Notes: Delta-like 1 homolog (DLK1) functions in cell differentiation during development in both a Notch-dependent and -independent manner. Here, the CheckMate™/Flexi® Mammalian Two-Hybrid System and Dual-Luciferase® Reporter Assay System are used to monitor DLK1-DLK1, DLK1-fibronectin, and DLK1-cysteine-rich FGF receptor interactions. This further illustrates the function of the Notch-independent mechanism in development. (5104)

Notes: Chickens were immunized with PT-gliadin and splenocytes were isolated. Anti-PT-gliadin-positive cells were enriched on magnetic beads coated with PT-gliadin. Total RNA was extracted from the bead-bound cells with the ReliaPrep™ RNA Cell Miniprep System and used to clone the chicken IgY hypervariable regions. The scFv expression vectors were purified with the PureYield™ Plasmid Miniprep or Midiprep System prior to transformation into BL21(DE3) cells. ELISA assays were developed with standard TMB substrate and read with the GloMax® Instrument. (4618)

Notes: These authors examined conformation of DNA bound to the DNA-gate of Bacillus subtilis gyrase as well as the conformation of the DNA-gate itself. Negatively supercoiled pUC18 plasmid was purified using the PureYield™ Plasmid Midiprep System and used in single-molecule FRET experiments. (4061)

Notes: These authors amplified and characterized a putative epoxide hydrolase gene from the jewel wasp Nasonia vitripennis. PCR fragments were amplified from genomic DNA, purified from gels using the Wizard^® SV Gel and PCR Clean Up System and then subcloned into the pGEM^®-T Easy Vector. The plasmid DNA was purified using the PureYield™ Midiprep System. Linearized plasmids were used for in vitro transcription of RNA for use in RNA interference experiments. (3903)

Notes: To amplify yeast mutarotase, S. cerevisiae was used, and E. coli strain JM109 (Promega Cat.# L2001) was used for plasmid propagation. Fungal mycelia were harvested by filtration, washed, frozen and ground under liquid nitrogen. Genomic DNA was extracted using the Wizard Genomic DNA Purification System (Promega Cat.# A1120). RNA for hybridization and RT-PCR was extracted from mycelia using the SV Total RNA Isolation System (Promega Cat.# Z3101) and plasmid DNA isolated using the PureYield(TM) Plasmid Midiprep System (Cat.# A2492). (3919)

Notes: Heterogeneous culture maturation over several days provided an opportunity to influence several different steps in the differentiation process from ES to mature, post-mitotic cells. The authors procured a mouse genomic committee rearrayed IRAV library of approximately 8,000 full-length mouse cDNAs in an expression vector under the CMV promoter. For an easy and efficient gene delivery method, they used liposome-based transfection and were able to reliably achieve greater than 80% transfection efficiency of monolayer cultures as detected by flow cytometry for CMV-eGFP expression. This high rate of transfection suggests that differentiating cells were accessible over the course of at least 4 days to exogenously expressed genes from plasmid DNA. DNA isolation was accomplished using PureYield™ Midiprep System (Cat. A2492). (3921)

Notes: These authors developed a strategy for screening large numbers of genes that influence the pluripotency and differentiation of embryonic stem cells to specific fates. A plasmid expression library was grown in deep-well plates, 32 clones were pooled and the plasmid isolated using the PureYield™ Plasmid Midiprep System. The purified plasmid DNA was used to transfect E14 ES cells and measure expression of a specific cell fate reporter construct. (3583)

Notes: In this study a 984bp fragment of the IRG1 5´ promoter region was cloned into the pGL3 Basic Vector. Transfection-quality plasmid DNA was purified using the PureYield™ Plasmid Midiprep System and used to transfect RAW264.7 cells. Twenty-four hours post-transfection, cells were stimulated with LPS or infected with Mycobacterium paratuberculosis or Mycobacterium smegmatis for an additional 24 hours. Relative luciferase activities in LPS-stimulated and infected macrophages were then assayed using the Dual-Luciferase^® Reporter Assay System. (3365)

Notes: This study investigated the effect of teichoic acid alanylation on biofilm formation, adhesion, sensitivity to antimicrobial peptides and resistance to neutrophil killing in Enterococcus faecalis. The dlt operon in E. faecalis controls D-alanylation of teichoic acids. A deletion mutant lacking a portion of the dltA gene, which encodes a putative D-alanine activating enzyme, was created. Compared to the wildtype strain, the E. faecalis dltA deletion mutant produced less biofilm, exhibited reduced adhesion to cultured epithelial cells, was more sensitive to various antimicrobial peptides and less sensitive to opsonic killing. Plasmids used during the mutagenesis procedure and for complementation studies were purified from E. coli or Enterococci using the PureYield™ Plasmid Midipreps System or the Wizard^® Plus SV Minipreps System. (3526)

Notes: LIM domains are double zinc finger motifs that mediate protein:protein interactions and play critical roles in vertebrate development and cell differentiation. LIM domain binding proteins (Ldbs) are nuclear cofactors that contain both dimerization domains and LIM-interaction domains, facilitating the formation of regulatory complexes. This study isolated and characterized various splice isoforms of Ldbs lacking the LIM interaction domain from several species including mouse, chicken, Xenopus, and Zebrafish. A role for these splice variants in regulation of Ldb function was postulated. During the course of the study, cDNAs encoding the various Ldb genes were amplified from total RNA and cloned into various vectors. All cloning vectors were purified using the PureYield™ Plasmid Midiprep System. (3527)

Notes: The authors note that DNA gyrase is involved in crucial cellular processes; however the active sites of DNA binding in GyrA C-terminal domain and the mechanisms of DNA binding are largely unknown. In this paper they investigated the DNA-binding sites in the GyrA C-terminal domain (CTD) of Mycobacterium tuberculosis gyrase through site-directed mutagenesis. The authors isolated supercoiled plasmid pBR322 DNA using the PureYield™ Plasmid Midipreps DNA Purification System (Cat.# A2492). This DNA, along with relaxed pBR322 DNA, was used to test the function of mutants of gyrases GyrA and GyrB, independently and combined. The DNA-binding ability of the GyrA and B mutants was tested, as well as their ability to relax or supercoil DNA. The authors were able to identify key DNA-binding residues in M. tuberculosis GyrA-CTD, and showed mutations that led to loss of supercoiling and relaxation activities. This is the first time that DNA-binding sites in GyrA-CTD have been identified.
(3619)