dna plasmids  (Thermo Fisher)

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

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

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

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