dna plasmids  (Thermo Fisher)


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    Name:
    Lipofectin Transfection Reagent
    Description:
    Lipofectin Transfection Reagent is the reagent of choice for transfection of endothelial cells Lipofectin Transfection Reagent is also suitable for transfecting DNA RNA and oligonucleotides into mammalian cells and DNA into plant protoplasts Lipofectin reagent has also been shown to work well in combination with PLUS Reagent for the transfection of HeLa cells Lipofectin Transfection Reagent is a 1 1 w w liposome formulation of the cationic lipid N 1 2 3 dioleyloxy propyl n n n trimethylammonium chloride DOTMA and dioleoyl phophotidylethanolamine DOPE in membrane filtered water
    Catalog Number:
    18292011
    Price:
    None
    Category:
    Cell Culture Transfection Reagents
    Applications:
    Cell Culture|Plasmid Transfection|Stem Cell & Primary Cell Transfections|Transfection
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    Structured Review

    Thermo Fisher dna plasmids
    Altered localization of cohesin and its regulators in Vpr-expressing cells. (A) Expression of the cohesin proteins Smc1, Smc3, and <t>hRad21</t> in DOX-treated MIT-23 versus ΔVpr cells at 48 h. Ch, isolated chromatin; Wh, whole-cell lysate. Loading controls: histone H3 (HH3) and β-tubulin (βTub). Data are representative of at least three independent experiments. (B) Anti-hRad21 staining in DOX-treated MIT-23 cells versus ΔVpr cells. Arrows indicate hRad21 signals at the interface of sister chromatids within the centromere, and arrowheads indicate undetectable hRad21 signals at loosely aligned sister chromatids. Blue, <t>DNA;</t> red, hRad21. Similar results were obtained in three independent experiments. (C) Anti-hSgo1 immunostaining. (D and E, top) Immunostaining of AurB (D) or INCENP (E) in early mitosis. (insets) Magnifications of the centromere. (bottom) Intensities of AurB (D) or INCENP (E) at the inner centromere region. *, P
    Lipofectin Transfection Reagent is the reagent of choice for transfection of endothelial cells Lipofectin Transfection Reagent is also suitable for transfecting DNA RNA and oligonucleotides into mammalian cells and DNA into plant protoplasts Lipofectin reagent has also been shown to work well in combination with PLUS Reagent for the transfection of HeLa cells Lipofectin Transfection Reagent is a 1 1 w w liposome formulation of the cationic lipid N 1 2 3 dioleyloxy propyl n n n trimethylammonium chloride DOTMA and dioleoyl phophotidylethanolamine DOPE in membrane filtered water
    https://www.bioz.com/result/dna plasmids/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna plasmids - by Bioz Stars, 2021-03
    86/100 stars

    Images

    1) Product Images from "Epigenetic displacement of HP1 from heterochromatin by HIV-1 Vpr causes premature sister chromatid separation"

    Article Title: Epigenetic displacement of HP1 from heterochromatin by HIV-1 Vpr causes premature sister chromatid separation

    Journal: The Journal of Cell Biology

    doi: 10.1083/jcb.201010118

    Altered localization of cohesin and its regulators in Vpr-expressing cells. (A) Expression of the cohesin proteins Smc1, Smc3, and hRad21 in DOX-treated MIT-23 versus ΔVpr cells at 48 h. Ch, isolated chromatin; Wh, whole-cell lysate. Loading controls: histone H3 (HH3) and β-tubulin (βTub). Data are representative of at least three independent experiments. (B) Anti-hRad21 staining in DOX-treated MIT-23 cells versus ΔVpr cells. Arrows indicate hRad21 signals at the interface of sister chromatids within the centromere, and arrowheads indicate undetectable hRad21 signals at loosely aligned sister chromatids. Blue, DNA; red, hRad21. Similar results were obtained in three independent experiments. (C) Anti-hSgo1 immunostaining. (D and E, top) Immunostaining of AurB (D) or INCENP (E) in early mitosis. (insets) Magnifications of the centromere. (bottom) Intensities of AurB (D) or INCENP (E) at the inner centromere region. *, P
    Figure Legend Snippet: Altered localization of cohesin and its regulators in Vpr-expressing cells. (A) Expression of the cohesin proteins Smc1, Smc3, and hRad21 in DOX-treated MIT-23 versus ΔVpr cells at 48 h. Ch, isolated chromatin; Wh, whole-cell lysate. Loading controls: histone H3 (HH3) and β-tubulin (βTub). Data are representative of at least three independent experiments. (B) Anti-hRad21 staining in DOX-treated MIT-23 cells versus ΔVpr cells. Arrows indicate hRad21 signals at the interface of sister chromatids within the centromere, and arrowheads indicate undetectable hRad21 signals at loosely aligned sister chromatids. Blue, DNA; red, hRad21. Similar results were obtained in three independent experiments. (C) Anti-hSgo1 immunostaining. (D and E, top) Immunostaining of AurB (D) or INCENP (E) in early mitosis. (insets) Magnifications of the centromere. (bottom) Intensities of AurB (D) or INCENP (E) at the inner centromere region. *, P

    Techniques Used: Expressing, Isolation, Staining, Immunostaining

    Altered localization of HP1-α, HP1-γ, and hMis12 proteins in Vpr-expressing cells. (A) HP1-α, HP1-β, and HP1-γ expression in DOX-treated MIT-23 and ΔVpr cells. Loading control (Cont): a major band of the chromosomal fraction corresponding to 15 kD by Coomassie brilliant blue staining. (B) Chronological changes in HP1-α and hRad21 in the chromosomal (Ch) versus whole-cell (Wh) fractions after DOX addition (days 0–3). βTub, β-tubulin; Vpr, Vpr expression. Data are representative of three independent experiments. (C) Cells were preextracted with PBS containing Triton X-100 and stained with anti–HP1-α antibody and Hoechst 33342. In the bottom right, the pictured cells are in interphase (top cell) and undergoing mitosis (bottom cell). Similar results were obtained in at least three independent experiments. (D) Chromosome spreads were immunostained for HP1 subtypes and CENP-H. Similar results were obtained in three independent experiments. (E) Mitotic cells were immunostained for hMis12 and CENP-A. (F) Chromosome spreads prepared from DOX-induced cells (48 h) were immunostained for hMis12 and CENP-A. (G, left) DOX-induced cells (48 h) were immunostained for hMis12 and CENP-A. (insets) Magnifications of the centromere. (right) The hMis12/CENP-A signal intensity ratio was measured during prophase and prometaphase. Gray bars, ΔVpr cells; black bars, MIT-23 cells. Values represent the mean ± SD (three experiments) of data generated using the cells shown on the left. *, P = 0.0008 versus Vpr-expressing cells in prophase. (H) Chromosome spreads immunostained with CENP-A and CENP-H antibodies and ACA antisera. Blue, DNA; red, CENP-A, ACA, or CENP-H. Vpr (−), DOX-treated ΔVpr cells at 48 h; Vpr (+), DOX-treated MIT-23 cells at 48 h. Bars: (C, E, and G) 10 µm; (D, F, and H) 5 µm; (G, insets) 0.5 µm.
    Figure Legend Snippet: Altered localization of HP1-α, HP1-γ, and hMis12 proteins in Vpr-expressing cells. (A) HP1-α, HP1-β, and HP1-γ expression in DOX-treated MIT-23 and ΔVpr cells. Loading control (Cont): a major band of the chromosomal fraction corresponding to 15 kD by Coomassie brilliant blue staining. (B) Chronological changes in HP1-α and hRad21 in the chromosomal (Ch) versus whole-cell (Wh) fractions after DOX addition (days 0–3). βTub, β-tubulin; Vpr, Vpr expression. Data are representative of three independent experiments. (C) Cells were preextracted with PBS containing Triton X-100 and stained with anti–HP1-α antibody and Hoechst 33342. In the bottom right, the pictured cells are in interphase (top cell) and undergoing mitosis (bottom cell). Similar results were obtained in at least three independent experiments. (D) Chromosome spreads were immunostained for HP1 subtypes and CENP-H. Similar results were obtained in three independent experiments. (E) Mitotic cells were immunostained for hMis12 and CENP-A. (F) Chromosome spreads prepared from DOX-induced cells (48 h) were immunostained for hMis12 and CENP-A. (G, left) DOX-induced cells (48 h) were immunostained for hMis12 and CENP-A. (insets) Magnifications of the centromere. (right) The hMis12/CENP-A signal intensity ratio was measured during prophase and prometaphase. Gray bars, ΔVpr cells; black bars, MIT-23 cells. Values represent the mean ± SD (three experiments) of data generated using the cells shown on the left. *, P = 0.0008 versus Vpr-expressing cells in prophase. (H) Chromosome spreads immunostained with CENP-A and CENP-H antibodies and ACA antisera. Blue, DNA; red, CENP-A, ACA, or CENP-H. Vpr (−), DOX-treated ΔVpr cells at 48 h; Vpr (+), DOX-treated MIT-23 cells at 48 h. Bars: (C, E, and G) 10 µm; (D, F, and H) 5 µm; (G, insets) 0.5 µm.

    Techniques Used: Expressing, Staining, Generated

    2) Product Images from "Quantifying the Antibody Binding on Protein Microarrays using Microarray Nonlinear Calibration"

    Article Title: Quantifying the Antibody Binding on Protein Microarrays using Microarray Nonlinear Calibration

    Journal: BioTechniques

    doi: 10.2144/000114028

    Quality control of NAPPA microarrays with 849 TB genes Expression clones encoding the target proteins fused to a C-terminal GST tag were printed along with a polyclonal anti-GST antibody in duplicate on the array surface. DNA capture was confirmed by PicoGreen (PG) staining (DNA, left), in situ protein expression and capture were assessed by GST detection using a monoclonal antibody (protein, middle). The correlation of duplicate spots in one slide (upper right) and between two different slides (lower right) is 0.96 and 0.81, respectively (GST color code: red > orange > yellow > green > blue).
    Figure Legend Snippet: Quality control of NAPPA microarrays with 849 TB genes Expression clones encoding the target proteins fused to a C-terminal GST tag were printed along with a polyclonal anti-GST antibody in duplicate on the array surface. DNA capture was confirmed by PicoGreen (PG) staining (DNA, left), in situ protein expression and capture were assessed by GST detection using a monoclonal antibody (protein, middle). The correlation of duplicate spots in one slide (upper right) and between two different slides (lower right) is 0.96 and 0.81, respectively (GST color code: red > orange > yellow > green > blue).

    Techniques Used: Expressing, Clone Assay, Staining, In Situ

    The detection principle of antibody assay using protein microarrays (A) is the schematic illustration of antibody assay using MiNC method. It consists of adding a series of features with different known amounts of IgG standards to a standard array. The IgG used should match the species from which the primary antibody came (lower graph, brown). The IgG standards are used to construct a nonlinear standard curve (upper graph, left) to interpolate the amount of IgG antibodies that bind to the surface of each protein spot after antibody incubation (upper graph, right), which is calculated and represented in fmol (red). The detection is performed with fluorescent dyes conjugated secondary antibody. SFI is the sum of fluorescent intensity within each protein spot (black). (B) is fluorescent images of the detection of different concentrations of mouse anti-p53 antibody. The left sub-array shows the mouse IgG standards (0, 3, 10, 30, 89, 266 fmol) and the right sub-array comprises different concentrations of p53 DNA plasmids (316, 474, 711, 1067, 1600 and 2400 ng/μl) in six replicates used to express proteins by the NAPPA method. The detection was performed with DyLight549 conjugated rabbit anti-mouse IgG secondary antibody.
    Figure Legend Snippet: The detection principle of antibody assay using protein microarrays (A) is the schematic illustration of antibody assay using MiNC method. It consists of adding a series of features with different known amounts of IgG standards to a standard array. The IgG used should match the species from which the primary antibody came (lower graph, brown). The IgG standards are used to construct a nonlinear standard curve (upper graph, left) to interpolate the amount of IgG antibodies that bind to the surface of each protein spot after antibody incubation (upper graph, right), which is calculated and represented in fmol (red). The detection is performed with fluorescent dyes conjugated secondary antibody. SFI is the sum of fluorescent intensity within each protein spot (black). (B) is fluorescent images of the detection of different concentrations of mouse anti-p53 antibody. The left sub-array shows the mouse IgG standards (0, 3, 10, 30, 89, 266 fmol) and the right sub-array comprises different concentrations of p53 DNA plasmids (316, 474, 711, 1067, 1600 and 2400 ng/μl) in six replicates used to express proteins by the NAPPA method. The detection was performed with DyLight549 conjugated rabbit anti-mouse IgG secondary antibody.

    Techniques Used: Construct, Incubation

    3) Product Images from "Role of NF-?B and Myc Proteins in Apoptosis Induced by Hepatitis B Virus HBx Protein"

    Article Title: Role of NF-?B and Myc Proteins in Apoptosis Induced by Hepatitis B Virus HBx Protein

    Journal: Journal of Virology

    doi: 10.1128/JVI.75.1.215-225.2001

    Suppression of NF-κB activation promotes HBx-mediated apoptosis. WT and RelA KO 3T3 cells were transfected by a plasmid expressing a superrepressor (S.R.) mutant form of IκBα, which blocks activation of NF-κB. Eight hours later, cells were transduced for 10 h with Ad-HBx or vector alone (Ad dl312, vec), a control virus vector that is functionally identical to control virus AdHBxo. At 10 h following transduction, cells were either mock treated or treated with murine TNF-α (10 ng/ml) for 16 h. Approximately 70% of cells were transfected by this approach, as determined by inclusion of a pGFP expression vector (data not shown). (A) Nuclear extracts were prepared and 10 μg of protein was analyzed for NF-κB DNA-binding complexes using a 32 P-labeled DNA probe and EMSA. Killing of (B) WT 3T3 cells and (C) RelA KO cells was quantified spectrophotometrically by the trypan blue dye exclusion assay. Results represent the means of three independent experiments, with calculated standard errors shown.
    Figure Legend Snippet: Suppression of NF-κB activation promotes HBx-mediated apoptosis. WT and RelA KO 3T3 cells were transfected by a plasmid expressing a superrepressor (S.R.) mutant form of IκBα, which blocks activation of NF-κB. Eight hours later, cells were transduced for 10 h with Ad-HBx or vector alone (Ad dl312, vec), a control virus vector that is functionally identical to control virus AdHBxo. At 10 h following transduction, cells were either mock treated or treated with murine TNF-α (10 ng/ml) for 16 h. Approximately 70% of cells were transfected by this approach, as determined by inclusion of a pGFP expression vector (data not shown). (A) Nuclear extracts were prepared and 10 μg of protein was analyzed for NF-κB DNA-binding complexes using a 32 P-labeled DNA probe and EMSA. Killing of (B) WT 3T3 cells and (C) RelA KO cells was quantified spectrophotometrically by the trypan blue dye exclusion assay. Results represent the means of three independent experiments, with calculated standard errors shown.

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Transduction, Binding Assay, Labeling, Exclusion Assay

    Related Articles

    Transfection:

    Article Title: Sirt-1 Is Required for the Inhibition of Apoptosis and Inflammatory Responses in Human Tenocytes
    Article Snippet: All ASO and SO were phosphorothioate-modified to protect them from the cell nucleases. .. Subconfluent tenocytes were transfected with ASO or SO by use of Lipofectin reagent (Invitrogen) according to the manufacturer's instructions by using 0.2, 0.5, 1, 5, and 10 μ m antisense or sense in serum-starved medium for 24 h. ..

    Article Title: Prevention of HIV-1 infection in human peripheral blood mononuclear cells by specific RNA interference
    Article Snippet: Twenty-four hours before transfection, adherent cells were seeded in 6-well multiwell plates at a density of 2 × 105 cells/well. .. COS cells were transfected with 2 µg dsRNA and 2 µg pNL4-3, using 2 µl of Lipofectin (Gibco BRL) and 3 µl of FuGENE 6 (Roche), according to the manufacturers’ optimized protocols. .. After a 72 h incubation, the virus replication was monitored in the culture supernatants with the HIV-1 p24 CLEIA assay.

    Article Title: Expression of RASSF1A, an epigenetically silenced tumor suppressor, overcomes resistance to apoptosis induction by interferons
    Article Snippet: To downregulate DNMT1, cells were transfected with MG98 (MethylGene, Quebec, Canada), a second-generation 4×4 2′O methyl (bold) phosphorothioate oligonucleotide antisense against the 3′ UTR of DNMT1 mRNA (5′- TTCA TGTCAGCCAAGG CCAC -3′) or mismatch (MM) control oligonucleotide of similar sequence (5′- TTAA TGTAACCTAAGG TCAA -3′) starting 1d after plating at cell concentrations that allowed at least doubling of number in 48 hr and optimal transfection efficiency (5000 and 15000 cells/cm 2 for SK-RC-45 and ACHN cells, respectively). .. Transfections were with 6.25 μg/ml Lipofectin in OptiMem (GIBCO, Invitrogen, Carlsbad, CA) over 4 hr. .. Before and after transfections cells were washed with PBS, every 2nd day cells were replated 4 hr after the end of the preceding transfection.

    Allele-specific Oligonucleotide:

    Article Title: Sirt-1 Is Required for the Inhibition of Apoptosis and Inflammatory Responses in Human Tenocytes
    Article Snippet: All ASO and SO were phosphorothioate-modified to protect them from the cell nucleases. .. Subconfluent tenocytes were transfected with ASO or SO by use of Lipofectin reagent (Invitrogen) according to the manufacturer's instructions by using 0.2, 0.5, 1, 5, and 10 μ m antisense or sense in serum-starved medium for 24 h. ..

    Plasmid Preparation:

    Article Title: Isolation of full-length ATM cDNA and correction of the ataxia-telangiectasia cellular phenotype
    Article Snippet: Transfection of EBV-transformed lymphoblastoid cells was carried out as described ( , ). .. Briefly, 2 × 106 exponentially growing cells were pelleted, rinsed twice with fetal calf serum-free medium (Opti-MEM, GIBCO/BRL), and resuspended in 1 ml of Opt-MEM medium containing 6 μg of plasmid DNA and 20 μg of Lipofectin (GIBCO/BRL). .. After incubation for 6–7 h at 37°C, 4 ml of serum-containing RPMI 1640 medium was added and the cells were further incubated.

    Inverted Microscopy:

    Article Title: Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mAb
    Article Snippet: The lipofection-mediated intracellular neutralization assay was based on the rotavirus DLP transfection method. .. Rotavirus DLPs diluted in M199 (to give approximately 50–100 foci per field in an “unneutralized” control using an inverted microscope) were mixed with lipofectin (GIBCO BRL; Life Technologies Inc.) (15% vol/vol) and incubated at room temperature for 40 minutes. .. Selected mAb’s (starting concentration 1 mg/ml) were diluted fourfold from 1:40 to 1:163,840 in M199 and added at 30 μl per well to 96-well plates.

    Incubation:

    Article Title: Inhibition of rotavirus replication by a non-neutralizing, rotavirus VP6-specific IgA mAb
    Article Snippet: The lipofection-mediated intracellular neutralization assay was based on the rotavirus DLP transfection method. .. Rotavirus DLPs diluted in M199 (to give approximately 50–100 foci per field in an “unneutralized” control using an inverted microscope) were mixed with lipofectin (GIBCO BRL; Life Technologies Inc.) (15% vol/vol) and incubated at room temperature for 40 minutes. .. Selected mAb’s (starting concentration 1 mg/ml) were diluted fourfold from 1:40 to 1:163,840 in M199 and added at 30 μl per well to 96-well plates.

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  • 86
    Thermo Fisher plasmid dna
    Standard curves of <t>Saccharibacteria</t> qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid <t>DNA</t> carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.
    Plasmid Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid dna/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    plasmid dna - by Bioz Stars, 2021-03
    86/100 stars
      Buy from Supplier

    96
    Thermo Fisher dna plasmids
    Suppression of NF-κB activation promotes HBx-mediated apoptosis. WT and RelA KO 3T3 cells were <t>transfected</t> by a plasmid expressing a superrepressor (S.R.) mutant form of IκBα, which blocks activation of NF-κB. Eight hours later, cells were transduced for 10 h with Ad-HBx or vector alone (Ad dl312, vec), a control virus vector that is functionally identical to control virus AdHBxo. At 10 h following transduction, cells were either mock treated or treated with murine TNF-α (10 ng/ml) for 16 h. Approximately 70% of cells were transfected by this approach, as determined by inclusion of a pGFP expression vector (data not shown). (A) Nuclear extracts were prepared and 10 μg of protein was analyzed for NF-κB <t>DNA-binding</t> complexes using a 32 P-labeled DNA probe and EMSA. Killing of (B) WT 3T3 cells and (C) RelA KO cells was quantified spectrophotometrically by the trypan blue dye exclusion assay. Results represent the means of three independent experiments, with calculated standard errors shown.
    Dna Plasmids, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna plasmids/product/Thermo Fisher
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna plasmids - by Bioz Stars, 2021-03
    96/100 stars
      Buy from Supplier

    93
    Thermo Fisher plasmid dna harboring microcystis its sequences
    Phylogenetic tree for the six <t>Microcystis</t> genotypes generated based on the internal transcribed spacer <t>(ITS)</t> region. The number at each node represents the posterior probability value. The scale bar indicates inferred nucleotide substitution rate. A portion of ITS sequence from Gloeocapsa sp. (GenBank accession number: KJ746508.1) was used as an outgroup for generating the rooted phylogenetic tree. The numbers in the parentheses are the internal reference ID number.
    Plasmid Dna Harboring Microcystis Its Sequences, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/plasmid dna harboring microcystis its sequences/product/Thermo Fisher
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    plasmid dna harboring microcystis its sequences - by Bioz Stars, 2021-03
    93/100 stars
      Buy from Supplier

    Image Search Results


    Standard curves of Saccharibacteria qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid DNA carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.

    Journal: Materials

    Article Title: Specificities and Efficiencies of Primers Targeting Candidatus Phylum Saccharibacteria in Activated Sludge

    doi: 10.3390/ma11071129

    Figure Lengend Snippet: Standard curves of Saccharibacteria qPCR for the measurement of activated sludge samples using 10-fold serial dilutions of plasmid DNA carrying Saccharibacteria 16S rRNA genes and the four primer sets: TM7314F and TM7-910R ( A ); TM7314F and TM7-1177R ( B ); TM7580F and TM7-910R ( C ); and TM7580F and TM7-1177R ( D ). The slope, coefficient of determination (R 2 ), and amplification efficiency are also shown in the figures.

    Article Snippet: Standard curves were constructed from 10-fold dilutions of plasmid DNA (pCR2.1-TOPO, Thermo Fisher Scientific) carrying partial Saccharibacteria 16S rRNA genes retrieved from a clone library described in section 3.4 with a known copy number.

    Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Amplification

    Suppression of NF-κB activation promotes HBx-mediated apoptosis. WT and RelA KO 3T3 cells were transfected by a plasmid expressing a superrepressor (S.R.) mutant form of IκBα, which blocks activation of NF-κB. Eight hours later, cells were transduced for 10 h with Ad-HBx or vector alone (Ad dl312, vec), a control virus vector that is functionally identical to control virus AdHBxo. At 10 h following transduction, cells were either mock treated or treated with murine TNF-α (10 ng/ml) for 16 h. Approximately 70% of cells were transfected by this approach, as determined by inclusion of a pGFP expression vector (data not shown). (A) Nuclear extracts were prepared and 10 μg of protein was analyzed for NF-κB DNA-binding complexes using a 32 P-labeled DNA probe and EMSA. Killing of (B) WT 3T3 cells and (C) RelA KO cells was quantified spectrophotometrically by the trypan blue dye exclusion assay. Results represent the means of three independent experiments, with calculated standard errors shown.

    Journal: Journal of Virology

    Article Title: Role of NF-?B and Myc Proteins in Apoptosis Induced by Hepatitis B Virus HBx Protein

    doi: 10.1128/JVI.75.1.215-225.2001

    Figure Lengend Snippet: Suppression of NF-κB activation promotes HBx-mediated apoptosis. WT and RelA KO 3T3 cells were transfected by a plasmid expressing a superrepressor (S.R.) mutant form of IκBα, which blocks activation of NF-κB. Eight hours later, cells were transduced for 10 h with Ad-HBx or vector alone (Ad dl312, vec), a control virus vector that is functionally identical to control virus AdHBxo. At 10 h following transduction, cells were either mock treated or treated with murine TNF-α (10 ng/ml) for 16 h. Approximately 70% of cells were transfected by this approach, as determined by inclusion of a pGFP expression vector (data not shown). (A) Nuclear extracts were prepared and 10 μg of protein was analyzed for NF-κB DNA-binding complexes using a 32 P-labeled DNA probe and EMSA. Killing of (B) WT 3T3 cells and (C) RelA KO cells was quantified spectrophotometrically by the trypan blue dye exclusion assay. Results represent the means of three independent experiments, with calculated standard errors shown.

    Article Snippet: Cells at 50% confluency were transfected with DNA plasmids using Lipofectin (Gibco) with a total of 10 μg of plasmid DNA per 10-cm plate of cells.

    Techniques: Activation Assay, Transfection, Plasmid Preparation, Expressing, Mutagenesis, Transduction, Binding Assay, Labeling, Exclusion Assay

    Phylogenetic tree for the six Microcystis genotypes generated based on the internal transcribed spacer (ITS) region. The number at each node represents the posterior probability value. The scale bar indicates inferred nucleotide substitution rate. A portion of ITS sequence from Gloeocapsa sp. (GenBank accession number: KJ746508.1) was used as an outgroup for generating the rooted phylogenetic tree. The numbers in the parentheses are the internal reference ID number.

    Journal: PLoS ONE

    Article Title: Biodiversity of cyanobacteria and other aquatic microorganisms across a freshwater to brackish water gradient determined by shotgun metagenomic sequencing analysis in the San Francisco Estuary, USA

    doi: 10.1371/journal.pone.0203953

    Figure Lengend Snippet: Phylogenetic tree for the six Microcystis genotypes generated based on the internal transcribed spacer (ITS) region. The number at each node represents the posterior probability value. The scale bar indicates inferred nucleotide substitution rate. A portion of ITS sequence from Gloeocapsa sp. (GenBank accession number: KJ746508.1) was used as an outgroup for generating the rooted phylogenetic tree. The numbers in the parentheses are the internal reference ID number.

    Article Snippet: Standard curves were generated by running reactions on serial dilutions of plasmid DNA harboring Microcystis ITS sequences, which were chemically synthesized and ligated into a cloning vector by a manufacturer (ThermoFisher Scientific).

    Techniques: Generated, Sequencing

    Dynamitin overexpression inhibits nuclear targeting of incoming Ad2, but not release of a fluid-phase endosomal marker into the cytosol. (A) TR-labeled wt virus was bound in the cold to HeLa cells transiently transfected with a plasmid DNA expressing eGFP (panels a–c, arrows) or with the eGFP-plasmid together with a dynamitin/p50 (Dyn)-expressing plasmid (panels d–f). In the latter case, 95% of the GFP expressing cells also overexpressed dynamitin. Virus was internalized for 60 min at 37°C and cells were processed for indirect immunofluorescence microscopy using anti-dynamitin mAb 50.1 and goat anti–mouse IgG-Cy5. (B) wt Ad2 (panels a–c) or ts1 (panels d–f) was bound at 50 μg/ml to the surface of HeLa cells, which had been transiently transfected with dynamitin/p50 DNA. Virus was internalized for 15 min in the presence of FITC-labeled 10 K dextran (5 mg/ml), followed by a 15-min incubation in the absence of dextran, fixed with pFA, and then stained for dynamitin using mAb 50.1 and anti-mouse IgG-Cy5 (panels b and e). The distribution of dextran was recorded in the FITC channel (panels a and d, transfected cells are indicated by an arrow) and DIC images of the same cells shown in panels c and f.

    Journal: The Journal of Cell Biology

    Article Title: Microtubule-dependent Plus- and Minus End-directed Motilities Are Competing Processes for Nuclear Targeting of Adenovirus

    doi:

    Figure Lengend Snippet: Dynamitin overexpression inhibits nuclear targeting of incoming Ad2, but not release of a fluid-phase endosomal marker into the cytosol. (A) TR-labeled wt virus was bound in the cold to HeLa cells transiently transfected with a plasmid DNA expressing eGFP (panels a–c, arrows) or with the eGFP-plasmid together with a dynamitin/p50 (Dyn)-expressing plasmid (panels d–f). In the latter case, 95% of the GFP expressing cells also overexpressed dynamitin. Virus was internalized for 60 min at 37°C and cells were processed for indirect immunofluorescence microscopy using anti-dynamitin mAb 50.1 and goat anti–mouse IgG-Cy5. (B) wt Ad2 (panels a–c) or ts1 (panels d–f) was bound at 50 μg/ml to the surface of HeLa cells, which had been transiently transfected with dynamitin/p50 DNA. Virus was internalized for 15 min in the presence of FITC-labeled 10 K dextran (5 mg/ml), followed by a 15-min incubation in the absence of dextran, fixed with pFA, and then stained for dynamitin using mAb 50.1 and anti-mouse IgG-Cy5 (panels b and e). The distribution of dextran was recorded in the FITC channel (panels a and d, transfected cells are indicated by an arrow) and DIC images of the same cells shown in panels c and f.

    Article Snippet: Plasmids and DNA Experiments GFP-encoding plasmid DNA (gift of S. Zimmermann; Invitrogen, Zürich, Switzerland) was transfected into TC7 or HeLa cells alone or together with dynamitin-encoding DNA (gift of R. Vallee, University of Massachusetts, Worcester, MA) ( ) at a molar ratio of 2:1 using Lipofectamine™ (5 μl/μg DNA; GIBCO BRL ) or TransFast™ (7 μl/μg DNA; Promega ).

    Techniques: Over Expression, Marker, Labeling, Transfection, Plasmid Preparation, Expressing, Immunofluorescence, Microscopy, Incubation, Staining