or Rad51-EED (EED) were incubated with 30 µM nt PhiX174 virion DNA (ssDNA; B ) or 20 µM nt of linearized PhiX174 RF I DNA (dsDNA; C ) in the presence or absence of ATP. Protein-DNA complexes were crosslinked with glutaraldehyde and then resolved by agarose gel electrophoresis. ( D ) Purified Rad51 (WT or E206K) was incubated with purified Swi5-Sfr1 (or the equivalent volume of protein storage buffer) and subjected to immunoprecipitation with an anti-Sfr1 antibody. For quantification, Rad51 signal was normalized to Sfr1 signal and expressed relative to wild type. Data in ( D ) are the means of two independent experiments with individual values shown. Relative co-IP of Rad51 (%) for Figure 6—figure supplement 1D .

    Techniques Used: Purification, SDS Page, Staining, Incubation, Agarose Gel Electrophoresis, Immunoprecipitation, Co-Immunoprecipitation Assay

    5) Product Images from "A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors"

    Article Title: A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors

    Journal: eLife

    doi: 10.7554/eLife.64131

    Rad51-EED binds both ssDNA and dsDNA like wild-type Rad51. ( A ) Purified Rad51-EED was examined by SDS-PAGE and coomassie staining. ( B, C ) The indicated concentrations of Rad51 (WT) or Rad51-EED (EED) were incubated with 30 µM nt PhiX174 virion DNA (ssDNA; B ) or 20 µM nt of linearized PhiX174 RF I DNA (dsDNA; C ) in the presence or absence of ATP. Protein-DNA complexes were crosslinked with glutaraldehyde and then resolved by agarose gel electrophoresis. ( D ) Purified Rad51 (WT or E206K) was incubated with purified Swi5-Sfr1 (or the equivalent volume of protein storage buffer) and subjected to immunoprecipitation with an anti-Sfr1 antibody. For quantification, Rad51 signal was normalized to Sfr1 signal and expressed relative to wild type. Data in ( D ) are the means of two independent experiments with individual values shown. Relative co-IP of Rad51 (%) for Figure 6—figure supplement 1D .
    Figure Legend Snippet: Rad51-EED binds both ssDNA and dsDNA like wild-type Rad51. ( A ) Purified Rad51-EED was examined by SDS-PAGE and coomassie staining. ( B, C ) The indicated concentrations of Rad51 (WT) or Rad51-EED (EED) were incubated with 30 µM nt PhiX174 virion DNA (ssDNA; B ) or 20 µM nt of linearized PhiX174 RF I DNA (dsDNA; C ) in the presence or absence of ATP. Protein-DNA complexes were crosslinked with glutaraldehyde and then resolved by agarose gel electrophoresis. ( D ) Purified Rad51 (WT or E206K) was incubated with purified Swi5-Sfr1 (or the equivalent volume of protein storage buffer) and subjected to immunoprecipitation with an anti-Sfr1 antibody. For quantification, Rad51 signal was normalized to Sfr1 signal and expressed relative to wild type. Data in ( D ) are the means of two independent experiments with individual values shown. Relative co-IP of Rad51 (%) for Figure 6—figure supplement 1D .

    Techniques Used: Purification, SDS Page, Staining, Incubation, Agarose Gel Electrophoresis, Immunoprecipitation, Co-Immunoprecipitation Assay

    6) Product Images from "Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis"

    Article Title: Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl553

    Cleavage of a phiX174-based oligonucleotide by Endo IV. A 45 base oligonucleotide based on the sequence of phiX174 ssDNA and labeled at the 5′ end with Cy5 was used as the substrate at a concentration of 10 μM for assay of the activity of Endo IV (6.6, 3.3, 1.3, 0.66 or 0.33 μg/ml). The reaction products were separated by electrophoresis on a 10% polyacrylamide gel containing 7 M urea and visualized with an image analyzer. Lane (–) represents a reaction mixture incubated in the absence of enzyme. Lane M represents a mixture of oligonucleotides labeled at the 5′ end with Cy5 and with sequences identical to those of residues 1–18, 1–27, 1–32, 1–34, 1–38, 1–41 and 1–45 of the substrate. Cleavage sites of the substrate are indicated by arrows with a size proportional to the relative extent of cleavage at the corresponding position. Each dC in the substrate sequence is shown in boldface.
    Figure Legend Snippet: Cleavage of a phiX174-based oligonucleotide by Endo IV. A 45 base oligonucleotide based on the sequence of phiX174 ssDNA and labeled at the 5′ end with Cy5 was used as the substrate at a concentration of 10 μM for assay of the activity of Endo IV (6.6, 3.3, 1.3, 0.66 or 0.33 μg/ml). The reaction products were separated by electrophoresis on a 10% polyacrylamide gel containing 7 M urea and visualized with an image analyzer. Lane (–) represents a reaction mixture incubated in the absence of enzyme. Lane M represents a mixture of oligonucleotides labeled at the 5′ end with Cy5 and with sequences identical to those of residues 1–18, 1–27, 1–32, 1–34, 1–38, 1–41 and 1–45 of the substrate. Cleavage sites of the substrate are indicated by arrows with a size proportional to the relative extent of cleavage at the corresponding position. Each dC in the substrate sequence is shown in boldface.

    Techniques Used: Sequencing, Labeling, Concentration Assay, Activity Assay, Electrophoresis, Incubation

    Substrate specificity of Endo IV. The activity of Endo IV (1, 0.5, 0.2, 0.1 or 0.05 μg/ml) was assayed with phiX174 RFI circular dsDNA ( A ), phiX174 RFI linear dsDNA ( B ), phiX174 virion circular ssDNA ( C ), linear ssRNA used for the in vitro synthesis of the GST-Endo IV fusion protein ( D ), heat-denatured T4 genomic dsDNA containing gluc-dHMC ( E ), or heat-denatured dC-substituted T4 (T4dC) genomic dsDNA ( F ) as the substrate (5 μg/ml). The reaction products were separated by electrophoresis on a 1.0% agarose gel and stained with SYBR Gold. Lanes M and (–) contain a 1 kb DNA ladder and a reaction mixture incubated in the absence of the enzyme, respectively.
    Figure Legend Snippet: Substrate specificity of Endo IV. The activity of Endo IV (1, 0.5, 0.2, 0.1 or 0.05 μg/ml) was assayed with phiX174 RFI circular dsDNA ( A ), phiX174 RFI linear dsDNA ( B ), phiX174 virion circular ssDNA ( C ), linear ssRNA used for the in vitro synthesis of the GST-Endo IV fusion protein ( D ), heat-denatured T4 genomic dsDNA containing gluc-dHMC ( E ), or heat-denatured dC-substituted T4 (T4dC) genomic dsDNA ( F ) as the substrate (5 μg/ml). The reaction products were separated by electrophoresis on a 1.0% agarose gel and stained with SYBR Gold. Lanes M and (–) contain a 1 kb DNA ladder and a reaction mixture incubated in the absence of the enzyme, respectively.

    Techniques Used: Activity Assay, In Vitro, Electrophoresis, Agarose Gel Electrophoresis, Staining, Incubation

    7) Product Images from "Expression, Purification and Biochemical Evaluation of Human RAD51 Protein"

    Article Title: Expression, Purification and Biochemical Evaluation of Human RAD51 Protein

    Journal: Methods in enzymology

    doi: 10.1016/bs.mie.2017.11.011

    Three Strand Exchange Recombinase Assay A Active RAD51 nucleoprotein filament is formed on ϕX174 Virion circular ssDNA in reaction conditions permitting ATP hydrolysis followed by addition of RPA which helps remove secondary structures in the ssDNA allowing stable nucleoprotein filament formation. The RAD51 nucleoprotein then invades ϕX174 RFI linear dsDNA to form nicked circular dsDNA products through several joint-molecule intermediates showing various stages of branch migration. The RPA also helps sequester any free displaced ssDNA following strand exchange thereby preventing a reverse reaction. B . Typical agarose gel image from a strand exchange assay used to resolve the substrates, intermediates and the products of the reaction over a time course of 0, 30, 60, 120 and 180 minutes. In reactions containing the wild type protein, nicked circular products can be observed as early as 30 minutes. C. Quantitation of the strand exchange assay gel showing appearance of nicked circular products (black) and joint molecules (blue) on the left Y axis. Disappearance of the linear dsDNA substrate (red) is quantitated on the right Y axis. Time in minutes is indicated on the X axis.
    Figure Legend Snippet: Three Strand Exchange Recombinase Assay A Active RAD51 nucleoprotein filament is formed on ϕX174 Virion circular ssDNA in reaction conditions permitting ATP hydrolysis followed by addition of RPA which helps remove secondary structures in the ssDNA allowing stable nucleoprotein filament formation. The RAD51 nucleoprotein then invades ϕX174 RFI linear dsDNA to form nicked circular dsDNA products through several joint-molecule intermediates showing various stages of branch migration. The RPA also helps sequester any free displaced ssDNA following strand exchange thereby preventing a reverse reaction. B . Typical agarose gel image from a strand exchange assay used to resolve the substrates, intermediates and the products of the reaction over a time course of 0, 30, 60, 120 and 180 minutes. In reactions containing the wild type protein, nicked circular products can be observed as early as 30 minutes. C. Quantitation of the strand exchange assay gel showing appearance of nicked circular products (black) and joint molecules (blue) on the left Y axis. Disappearance of the linear dsDNA substrate (red) is quantitated on the right Y axis. Time in minutes is indicated on the X axis.

    Techniques Used: Recombinase Polymerase Amplification, Migration, Agarose Gel Electrophoresis, Quantitation Assay

    8) Product Images from "Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues"

    Article Title: Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues

    Journal: mBio

    doi: 10.1128/mBio.01714-14

    PathoChip assay performance assessed using positive-control DNA. (A) Whole-genome amplification kits that feature three different enzymatic processes were compared in their abilities to detect phiX174 bacteriophage genomic DNA spiked into human DNA. 1× DNA was equivalent to the molarity for a single-copy locus in the human genome. Green bars are the median Cy3 signal for the 14 phiX174 probes hybridized to test samples, and red bars show the median Cy5 signal from control samples (human DNA only). Error bars indicate standard deviations across probes. (B) Detection responses for three viruses were measured over a dilution series from 10,000 to 10 genomic copies per sample. Genomic DNA for each virus was spiked into a reference amount of human DNA. Blue bars are the average Cy3 signals for all probes to the indicated viruses hybridized to test samples, and white lines indicate the probes’ Cy5 average from control samples (human DNA only). (C) Human cytomegalovirus (CMV) DNA was hybridized to a PathoChip containing 299 probes for saturation tiling across the reference CMV genome (NCBI accession NC_006273 ). The DNA was from CMV AD169, a strain that differs from the reference sequence at several locations, and was spiked into a background of human DNA for cohybridization with reference human DNA only (xhh). Red numerals indicate example probes for positive detection (1), low signal due to sequence polymorphisms (2, 3, and 4), and missing signal due to deletion in AD169 (5 and 6).
    Figure Legend Snippet: PathoChip assay performance assessed using positive-control DNA. (A) Whole-genome amplification kits that feature three different enzymatic processes were compared in their abilities to detect phiX174 bacteriophage genomic DNA spiked into human DNA. 1× DNA was equivalent to the molarity for a single-copy locus in the human genome. Green bars are the median Cy3 signal for the 14 phiX174 probes hybridized to test samples, and red bars show the median Cy5 signal from control samples (human DNA only). Error bars indicate standard deviations across probes. (B) Detection responses for three viruses were measured over a dilution series from 10,000 to 10 genomic copies per sample. Genomic DNA for each virus was spiked into a reference amount of human DNA. Blue bars are the average Cy3 signals for all probes to the indicated viruses hybridized to test samples, and white lines indicate the probes’ Cy5 average from control samples (human DNA only). (C) Human cytomegalovirus (CMV) DNA was hybridized to a PathoChip containing 299 probes for saturation tiling across the reference CMV genome (NCBI accession NC_006273 ). The DNA was from CMV AD169, a strain that differs from the reference sequence at several locations, and was spiked into a background of human DNA for cohybridization with reference human DNA only (xhh). Red numerals indicate example probes for positive detection (1), low signal due to sequence polymorphisms (2, 3, and 4), and missing signal due to deletion in AD169 (5 and 6).

    Techniques Used: Positive Control, Whole Genome Amplification, Sequencing

    Confirmation of HPV16 detection. (A) The heat map indicates test minus xhh signals for every HPV16 probe (columns) from PathoChip assays of the OSCC samples (rows). Row numbers are indicated for samples that are examples of no HPV16 signal (2021 and 2023), hybridization to nearly all probes (2022 and 2024), or hybridization to a smaller subset of probes (2032, 2035, 2053, and 2061). Probe locations are indicated relative to the transcript map for early (E) and late (L) genes, and black arrows show the positions of forward (f) and reverse (r) PCR primers. Probe names in boxes correspond to the oligomers used for capture bead enrichment and deep sequencing (cap-seq) of samples that were pooled as marked by the right axis bars. The histogram shows the sum of cap-seq reads that mapped to the HPV16 genome from all sample pools; the x axis shows map coordinates scaled to match the transcript map. (B) PCR using the forward (fwd) and reverse (rev) primers shown in panel a detected at least one HPV16 region in samples with hybridization to most or some PathoChip HPV16 probes and no detection in samples that were negative for PathoChip signal or were no-template controls. The m1 marker is phiX174 HaeIII digest, and the m2 marker contains the four amplicons produced from a plasmid carrying the HPV16 genome. (C) The individual reads obtained from cap-seq are shown for the sample pools from panel A. Pool 1 contained seven samples with low or no hybridization signal to HPV16 probes in PathoChip screening assays; 71% of the remaining samples were positive for PathoChip HPV16 detection. Whole-genome amplified DNA plus cDNA was hybridized to a set of six biotinylated HPV16 probes, captured on streptavidin beads, and used for tagmentation library preparation and deep sequencing with paired-end 250-nt reads. (Tagmentation is the process of tagging the fragmented DNA generated during library perpetration.) Reads (gray arrows) that map to the HPV16 reference genome sequence (blue) cluster around the capture probe locations (red segments in the 1-kb coordinate map), but templates up to 3 kb away from a capture probe were also recovered.
    Figure Legend Snippet: Confirmation of HPV16 detection. (A) The heat map indicates test minus xhh signals for every HPV16 probe (columns) from PathoChip assays of the OSCC samples (rows). Row numbers are indicated for samples that are examples of no HPV16 signal (2021 and 2023), hybridization to nearly all probes (2022 and 2024), or hybridization to a smaller subset of probes (2032, 2035, 2053, and 2061). Probe locations are indicated relative to the transcript map for early (E) and late (L) genes, and black arrows show the positions of forward (f) and reverse (r) PCR primers. Probe names in boxes correspond to the oligomers used for capture bead enrichment and deep sequencing (cap-seq) of samples that were pooled as marked by the right axis bars. The histogram shows the sum of cap-seq reads that mapped to the HPV16 genome from all sample pools; the x axis shows map coordinates scaled to match the transcript map. (B) PCR using the forward (fwd) and reverse (rev) primers shown in panel a detected at least one HPV16 region in samples with hybridization to most or some PathoChip HPV16 probes and no detection in samples that were negative for PathoChip signal or were no-template controls. The m1 marker is phiX174 HaeIII digest, and the m2 marker contains the four amplicons produced from a plasmid carrying the HPV16 genome. (C) The individual reads obtained from cap-seq are shown for the sample pools from panel A. Pool 1 contained seven samples with low or no hybridization signal to HPV16 probes in PathoChip screening assays; 71% of the remaining samples were positive for PathoChip HPV16 detection. Whole-genome amplified DNA plus cDNA was hybridized to a set of six biotinylated HPV16 probes, captured on streptavidin beads, and used for tagmentation library preparation and deep sequencing with paired-end 250-nt reads. (Tagmentation is the process of tagging the fragmented DNA generated during library perpetration.) Reads (gray arrows) that map to the HPV16 reference genome sequence (blue) cluster around the capture probe locations (red segments in the 1-kb coordinate map), but templates up to 3 kb away from a capture probe were also recovered.

    Techniques Used: Hybridization, Polymerase Chain Reaction, Sequencing, Marker, Produced, Plasmid Preparation, Amplification, Generated

    Related Articles

    Purification:

    Article Title: Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis
    Article Snippet: Endo IV assay Assay of Endo IV activity was performed by incubation of enzyme and substrate for 30 min at 37°C in a reaction mixture (20 μl) containing 10 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 1 mM DTT and BSA (0.1 mg/ml). .. Purified Endo IV was diluted in a solution containing 10 mM Tris–HCl (pH 8.0), 1 mM DTT and BSA (0.1 mg/ml) and was included in the reaction mixture in a volume of 1 μl. phiX174 replicative form I (RFI) circular dsDNA (New England BioLabs), phiX174 RFI linear dsDNA prepared by PstI digestion, phiX174 virion circular ssDNA (New England BioLabs), linear ssRNA used for synthesis of the GST-Endo IV fusion protein, and heat-denatured T4 or T4dC genomic dsDNA were used as substrates at a final concentration of 5 μg/ml. .. The reaction was stopped by the addition of 20 μl of 25 mM EDTA (pH 8.0), and the reaction products were separated by electrophoresis on a 1.0% agarose gel and stained with SYBR Gold (Invitrogen).

    Article Title: Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues
    Article Snippet: .. Purified phiX174 virion DNA was purchased from New England Biolabs (N3023S; Ipswich, MA, USA), total DNA from human MRC-5 cells infected with cytomegalovirus (human herpesvirus 5 strain AD169) was ATCC VR-538D, total DNA from human A549 cells infected with adenovirus type 5 (HAdV-5 strain Adenoid 75) was ATCC VR-5D, and total RNA from human HEp-2 cells infected with respiratory syncytial virus (HRSV strain Long) was ATCC VR-26D, all purchased from ATCC (Manassas, VA, USA). .. Plasmid minipreps were prepared from pBR322 subclones carrying JC or BK polyomavirus genomes (J. C. Alwine, University of Pennsylvania, Philadelphia, PA) and from pUC19 carrying the human papillomavirus 16 (HPV16) genome (obtained from Peter Howley, Harvard Medical School, Boston, MA).

    Concentration Assay:

    Article Title: Biochemical analysis of the substrate specificity and sequence preference of endonuclease IV from bacteriophage T4, a dC-specific endonuclease implicated in restriction of dC-substituted T4 DNA synthesis
    Article Snippet: Endo IV assay Assay of Endo IV activity was performed by incubation of enzyme and substrate for 30 min at 37°C in a reaction mixture (20 μl) containing 10 mM Tris–HCl (pH 8.0), 10 mM MgCl2 , 1 mM DTT and BSA (0.1 mg/ml). .. Purified Endo IV was diluted in a solution containing 10 mM Tris–HCl (pH 8.0), 1 mM DTT and BSA (0.1 mg/ml) and was included in the reaction mixture in a volume of 1 μl. phiX174 replicative form I (RFI) circular dsDNA (New England BioLabs), phiX174 RFI linear dsDNA prepared by PstI digestion, phiX174 virion circular ssDNA (New England BioLabs), linear ssRNA used for synthesis of the GST-Endo IV fusion protein, and heat-denatured T4 or T4dC genomic dsDNA were used as substrates at a final concentration of 5 μg/ml. .. The reaction was stopped by the addition of 20 μl of 25 mM EDTA (pH 8.0), and the reaction products were separated by electrophoresis on a 1.0% agarose gel and stained with SYBR Gold (Invitrogen).

    Article Title: A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors
    Article Snippet: After a further 5 min at 37°C, the reaction was initiated with 10 µM nt of PhiX RFI DNA (NEB) linearized with ApaLI and incubated for 120 min at 37°C. .. In strand exchange assays containing Rad54, 7 µM of Rad51 (wild type, Rad51-E206A, or Rad51-EED) was incubated with 7 µM nt PhiX174 virion DNA (NEB) for 8 min at 37°C in strand exchange buffer (same as above except the total salt concentration was 100 mM KCl). ..

    Article Title: A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors
    Article Snippet: The gel was then stained with SYBR Gold (Thermo Fisher Scientific) and imaged using a LAS4000 mini (GE Healthcare). .. In strand exchange assays containing Swi5-Sfr1, 5 µM of Rad51 (wild type, Rad51-E206A, or Rad51-EED) was incubated with 10 µM nt PhiX174 virion DNA (NEB) for 5 min at 37°C in strand exchange buffer (same as above except the total salt concentration was 100 mM KCl). .. Next, 0.5 µM of Swi5-Sfr1 (or the indicated concentration) was added and following a 5 min incubation at 37°C, 1 µM of RPA was included.

    Infection:

    Article Title: Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues
    Article Snippet: .. Purified phiX174 virion DNA was purchased from New England Biolabs (N3023S; Ipswich, MA, USA), total DNA from human MRC-5 cells infected with cytomegalovirus (human herpesvirus 5 strain AD169) was ATCC VR-538D, total DNA from human A549 cells infected with adenovirus type 5 (HAdV-5 strain Adenoid 75) was ATCC VR-5D, and total RNA from human HEp-2 cells infected with respiratory syncytial virus (HRSV strain Long) was ATCC VR-26D, all purchased from ATCC (Manassas, VA, USA). .. Plasmid minipreps were prepared from pBR322 subclones carrying JC or BK polyomavirus genomes (J. C. Alwine, University of Pennsylvania, Philadelphia, PA) and from pUC19 carrying the human papillomavirus 16 (HPV16) genome (obtained from Peter Howley, Harvard Medical School, Boston, MA).

    Incubation:

    Article Title: A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors
    Article Snippet: After a further 5 min at 37°C, the reaction was initiated with 10 µM nt of PhiX RFI DNA (NEB) linearized with ApaLI and incubated for 120 min at 37°C. .. In strand exchange assays containing Rad54, 7 µM of Rad51 (wild type, Rad51-E206A, or Rad51-EED) was incubated with 7 µM nt PhiX174 virion DNA (NEB) for 8 min at 37°C in strand exchange buffer (same as above except the total salt concentration was 100 mM KCl). ..

    Article Title: A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors
    Article Snippet: The concentration of inorganic phosphate was then measured with a commercial malachite green phosphate detection kit (BioAssay Systems) according to the manufacturer’s instructions. .. Three-strand exchange assay To examine the intrinsic strand exchange activity of Rad51, 15 µM of Rad51 (wild type, Rad51-E206A, or Rad51-EED) was incubated with 30 µM nt PhiX174 virion DNA (NEB) for 5 min at 37°C in strand exchange buffer (30 mM Tris-HCl [pH7.5], 1 mM DTT, 150 mM KCl, 3.5 mM MgCl2, 2 mM ATP, 8 mM phosphocreatine, 8 units/mL creatine phosphokinase and 2.5% glycerol). .. Next, 1 µM of RPA was added to the reaction and after a 5 min incubation, the reaction was initiated with 20 µM nt of PhiX RFI DNA (NEB) linearized with ApaLI.

    Article Title: A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors
    Article Snippet: The gel was then stained with SYBR Gold (Thermo Fisher Scientific) and imaged using a LAS4000 mini (GE Healthcare). .. In strand exchange assays containing Swi5-Sfr1, 5 µM of Rad51 (wild type, Rad51-E206A, or Rad51-EED) was incubated with 10 µM nt PhiX174 virion DNA (NEB) for 5 min at 37°C in strand exchange buffer (same as above except the total salt concentration was 100 mM KCl). .. Next, 0.5 µM of Swi5-Sfr1 (or the indicated concentration) was added and following a 5 min incubation at 37°C, 1 µM of RPA was included.

    Activity Assay:

    Article Title: A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors
    Article Snippet: The concentration of inorganic phosphate was then measured with a commercial malachite green phosphate detection kit (BioAssay Systems) according to the manufacturer’s instructions. .. Three-strand exchange assay To examine the intrinsic strand exchange activity of Rad51, 15 µM of Rad51 (wild type, Rad51-E206A, or Rad51-EED) was incubated with 30 µM nt PhiX174 virion DNA (NEB) for 5 min at 37°C in strand exchange buffer (30 mM Tris-HCl [pH7.5], 1 mM DTT, 150 mM KCl, 3.5 mM MgCl2, 2 mM ATP, 8 mM phosphocreatine, 8 units/mL creatine phosphokinase and 2.5% glycerol). .. Next, 1 µM of RPA was added to the reaction and after a 5 min incubation, the reaction was initiated with 20 µM nt of PhiX RFI DNA (NEB) linearized with ApaLI.

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    New England Biolabs nt phix174 virion dna
    Rad51-EED binds both ssDNA and dsDNA like wild-type Rad51. ( A ) Purified Rad51-EED was examined by SDS-PAGE and coomassie staining. ( B, C ) The indicated concentrations of Rad51 (WT) or Rad51-EED (EED) were incubated with 30 µM nt <t>PhiX174</t> virion DNA (ssDNA; B ) or 20 µM nt of linearized PhiX174 RF I DNA (dsDNA; C ) in the presence or absence of ATP. Protein-DNA complexes were crosslinked with glutaraldehyde and then resolved by agarose gel electrophoresis. ( D ) Purified Rad51 (WT or E206K) was incubated with purified Swi5-Sfr1 (or the equivalent volume of protein storage buffer) and subjected to immunoprecipitation with an anti-Sfr1 antibody. For quantification, Rad51 signal was normalized to Sfr1 signal and expressed relative to wild type. Data in ( D ) are the means of two independent experiments with individual values shown. Relative co-IP of Rad51 (%) for Figure 6—figure supplement 1D .
    Nt Phix174 Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Rad51-EED binds both ssDNA and dsDNA like wild-type Rad51. ( A ) Purified Rad51-EED was examined by SDS-PAGE and coomassie staining. ( B, C ) The indicated concentrations of Rad51 (WT) or Rad51-EED (EED) were incubated with 30 µM nt PhiX174 virion DNA (ssDNA; B ) or 20 µM nt of linearized PhiX174 RF I DNA (dsDNA; C ) in the presence or absence of ATP. Protein-DNA complexes were crosslinked with glutaraldehyde and then resolved by agarose gel electrophoresis. ( D ) Purified Rad51 (WT or E206K) was incubated with purified Swi5-Sfr1 (or the equivalent volume of protein storage buffer) and subjected to immunoprecipitation with an anti-Sfr1 antibody. For quantification, Rad51 signal was normalized to Sfr1 signal and expressed relative to wild type. Data in ( D ) are the means of two independent experiments with individual values shown. Relative co-IP of Rad51 (%) for Figure 6—figure supplement 1D .

    Journal: eLife

    Article Title: A novel motif of Rad51 serves as an interaction hub for recombination auxiliary factors

    doi: 10.7554/eLife.64131

    Figure Lengend Snippet: Rad51-EED binds both ssDNA and dsDNA like wild-type Rad51. ( A ) Purified Rad51-EED was examined by SDS-PAGE and coomassie staining. ( B, C ) The indicated concentrations of Rad51 (WT) or Rad51-EED (EED) were incubated with 30 µM nt PhiX174 virion DNA (ssDNA; B ) or 20 µM nt of linearized PhiX174 RF I DNA (dsDNA; C ) in the presence or absence of ATP. Protein-DNA complexes were crosslinked with glutaraldehyde and then resolved by agarose gel electrophoresis. ( D ) Purified Rad51 (WT or E206K) was incubated with purified Swi5-Sfr1 (or the equivalent volume of protein storage buffer) and subjected to immunoprecipitation with an anti-Sfr1 antibody. For quantification, Rad51 signal was normalized to Sfr1 signal and expressed relative to wild type. Data in ( D ) are the means of two independent experiments with individual values shown. Relative co-IP of Rad51 (%) for Figure 6—figure supplement 1D .

    Article Snippet: In strand exchange assays containing Swi5-Sfr1, 5 µM of Rad51 (wild type, Rad51-E206A, or Rad51-EED) was incubated with 10 µM nt PhiX174 virion DNA (NEB) for 5 min at 37°C in strand exchange buffer (same as above except the total salt concentration was 100 mM KCl).

    Techniques: Purification, SDS Page, Staining, Incubation, Agarose Gel Electrophoresis, Immunoprecipitation, Co-Immunoprecipitation Assay

    Bcl2 directly inhibits MRN-mediated DNA resection. ( A ) DNA resection activity of purified Mre11, Rad50, NBS1 and MRN complex were measured using PhiX174 circular ssDNA as substrate (left panel: endonuclease activity) and dsDNA as substrate (right panel: exonuclease activity). ( B ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified Bcl2. BSA was used as control. ( C ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified WT Bcl2 or Bcl2 BH deletion mutant protein(s).

    Journal: Nucleic Acids Research

    Article Title: Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation

    doi: 10.1093/nar/gku1358

    Figure Lengend Snippet: Bcl2 directly inhibits MRN-mediated DNA resection. ( A ) DNA resection activity of purified Mre11, Rad50, NBS1 and MRN complex were measured using PhiX174 circular ssDNA as substrate (left panel: endonuclease activity) and dsDNA as substrate (right panel: exonuclease activity). ( B ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified Bcl2. BSA was used as control. ( C ) DNA resection activity of MRN complex was measured in the absence or presence of increasing concentrations of purified WT Bcl2 or Bcl2 BH deletion mutant protein(s).

    Article Snippet: The PhiX174 circular single-stranded virion DNA substrate was purchased from New England Biolabs (Ipswich, MA, USA).

    Techniques: Activity Assay, Purification, Mutagenesis

    EXO1-dependent ATR Activation. A , a model system for excision repair-checkpoint coupling. ATR kinase reactions were carried out with ATR-ATRIP, TopBP1, RPA, and EXO1 as indicated. 0.6 ng (27 p m ) single-stranded φX174 DNA ( ssDNA ), plasmid DNA (

    Journal: The Journal of Biological Chemistry

    Article Title: Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System *

    doi: 10.1074/jbc.M113.542787

    Figure Lengend Snippet: EXO1-dependent ATR Activation. A , a model system for excision repair-checkpoint coupling. ATR kinase reactions were carried out with ATR-ATRIP, TopBP1, RPA, and EXO1 as indicated. 0.6 ng (27 p m ) single-stranded φX174 DNA ( ssDNA ), plasmid DNA (

    Article Snippet: The φX174 ssDNA was purchased from New England Biolabs (N3023).

    Techniques: Activation Assay, Recombinase Polymerase Amplification, Plasmid Preparation