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
    Size:
    250 ug
    Category:
    Genomic DNA
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    Structured Review

    New England Biolabs phix174 virion dna
    PhiX174 Virion DNA
    PhiX174 Virion DNA 250 ug
    https://www.bioz.com/result/phix174 virion dna/product/New England Biolabs
    Average 94 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    phix174 virion dna - by Bioz Stars, 2020-02
    94/100 stars

    Images

    1) 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

    2) Product Images from "MutS inhibits RecA-mediated strand exchange with platinated DNA substrates"

    Article Title: MutS inhibits RecA-mediated strand exchange with platinated DNA substrates

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

    doi: 10.1073/pnas.0406104101

    Survival of platinated phiX174 RFI in wild-type and NER-deficient E. coli . RFI molecules with the indicated number of cisplatin adducts were mixed with an excess of wild-type (▪) or uvrA6 (•) bacteria and the number of plaque-forming units was determined.
    Figure Legend Snippet: Survival of platinated phiX174 RFI in wild-type and NER-deficient E. coli . RFI molecules with the indicated number of cisplatin adducts were mixed with an excess of wild-type (▪) or uvrA6 (•) bacteria and the number of plaque-forming units was determined.

    Techniques Used:

    Related Articles

    Positive Control:

    Article Title: Quality control of purified proteins involved in homologous recombination
    Article Snippet: Commercial Form I ΦX174 (NEB, N3021L) and virion DNA (circular ssDNA, NEB, N3023L). .. RecA protein (NEB, M0249S) as positive control.

    Blocking Assay:

    Article Title: Quality control of purified proteins involved in homologous recombination
    Article Snippet: Commercial RFI ΦX174 (NEB, N3021L) and virion DNA (circular ssDNA, NEB, N3023L) (see ). .. 37 °C and 30 °C water baths, heat block.

    Electrophoresis:

    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: 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: The CRISPR Associated Protein Cas4 Is a 5′ to 3′ DNA Exonuclease with an Iron-Sulfur Cluster
    Article Snippet: Circular phiX174 virion DNA was purchased from New England Biolabs. .. On completion of the reaction the activity was quenched by the addition of EDTA to 20 mM final concentration and DNA was separated by electrophoresis on an agarose gel and visualised using ethidium bromide on a UV transilluminator.

    Incubation:

    Article Title: Ctp1 protein–DNA filaments promote DNA bridging and DNA double-strand break repair
    Article Snippet: 500-bp dsDNA was generated by PCR using a phiX174 Virion DNA template (New England BioLabs Inc., Ipswich, MA) and primers 5′-FAM-AGTTTTATCGCTTCCATGAC-3′ (where FAM represents fluorescein amidite) and 5′-TCAGAAAATCGAAATCATCTTC-3′ (Integrated DNA Technologies, Coralville, IA). .. Serial dilutions of Ctp1, diluted in 20 m m Tris, pH 7.5, 0.1 m m tris(2-carboxyethyl)phosphine, 50 m m potassium acetate, and 10% glycerol, were incubated with 15 n m DNA substrate, 5% glycerol, and 1× reaction buffer (20 m m Tris, pH 7.5, 250 m m potassium acetate, 0.1 m m DTT, 10 μg μl−1 BSA, 0.5% glycerol) for 20 min at 20 °C.

    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.

    Article Title: The CRISPR Associated Protein Cas4 Is a 5′ to 3′ DNA Exonuclease with an Iron-Sulfur Cluster
    Article Snippet: The reaction was incubated at 55 or 75°C for the time indicated and quenched by the addition of EDTA to a final concentration of 20 mM. .. Circular phiX174 virion DNA was purchased from New England Biolabs.

    Article Title: Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1
    Article Snippet: .. Substrates for the recombinant GBSV1-NSN nuclease To determine the specificity of substrate for the recombinant GBSV1-NSN nuclease, 1 μg of the circular pGEX-4T-2 plasmid dsDNA, the linear pGEX-4T-2 plasmid dsDNA, the PhiX174 virion ssDNA (New England Biolabs, UK) or baker's yeast RNA (Sigma-Aldrich, USA) were respectively incubated with 1.5 μg of the purified GBSV1-NSN protein in 20 μl of a reaction buffer (20 mM Hepes, 50 mM KCl, 2 mM MgCl2 , 0.2 mM EDTA, 0.1 mg/ml BSA and 10% glycerol, pH 7.5) at 37°C. ..

    Activity 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.

    Article Title: The CRISPR Associated Protein Cas4 Is a 5′ to 3′ DNA Exonuclease with an Iron-Sulfur Cluster
    Article Snippet: Paragraph title: Nuclease activity of Sso0001 ... Circular phiX174 virion DNA was purchased from New England Biolabs.

    Cell Culture:

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: PCC 7002 (WT and ΔA2542) were cultured overnight in 20 ml media A+ in bubble tubes and diluted to an OD730 nm = 0.01 in 150 ml media A+ in 250 ml pyrex bottle with glass rod for bubbling (in quintuple). .. A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation.

    Shift Assay:

    Article Title: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
    Article Snippet: Paragraph title: Degradation assay, nuclei spin down assay, and phosphorylation shift assay ... Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S).

    Recombinase Polymerase Amplification:

    Article Title: In vitro assays for DNA pairing and recombination-associated DNA synthesis
    Article Snippet: Purified proteins from Saccharomyces cerevisiae : Rad51 and RPA ( see ) ( ). .. Store at 4 °C. ϕX174 ssDNA (virion) is purchased from NEB (catalog number: N3023S) ( see ).

    Competitive Binding Assay:

    Article Title: The CRISPR Associated Protein Cas4 Is a 5′ to 3′ DNA Exonuclease with an Iron-Sulfur Cluster
    Article Snippet: Circular phiX174 virion DNA was purchased from New England Biolabs. .. For the competition assay, 300 nM Sso0001 was incubated with 500 nM oligonucleotide 3-Fl-50mer in reaction buffer at 55°C.

    Spin Down Assay:

    Article Title: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
    Article Snippet: Paragraph title: Degradation assay, nuclei spin down assay, and phosphorylation shift assay ... Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S).

    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).

    Generated:

    Article Title: Ctp1 protein–DNA filaments promote DNA bridging and DNA double-strand break repair
    Article Snippet: .. 500-bp dsDNA was generated by PCR using a phiX174 Virion DNA template (New England BioLabs Inc., Ipswich, MA) and primers 5′-FAM-AGTTTTATCGCTTCCATGAC-3′ (where FAM represents fluorescein amidite) and 5′-TCAGAAAATCGAAATCATCTTC-3′ (Integrated DNA Technologies, Coralville, IA). .. The 500-bp dsDNA was gel-purified using a QiaQuick gel extraction kit (Qiagen, Hilden, Germany).

    Imaging:

    Article Title: In vitro assays for DNA pairing and recombination-associated DNA synthesis
    Article Snippet: Store at 4 °C. ϕX174 ssDNA (virion) is purchased from NEB (catalog number: N3023S) ( see ). .. TBE-Agarose gel running buffer, apparatus, and imaging system, as listed in Section 2.1.

    Polymerase Chain Reaction:

    Article Title: Ctp1 protein–DNA filaments promote DNA bridging and DNA double-strand break repair
    Article Snippet: .. 500-bp dsDNA was generated by PCR using a phiX174 Virion DNA template (New England BioLabs Inc., Ipswich, MA) and primers 5′-FAM-AGTTTTATCGCTTCCATGAC-3′ (where FAM represents fluorescein amidite) and 5′-TCAGAAAATCGAAATCATCTTC-3′ (Integrated DNA Technologies, Coralville, IA). .. The 500-bp dsDNA was gel-purified using a QiaQuick gel extraction kit (Qiagen, Hilden, Germany).

    Recombinant:

    Article Title: Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation
    Article Snippet: Purified recombinant Ku70 was purchased from Trevigen Inc. (Gaithersburg, MD, USA). .. The PhiX174 circular single-stranded virion DNA substrate was purchased from New England Biolabs (Ipswich, MA, USA).

    Article Title: Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1
    Article Snippet: .. Substrates for the recombinant GBSV1-NSN nuclease To determine the specificity of substrate for the recombinant GBSV1-NSN nuclease, 1 μg of the circular pGEX-4T-2 plasmid dsDNA, the linear pGEX-4T-2 plasmid dsDNA, the PhiX174 virion ssDNA (New England Biolabs, UK) or baker's yeast RNA (Sigma-Aldrich, USA) were respectively incubated with 1.5 μg of the purified GBSV1-NSN protein in 20 μl of a reaction buffer (20 mM Hepes, 50 mM KCl, 2 mM MgCl2 , 0.2 mM EDTA, 0.1 mg/ml BSA and 10% glycerol, pH 7.5) at 37°C. ..

    Radioactivity:

    Article Title: Quality control of purified proteins involved in homologous recombination
    Article Snippet: Equipment and safety measures for working with radioactivity (see ). .. Commercial Form I ΦX174 (NEB, N3021L) and virion DNA (circular ssDNA, NEB, N3023L).

    Isolation:

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation. .. DNA was isolated from four replicates as in , except that the pH of the phenol solution was adjusted to 8 using Tris alkaline buffer and purified DNA was diluted 1:1000.

    Labeling:

    Article Title: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
    Article Snippet: .. Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S). .. The reactions were analyzed by PhosphorImager and quantitation was performed using ImageQuant™ software (Molecular Dynamics).

    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: .. 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: Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation
    Article Snippet: Purified recombinant Ku70 was purchased from Trevigen Inc. (Gaithersburg, MD, USA). .. The PhiX174 circular single-stranded virion DNA substrate was purchased from New England Biolabs (Ipswich, MA, USA).

    Article Title: The CRISPR Associated Protein Cas4 Is a 5′ to 3′ DNA Exonuclease with an Iron-Sulfur Cluster
    Article Snippet: Nuclease activity of Sso0001 Purified Sso0001 wild-type protein or D99A variant was mixed with 1 µM oligonucleotide substrates in reaction buffer (20 mM MES pH 6.0, 10 mM DTT, 100 mM potassium glutamate, supplemented with 10 mM EDTA, 10 mM MgCl2 or 10 mM MnCl2 as indicated). .. Circular phiX174 virion DNA was purchased from New England Biolabs.

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation. .. DNA was isolated from four replicates as in , except that the pH of the phenol solution was adjusted to 8 using Tris alkaline buffer and purified DNA was diluted 1:1000.

    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).

    Article Title: MutS inhibits RecA-mediated strand exchange with platinated DNA substrates
    Article Snippet: MutSΔ680 protein was purified as described ( ). .. PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form.

    Article Title: Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1
    Article Snippet: .. Substrates for the recombinant GBSV1-NSN nuclease To determine the specificity of substrate for the recombinant GBSV1-NSN nuclease, 1 μg of the circular pGEX-4T-2 plasmid dsDNA, the linear pGEX-4T-2 plasmid dsDNA, the PhiX174 virion ssDNA (New England Biolabs, UK) or baker's yeast RNA (Sigma-Aldrich, USA) were respectively incubated with 1.5 μg of the purified GBSV1-NSN protein in 20 μl of a reaction buffer (20 mM Hepes, 50 mM KCl, 2 mM MgCl2 , 0.2 mM EDTA, 0.1 mg/ml BSA and 10% glycerol, pH 7.5) at 37°C. ..

    Article Title: In vitro assays for DNA pairing and recombination-associated DNA synthesis
    Article Snippet: Purified proteins from Saccharomyces cerevisiae : Rad51 and RPA ( see ) ( ). .. Store at 4 °C. ϕX174 ssDNA (virion) is purchased from NEB (catalog number: N3023S) ( see ).

    Degradation Assay:

    Article Title: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
    Article Snippet: Paragraph title: Degradation assay, nuclei spin down assay, and phosphorylation shift assay ... Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S).

    Construct:

    Article Title: Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation
    Article Snippet: [γ-32 P]-ATP was obtained from PerkinElmer (Waltham, MA, USA). pCBASce and DR-GFP (green fluorescent protein) constructs were purchased from Addgene (Cambridge, MA, USA). .. The PhiX174 circular single-stranded virion DNA substrate was purchased from New England Biolabs (Ipswich, MA, USA).

    Periodic Counter-current Chromatography:

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: PCC 7002 (WT and ΔA2542) were cultured overnight in 20 ml media A+ in bubble tubes and diluted to an OD730 nm = 0.01 in 150 ml media A+ in 250 ml pyrex bottle with glass rod for bubbling (in quintuple). .. A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation.

    Staining:

    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: 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).

    IA:

    Article Title: Ctp1 protein–DNA filaments promote DNA bridging and DNA double-strand break repair
    Article Snippet: .. 500-bp dsDNA was generated by PCR using a phiX174 Virion DNA template (New England BioLabs Inc., Ipswich, MA) and primers 5′-FAM-AGTTTTATCGCTTCCATGAC-3′ (where FAM represents fluorescein amidite) and 5′-TCAGAAAATCGAAATCATCTTC-3′ (Integrated DNA Technologies, Coralville, IA). .. The 500-bp dsDNA was gel-purified using a QiaQuick gel extraction kit (Qiagen, Hilden, Germany).

    Article Title: Bcl2 inhibits recruitment of Mre11 complex to DNA double-strand breaks in response to high-linear energy transfer radiation
    Article Snippet: The PhiX174 circular single-stranded virion DNA substrate was purchased from New England Biolabs (Ipswich, MA, USA). .. The PhiX174 circular single-stranded virion DNA substrate was purchased from New England Biolabs (Ipswich, MA, USA).

    Agarose Gel Electrophoresis:

    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: 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: The CRISPR Associated Protein Cas4 Is a 5′ to 3′ DNA Exonuclease with an Iron-Sulfur Cluster
    Article Snippet: Circular phiX174 virion DNA was purchased from New England Biolabs. .. On completion of the reaction the activity was quenched by the addition of EDTA to 20 mM final concentration and DNA was separated by electrophoresis on an agarose gel and visualised using ethidium bromide on a UV transilluminator.

    ATPase Assay:

    Article Title: Quality control of purified proteins involved in homologous recombination
    Article Snippet: ATPase assay buffer: 66 mM Tris-HCl, pH 7.5, 26 mM MgCl2 , 3.6 mM DTT, 2 mM ATP, 180 μg/ml BSA. .. Commercial Form I ΦX174 (NEB, N3021L) and virion DNA (circular ssDNA, NEB, N3023L).

    SDS Page:

    Article Title: MutS inhibits RecA-mediated strand exchange with platinated DNA substrates
    Article Snippet: Analysis of the purified protein by SDS/PAGE showed no visible contaminants. .. PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form.

    Plasmid Preparation:

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: Paragraph title: Plasmid copy number quantification ... A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation.

    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).

    Article Title: Characterization of a novel non-specific nuclease from thermophilic bacteriophage GBSV1
    Article Snippet: .. Substrates for the recombinant GBSV1-NSN nuclease To determine the specificity of substrate for the recombinant GBSV1-NSN nuclease, 1 μg of the circular pGEX-4T-2 plasmid dsDNA, the linear pGEX-4T-2 plasmid dsDNA, the PhiX174 virion ssDNA (New England Biolabs, UK) or baker's yeast RNA (Sigma-Aldrich, USA) were respectively incubated with 1.5 μg of the purified GBSV1-NSN protein in 20 μl of a reaction buffer (20 mM Hepes, 50 mM KCl, 2 mM MgCl2 , 0.2 mM EDTA, 0.1 mg/ml BSA and 10% glycerol, pH 7.5) at 37°C. ..

    Software:

    Article Title: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
    Article Snippet: Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S). .. The reactions were analyzed by PhosphorImager and quantitation was performed using ImageQuant™ software (Molecular Dynamics).

    Article Title: In vitro assays for DNA pairing and recombination-associated DNA synthesis
    Article Snippet: Store at 4 °C. ϕX174 ssDNA (virion) is purchased from NEB (catalog number: N3023S) ( see ). .. ImageQuant software (version 5.1, GE Healthcare).

    Binding Assay:

    Article Title: MutS inhibits RecA-mediated strand exchange with platinated DNA substrates
    Article Snippet: MutL protein was a gift from F. J. Lopez de Saro and M. ODonnell (The Rockefeller University, New York) and MutL and single-strand binding protein were obtained from United States Biological (Cleveland, OH). .. PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form.

    Sample Prep:

    Article Title: Metagenomic Assay for Identification of Microbial Pathogens in Tumor Tissues
    Article Snippet: Paragraph title: Sample preparation. ... 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).

    Ethanol Precipitation:

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: .. A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation. .. The spike-in was added immediately after addition of the TE (with lysozyme) to the frozen cell pellets at a concentration of 10 copies per cell.

    Quantitation Assay:

    Article Title: Proteolysis of Xenopus Cip-type CDK inhibitor, p16Xic2, is regulated by PCNA binding and CDK2 phosphorylation
    Article Snippet: Proteins labeled with 35 S-methionine were added to extracts at a final dilution of 1:15 in the presence or absence of 10 ng/ul demembranated XSC or ΦX174 single-stranded DNA (New England Biolab, N3023S). .. The reactions were analyzed by PhosphorImager and quantitation was performed using ImageQuant™ software (Molecular Dynamics).

    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: .. 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: The CRISPR Associated Protein Cas4 Is a 5′ to 3′ DNA Exonuclease with an Iron-Sulfur Cluster
    Article Snippet: The reaction was incubated at 55 or 75°C for the time indicated and quenched by the addition of EDTA to a final concentration of 20 mM. .. Circular phiX174 virion DNA was purchased from New England Biolabs.

    Article Title: Distinct and redundant functions of three homologs of RNase III in the cyanobacterium Synechococcus sp. strain PCC 7002
    Article Snippet: A linear DNA spike-in was made by amplifying ΦX174 Virion DNA (NEB, N3023S) with primer JCC425 and primer JCC426 and purifying the product via ethanol precipitation. .. The spike-in was added immediately after addition of the TE (with lysozyme) to the frozen cell pellets at a concentration of 10 copies per cell.

    Article Title: MutS inhibits RecA-mediated strand exchange with platinated DNA substrates
    Article Snippet: The concentration of RecA was determined by the ninhydrin protein assay ( ). .. PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form.

    Thin Layer Chromatography:

    Article Title: Quality control of purified proteins involved in homologous recombination
    Article Snippet: Paragraph title: 2.2.2. Thin-layer chromatography (TLC) method ... Commercial Form I ΦX174 (NEB, N3021L) and virion DNA (circular ssDNA, NEB, N3023L).

    Gel Extraction:

    Article Title: Ctp1 protein–DNA filaments promote DNA bridging and DNA double-strand break repair
    Article Snippet: 500-bp dsDNA was generated by PCR using a phiX174 Virion DNA template (New England BioLabs Inc., Ipswich, MA) and primers 5′-FAM-AGTTTTATCGCTTCCATGAC-3′ (where FAM represents fluorescein amidite) and 5′-TCAGAAAATCGAAATCATCTTC-3′ (Integrated DNA Technologies, Coralville, IA). .. The 500-bp dsDNA was gel-purified using a QiaQuick gel extraction kit (Qiagen, Hilden, Germany).

    Variant Assay:

    Article Title: The CRISPR Associated Protein Cas4 Is a 5′ to 3′ DNA Exonuclease with an Iron-Sulfur Cluster
    Article Snippet: Nuclease activity of Sso0001 Purified Sso0001 wild-type protein or D99A variant was mixed with 1 µM oligonucleotide substrates in reaction buffer (20 mM MES pH 6.0, 10 mM DTT, 100 mM potassium glutamate, supplemented with 10 mM EDTA, 10 mM MgCl2 or 10 mM MnCl2 as indicated). .. Circular phiX174 virion DNA was purchased from New England Biolabs.

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    New England Biolabs phix174 virion dna
    PathoChip assay performance assessed using positive-control <t>DNA.</t> (A) Whole-genome amplification kits that feature three different enzymatic processes were compared in their abilities to detect <t>phiX174</t> 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).
    Phix174 Virion Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phix174 virion dna/product/New England Biolabs
    Average 94 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    phix174 virion dna - by Bioz Stars, 2020-02
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    93
    New England Biolabs ϕx174 virion ssdna
    Three Strand Exchange Recombinase Assay A Active RAD51 nucleoprotein filament is formed on <t>ϕX174</t> Virion circular <t>ssDNA</t> 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.
    ϕx174 Virion Ssdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ϕx174 virion ssdna/product/New England Biolabs
    Average 93 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    ϕx174 virion ssdna - by Bioz Stars, 2020-02
    93/100 stars
      Buy from Supplier

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    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).

    Journal: mBio

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

    doi: 10.1128/mBio.01714-14

    Figure Lengend 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).

    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).

    Techniques: 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.

    Journal: mBio

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

    doi: 10.1128/mBio.01714-14

    Figure Lengend 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.

    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).

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

    Survival of platinated phiX174 RFI in wild-type and NER-deficient E. coli . RFI molecules with the indicated number of cisplatin adducts were mixed with an excess of wild-type (▪) or uvrA6 (•) bacteria and the number of plaque-forming units was determined.

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

    Article Title: MutS inhibits RecA-mediated strand exchange with platinated DNA substrates

    doi: 10.1073/pnas.0406104101

    Figure Lengend Snippet: Survival of platinated phiX174 RFI in wild-type and NER-deficient E. coli . RFI molecules with the indicated number of cisplatin adducts were mixed with an excess of wild-type (▪) or uvrA6 (•) bacteria and the number of plaque-forming units was determined.

    Article Snippet: PhiX174 replicative form (RFI) and virion DNA forms were from New England Biolabs and the RFI form was digested with Xho I restriction endonuclease (New England Biolabs) to produce the linear duplex form.

    Techniques:

    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.

    Journal: Methods in enzymology

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

    doi: 10.1016/bs.mie.2017.11.011

    Figure Lengend 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.

    Article Snippet: The oligonucleotides are resuspended in 10mM Tris (pH8.0) and stored at −20°C. ϕX174 Virion ssDNA (NEB# N3023) ϕX174 RFI dsDNA (NEB# N3021)

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