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 93 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    phix174 virion dna - by Bioz Stars, 2020-05
    93/100 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 "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

    3) 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

    Related Articles

    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.

    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: 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).

    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).

    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).

    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).

    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).

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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: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phix174 virion dna/product/New England Biolabs
    Average 93 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    phix174 virion dna - by Bioz Stars, 2020-05
    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

    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