control input dna  (Thermo Fisher)


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    Name:
    Control DNA from CEPH Individual 1347 02
    Description:
    The Control DNA can be used with Linkage Mapping Sets MD10 HD5 and LD20 • Functions as a control template to monitor PCR amplification • Serves as sizing reference for controlling gel to gel or capillary to capillary variation • Aids in the comparison of alleles sizes from different gels allele binning • Enables correlation with allele frequency data from external sources such as others databases and laboratories For Research Use Only Not for use in diagnostics procedures
    Catalog Number:
    403062
    Price:
    None
    Applications:
    Genotyping & Genomic Profiling|PCR|PCR & Real-Time PCR|PCR Genotyping|Gene Expression Analysis & Genotyping|Microsatellite Analysis
    Category:
    Standards Ladders Controls
    Buy from Supplier


    Structured Review

    Thermo Fisher control input dna
    The Control DNA can be used with Linkage Mapping Sets MD10 HD5 and LD20 • Functions as a control template to monitor PCR amplification • Serves as sizing reference for controlling gel to gel or capillary to capillary variation • Aids in the comparison of alleles sizes from different gels allele binning • Enables correlation with allele frequency data from external sources such as others databases and laboratories For Research Use Only Not for use in diagnostics procedures
    https://www.bioz.com/result/control input dna/product/Thermo Fisher
    Average 99 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    control input dna - by Bioz Stars, 2020-07
    99/100 stars

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    Related Articles

    Clone Assay:

    Article Title: Functional and Structural Analysis of Five Mutations Identified in Methylmalonic Aciduria cbIB Type
    Article Snippet: .. Gene fragments corresponding to exons and flanking intronic regions were amplified from patients P2 and P4 and from a DNA control and cloned into the TOPO vector (Invitrogen, Carlsbad, CA) as previously described ( ). .. 2 μg of the wild-type or mutant minigenes were transfected into the following cell lines: Hep3B, HEK293 or COS7 using JetPEI, following the manufacturer’s recommendations.

    Amplification:

    Article Title: Functional and Structural Analysis of Five Mutations Identified in Methylmalonic Aciduria cbIB Type
    Article Snippet: .. Gene fragments corresponding to exons and flanking intronic regions were amplified from patients P2 and P4 and from a DNA control and cloned into the TOPO vector (Invitrogen, Carlsbad, CA) as previously described ( ). .. 2 μg of the wild-type or mutant minigenes were transfected into the following cell lines: Hep3B, HEK293 or COS7 using JetPEI, following the manufacturer’s recommendations.

    Pyrolysis Gas Chromatography:

    Article Title: Identification of RegIV as a Novel GLI1 Target Gene in Human Pancreatic Cancer
    Article Snippet: .. Cells were then exposed to 5 mL medium (Opti-MEM; Invitrogen) with complexes containing packaging helper construct (GeneChem Company, Shanghai, China), 20 µg expression plasmid DNA (pGC-FU-EGFP-3FLAG-GLI1), or control plasmid DNA (pGC-FU-EGFP-3FLAG) with 100 µl lipofectamine 2000 (Invitrogen, USA) in the presence of polybrene (8 µg/mL, Sigma-Aldrich, St. Louis, MO, USA). ..

    Construct:

    Article Title: Identification of RegIV as a Novel GLI1 Target Gene in Human Pancreatic Cancer
    Article Snippet: .. Cells were then exposed to 5 mL medium (Opti-MEM; Invitrogen) with complexes containing packaging helper construct (GeneChem Company, Shanghai, China), 20 µg expression plasmid DNA (pGC-FU-EGFP-3FLAG-GLI1), or control plasmid DNA (pGC-FU-EGFP-3FLAG) with 100 µl lipofectamine 2000 (Invitrogen, USA) in the presence of polybrene (8 µg/mL, Sigma-Aldrich, St. Louis, MO, USA). ..

    Article Title: Akt, a Target of Phosphatidylinositol 3-Kinase, Inhibits Apoptosis in a Differentiating Neuronal Cell Line
    Article Snippet: .. Control vector DNA or Akt construct DNA was mixed with a green fluorescent protein (GFP) vector DNA (Green Lantern; Life Technologies, Gaithersburg, Md.), and the mixtures were injected into equal numbers of cells for each experimental construct. .. The final DNA concentration was 1 μg/μl in 50 mM HEPES (pH 7.4)–40 mM NaCl.

    Purification:

    Article Title: Development of a DNA Microarray for Detection and Identification of Fungal Pathogens Involved in Invasive Mycoses
    Article Snippet: .. The purified target DNA (0.5 pmol) together with control DNA (Cy3-GCTCCTGACTCGTCCAATC; 0.05 pmol) was hybridized within a gene frame (15 by 16 mm) closed with a coverslip (Abgene House, Hamburg, Germany) in 70 μl of 6× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaH2 PO4 , and 1 mM EDTA [pH 7.7]). .. Prior to hybridization, the hybridization mixture was incubated for 10 min at 95°C, stored for 1 min on ice, and immediately used.

    Real-time Polymerase Chain Reaction:

    Article Title: Rapid molecular detection of macrolide resistance
    Article Snippet: .. qPCR assay Primers F1 and R1 (Table ) were combined at a final concentration of 176 nM with control DNA (MGAS10394) dilutions at indicated concentrations, in 1X PowerSYBR (ThermoFisher Cat # 4367659) and run on an Agilent Stratagene Mx3005P. ..

    Concentration Assay:

    Article Title: Rapid molecular detection of macrolide resistance
    Article Snippet: .. qPCR assay Primers F1 and R1 (Table ) were combined at a final concentration of 176 nM with control DNA (MGAS10394) dilutions at indicated concentrations, in 1X PowerSYBR (ThermoFisher Cat # 4367659) and run on an Agilent Stratagene Mx3005P. ..

    other:

    Article Title: Developmental Validation of the Huaxia Platinum System and application in 3 main ethnic groups of China
    Article Snippet: Control DNA 007 and the prepared 30 case-type samples were tested in two different accredited laboratories.

    Article Title: Developmental Validation of the Huaxia Platinum System and application in 3 main ethnic groups of China
    Article Snippet: The representative electropherogram of Control DNA 007 is shown in .

    Expressing:

    Article Title: Identification of RegIV as a Novel GLI1 Target Gene in Human Pancreatic Cancer
    Article Snippet: .. Cells were then exposed to 5 mL medium (Opti-MEM; Invitrogen) with complexes containing packaging helper construct (GeneChem Company, Shanghai, China), 20 µg expression plasmid DNA (pGC-FU-EGFP-3FLAG-GLI1), or control plasmid DNA (pGC-FU-EGFP-3FLAG) with 100 µl lipofectamine 2000 (Invitrogen, USA) in the presence of polybrene (8 µg/mL, Sigma-Aldrich, St. Louis, MO, USA). ..

    Polymerase Chain Reaction:

    Article Title: Developmental Validation of the Huaxia Platinum System and application in 3 main ethnic groups of China
    Article Snippet: .. Control DNA 007 was also applied to concordance, reproducibility, PCR-based studies, sensitivity, stability and mixture study. .. Pre-PCR sample preparation Prior to PCR amplification, blood on plain paper (untreated paper substrates, sterile filter paper in this study) must be lysed efficiently.

    Injection:

    Article Title: Akt, a Target of Phosphatidylinositol 3-Kinase, Inhibits Apoptosis in a Differentiating Neuronal Cell Line
    Article Snippet: .. Control vector DNA or Akt construct DNA was mixed with a green fluorescent protein (GFP) vector DNA (Green Lantern; Life Technologies, Gaithersburg, Md.), and the mixtures were injected into equal numbers of cells for each experimental construct. .. The final DNA concentration was 1 μg/μl in 50 mM HEPES (pH 7.4)–40 mM NaCl.

    Plasmid Preparation:

    Article Title: Identification of RegIV as a Novel GLI1 Target Gene in Human Pancreatic Cancer
    Article Snippet: .. Cells were then exposed to 5 mL medium (Opti-MEM; Invitrogen) with complexes containing packaging helper construct (GeneChem Company, Shanghai, China), 20 µg expression plasmid DNA (pGC-FU-EGFP-3FLAG-GLI1), or control plasmid DNA (pGC-FU-EGFP-3FLAG) with 100 µl lipofectamine 2000 (Invitrogen, USA) in the presence of polybrene (8 µg/mL, Sigma-Aldrich, St. Louis, MO, USA). ..

    Article Title: Akt, a Target of Phosphatidylinositol 3-Kinase, Inhibits Apoptosis in a Differentiating Neuronal Cell Line
    Article Snippet: .. Control vector DNA or Akt construct DNA was mixed with a green fluorescent protein (GFP) vector DNA (Green Lantern; Life Technologies, Gaithersburg, Md.), and the mixtures were injected into equal numbers of cells for each experimental construct. .. The final DNA concentration was 1 μg/μl in 50 mM HEPES (pH 7.4)–40 mM NaCl.

    Article Title: Functional and Structural Analysis of Five Mutations Identified in Methylmalonic Aciduria cbIB Type
    Article Snippet: .. Gene fragments corresponding to exons and flanking intronic regions were amplified from patients P2 and P4 and from a DNA control and cloned into the TOPO vector (Invitrogen, Carlsbad, CA) as previously described ( ). .. 2 μg of the wild-type or mutant minigenes were transfected into the following cell lines: Hep3B, HEK293 or COS7 using JetPEI, following the manufacturer’s recommendations.

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  • 99
    Thermo Fisher pcr mix
    Graphical overview of the methods used to detect latent viral forms. (A) Method used to detect episomal genomic <t>DNA.</t> On the top the genome organization of the Sl.L-PV-1, on the bottom a representation of the predicted circular form. Red arrows represent primer binding sites. The use of reverse primer annealing at the beginning of NS1 and forward primers annealing at the end of VP1 allows the amplification the connecting genomic sequence. (B) Restriction enzyme based methods to detect integrated virus. The purple lines represent the host genomic sequence, the orange flashes indicate restriction enzyme sites and the orange boxed indicate artificially ligated adaptors. (B1) It is shown how, after overnight ligation of the digested DNA, the obtained fragment would self-ligate in a circular form and how a specific amplification would allow to amplify the integration site. (B2) It is shown how, after the ligation of specific adaptors to the obtained digested fragments, a semi specific <t>PCR</t> would allow the amplification of the integration site. (C) Method to detect the integrated virus based on repeated Alu sequences in the host genome. Purple boxes represent Alu sequences and red arrows represent primer binding sites. A comparison of the PCR results obtained amplifying the template with either a mixture of Alu binding primers and specific primers or Alu binding primers alone (negative control) would allow to amplify the integration site and determine which amplified fragment on gel corresponds to it.
    Pcr Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 944 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr mix/product/Thermo Fisher
    Average 99 stars, based on 944 article reviews
    Price from $9.99 to $1999.99
    pcr mix - by Bioz Stars, 2020-07
    99/100 stars
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    94
    Thermo Fisher input dna
    Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic <t>DNA</t> was extracted 3 and 5 days after the transfection, linearized with <t>BglI</t> and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.
    Input Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 317 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/input dna/product/Thermo Fisher
    Average 94 stars, based on 317 article reviews
    Price from $9.99 to $1999.99
    input dna - by Bioz Stars, 2020-07
    94/100 stars
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    93
    Thermo Fisher biotinylated dna
    PcG proteins bind to replicating HCMV genomes. (A) HFF cells were infected with AD169 at an MOI of 1.5. At 72 hpi, newly synthesized <t>DNA</t> was labeled with EdU (10 μM) for 4 h, and Alexa 488 azide was conjugated by click chemistry. The viral protein pUL44 was visualized by antibody staining. The samples were analyzed by confocal microscopy. (B) Graph representing the percentage of cells positive for EdU only or for both EdU and pUL44 (mock, n = 150; infected, n = 150). (C) HFF cells were infected with AD169 at an MOI of 1.5 and used for accelerated native isolation of proteins on nascent DNA (aniPOND). At 72 hpi, cells were labeled for 4 h and <t>biotinylated</t> by click chemistry for affinity purification. No click reaction served as the negative control.
    Biotinylated Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/biotinylated dna/product/Thermo Fisher
    Average 93 stars, based on 25 article reviews
    Price from $9.99 to $1999.99
    biotinylated dna - by Bioz Stars, 2020-07
    93/100 stars
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    Image Search Results


    Graphical overview of the methods used to detect latent viral forms. (A) Method used to detect episomal genomic DNA. On the top the genome organization of the Sl.L-PV-1, on the bottom a representation of the predicted circular form. Red arrows represent primer binding sites. The use of reverse primer annealing at the beginning of NS1 and forward primers annealing at the end of VP1 allows the amplification the connecting genomic sequence. (B) Restriction enzyme based methods to detect integrated virus. The purple lines represent the host genomic sequence, the orange flashes indicate restriction enzyme sites and the orange boxed indicate artificially ligated adaptors. (B1) It is shown how, after overnight ligation of the digested DNA, the obtained fragment would self-ligate in a circular form and how a specific amplification would allow to amplify the integration site. (B2) It is shown how, after the ligation of specific adaptors to the obtained digested fragments, a semi specific PCR would allow the amplification of the integration site. (C) Method to detect the integrated virus based on repeated Alu sequences in the host genome. Purple boxes represent Alu sequences and red arrows represent primer binding sites. A comparison of the PCR results obtained amplifying the template with either a mixture of Alu binding primers and specific primers or Alu binding primers alone (negative control) would allow to amplify the integration site and determine which amplified fragment on gel corresponds to it.

    Journal: Frontiers in Microbiology

    Article Title: Persistent viremia by a novel parvovirus in a slow loris (Nycticebus coucang) with diffuse histiocytic sarcoma

    doi: 10.3389/fmicb.2014.00655

    Figure Lengend Snippet: Graphical overview of the methods used to detect latent viral forms. (A) Method used to detect episomal genomic DNA. On the top the genome organization of the Sl.L-PV-1, on the bottom a representation of the predicted circular form. Red arrows represent primer binding sites. The use of reverse primer annealing at the beginning of NS1 and forward primers annealing at the end of VP1 allows the amplification the connecting genomic sequence. (B) Restriction enzyme based methods to detect integrated virus. The purple lines represent the host genomic sequence, the orange flashes indicate restriction enzyme sites and the orange boxed indicate artificially ligated adaptors. (B1) It is shown how, after overnight ligation of the digested DNA, the obtained fragment would self-ligate in a circular form and how a specific amplification would allow to amplify the integration site. (B2) It is shown how, after the ligation of specific adaptors to the obtained digested fragments, a semi specific PCR would allow the amplification of the integration site. (C) Method to detect the integrated virus based on repeated Alu sequences in the host genome. Purple boxes represent Alu sequences and red arrows represent primer binding sites. A comparison of the PCR results obtained amplifying the template with either a mixture of Alu binding primers and specific primers or Alu binding primers alone (negative control) would allow to amplify the integration site and determine which amplified fragment on gel corresponds to it.

    Article Snippet: All amplifications were performed by using 5 μl of DNA as input in a 50 μl PCR mix containing DreamTaq DNA polymerase (Thermo Scientific) and 0.2 μM primers: PCRs were performed according to the following thermo profile: initial denaturation at 95°C for 5 min, followed by 35 cycles (25 during the nested PCR) at 95°C for 30 s, 60°C for 30 s and 72°C for 1.5 min (for the episomal DNA test) or 3 min (for the integration tests) and a final elongation cycle at 72°C for 7 min.

    Techniques: Binding Assay, Amplification, Sequencing, Ligation, Polymerase Chain Reaction, Negative Control

    Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.

    Journal: PLoS ONE

    Article Title: The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells

    doi: 10.1371/journal.pone.0116151

    Figure Lengend Snippet: Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.

    Article Snippet: The samples were linearized by digestion with BglI (Thermo Scientific), and the bacterially produced input DNA was fragmented by digestion with DpnI (Thermo Scientific).

    Techniques: Southern Blot, Transfection, Hybridization, Real-time Polymerase Chain Reaction, Standard Deviation, Expressing, Western Blot, Plasmid Preparation, Over Expression

    Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.

    Journal: PLoS ONE

    Article Title: The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells

    doi: 10.1371/journal.pone.0116151

    Figure Lengend Snippet: Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.

    Article Snippet: The samples were linearized by digestion with BglI (Thermo Scientific), and the bacterially produced input DNA was fragmented by digestion with DpnI (Thermo Scientific).

    Techniques: Southern Blot, Transfection, Produced, Plasmid Preparation, Hybridization, DNA Extraction, Negative Control

    PcG proteins bind to replicating HCMV genomes. (A) HFF cells were infected with AD169 at an MOI of 1.5. At 72 hpi, newly synthesized DNA was labeled with EdU (10 μM) for 4 h, and Alexa 488 azide was conjugated by click chemistry. The viral protein pUL44 was visualized by antibody staining. The samples were analyzed by confocal microscopy. (B) Graph representing the percentage of cells positive for EdU only or for both EdU and pUL44 (mock, n = 150; infected, n = 150). (C) HFF cells were infected with AD169 at an MOI of 1.5 and used for accelerated native isolation of proteins on nascent DNA (aniPOND). At 72 hpi, cells were labeled for 4 h and biotinylated by click chemistry for affinity purification. No click reaction served as the negative control.

    Journal: Journal of Virology

    Article Title: A Noncanonical Function of Polycomb Repressive Complexes Promotes Human Cytomegalovirus Lytic DNA Replication and Serves as a Novel Cellular Target for Antiviral Intervention

    doi: 10.1128/JVI.02143-18

    Figure Lengend Snippet: PcG proteins bind to replicating HCMV genomes. (A) HFF cells were infected with AD169 at an MOI of 1.5. At 72 hpi, newly synthesized DNA was labeled with EdU (10 μM) for 4 h, and Alexa 488 azide was conjugated by click chemistry. The viral protein pUL44 was visualized by antibody staining. The samples were analyzed by confocal microscopy. (B) Graph representing the percentage of cells positive for EdU only or for both EdU and pUL44 (mock, n = 150; infected, n = 150). (C) HFF cells were infected with AD169 at an MOI of 1.5 and used for accelerated native isolation of proteins on nascent DNA (aniPOND). At 72 hpi, cells were labeled for 4 h and biotinylated by click chemistry for affinity purification. No click reaction served as the negative control.

    Article Snippet: Input controls were taken before biotinylated DNA was captured by incubation with Dynabeads MyOne streptavidin T1 (Thermo Fisher Scientific, Waltham, MA, USA) for 16 h. After extensive washing, the bound DNA was resolved with Roti-Load Laemmli buffer (Roth GmbH, Karlsruhe, Germany) by boiling at 95°C for 15 min.

    Techniques: Infection, Synthesized, Labeling, Staining, Confocal Microscopy, Isolation, Affinity Purification, Negative Control