Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Halanaerobacter jeridensis. (4337)

Notes: This paper provides an overview of the many applications of HaloTag® Technology. The authors describe the development of the technology, focusing on it's multifunctional utility for protein imaging, protein isolation and display, and in the study of protein complexes and interactions. They also discuss it's potential to facilitate proteomics research studies across complex biological systems at the biochemical, cell-based and whole animal level. (4325)

Notes: GoTaq® Green Master Mix was used in the PCR amplification of genomic DNA template for pyrosequencing. (4541)

Notes: The authors transiently transfected 2 × 10⁴ HEK293 cells per well of a 96-well plate using the FuGene® HD Transfection Reagent, 0.1µg of DNA and a reagent:DNA ratio of 3:1. (4430)

Notes: Caspase-Glo® 3/7 and Caspase-Glo® 8 Assays were used to assess activation of apoptosis pathways in MC3T3-E1 cells (clonal mouse), U-2 OS human osteoscarcoma cell line and CCL-51 cells (mouse mammary gland tumor cells). Although ibandronate reduced cell proliferation in all cell lines, its effect on activation of caspases was different in neoplastic versus non-neoplastic cells. Caspase-8 and caspase-3/7 activities were reduced in MC3T3-E1 cells after 72 hours treatment with ibandronate. In two tumor cell lines assayed, opposite results were seen: caspase-8 and caspase-3/7 activities increased in U-2 OS cells and in CCL-51 cells. Luminescence was detected using a GloMax® 96 Microplate Luminometer. (Figure 1 in the paper)

To analyze FAS promoter methylation levels, fragments of the targeted promoter regions were generated by digestion of genomic DNA using CpG methylation insensitive restriction enzymes MboII and PstI from cells cultured in the presence of ibandronate for varying lengths of time. The authors demonstrated that FAS promoter methylation is altered in tumor cells in the presence of ibandronate. (4253)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Campylobacter coli. (4316)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Bartonella. (4314)

Notes: Specific neuronal connectivity is thought to be based on the expression of the protocadherins (Pcdh)--a family of membrane adhesion proteins. Each neuron expresses only a specific subset of the Pcdh genes. The authors of this paper used a bioinformatics approach to identify conserved, gene-specific regions upstream of the Pcdh genes. They showed that this specific sequence element (SSE) is involved in transcription regulation together with a conserved sequence element (CSE), and identified a potential interacting protein partner. To demonstrate promoter activity, the SSE-CSE region was cloned upstream of the luciferase gene in the pGL3 Basic Vector and its effect on luciferase expression was evaluated. The authors then isolated protein complexes that bound the SSE-CSE region and characterized the interacting proteins by mass spectrometry. The CCTC binding-factor (CTCF) was identified as a key molecule that binds and activates Pcdh promoters. As part of the study, human CTCF  and the CTCF-binding domain were expressed in the TNT® T7 Quick Coupled Transcription/Translation System. The in vitro expressed proteins were fluorescently labeled using the FluoroTect™ Green_Lys System and were used in EMSA to confirm interaction with the SSE-CSE region. (4249)

Notes: The authors used the Roche 454 sequencing platform to perform a differential transcriptome analysis and identify genes that are differentially expressed in human buccal cancer and normal tissue. The quantity of first-strand and second-strand cDNA products was estimated using the QuantiFluor™-ST Fluorometer as well as the High Sensitivity DNA Chip kit and Agilent Bioanalyzer 2100. (4238)

Notes: Oropouche fever is a arboviral infection in Brazil, surpassed in frequency only by dengue. Oropouche virus (OROV) causes large outbreaks of acute febrile illness in areas along the Amazon and Central-Plateau regions. RNA was extracted from CSF and underwent reverse transcription-polymerase chain reaction and sequencing to identify OROV. Reverse transcription was performed with 5ml of the random primers, using the AccessQuick™ RT-PCR System. (4320)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Campylobacter jejuni. (4317)

Notes: This study examined how well primers developed in 1990 and 2004 for type A rotavirus (RVA) were able to genotype (G type) currently circulating RVAs in Asia. The VP7 gene from RVA was amplified using 2µl of dsRNA template with the AccessQuick™ RT-PCR System in a total volume of 50µl. The G type was determined using hemi-nested multiplex PCR using 1µl of the VP7 cDNA and PCR Master Mix in a final volume of 50µl. The final products were sequenced. (4340)

Notes: Fugene HD was used with HeLa-TetOff cells, seeded at a density of 100,000 cells/well of a 12-well culture plate. On day 2, cells were transfected with 0.5 μg total DNA/well and FuGene HD reagent with a DNA to FuGENE HD ratio of 1:3 (w/v). (4423)

Notes: These authors investigated the effects of various anesthetics on bioluminescence imaging with firefly luciferase. They observed decreases in luminescence with volatile anaethetics, and found increased luciferase expression with injectable anaethetics in intact cells, but not in cell lysates in vitro. They concluded that the decreases in luciferase activity observed with volatile anaesthesia were due to hemodynamic effects, and not due to a direct inhibitory effect on luciferase enzyme itself. The apparent enhancement of luciferase activity with certain injectable anaesthetics appeared to be due to cytotoxic effects that resulted in increased permeablity to luciferase, as the same enhancement was not observed in cell lysates. D-Luciferin was used for in vivo imaging experiments. The pGL4.10 vector (encoding firefly luciferase), Luciferase Assay Reagent, and the GloMax® 96 Microplate Luminometer were used for in vitro assays using cell lysates. (4190)

Notes: In this paper, FuGENE® HD reagent was used to transiently transfect HeLa and MEF cells. Transfection conditions were as follows: HeLa Tet-On cells were plated on glass coverslips at 1-1.3 X 10e5 cells/well in 12-well plates 1 day prior to transfection with 0.2µg plasmid DNA. Mouse embryonic fibroblast (MEF) cells were plated at 5x10e4 cells/well in 12-well plates 1 day prior to transfection with 2µg plasmid DNA.

  (4414)

Notes: In this study, the L. pneumophila protein Lem3 was expressed as a HaloTag^® fusion protein and purified using HaloLink™ Resin. Lem3 was first cloned into the pFN22K HaloTag^® Vector and the resultant HaloTag-Lem3 protein was expressed in Single-Step (KRX) competent cells before purification using the HaloTag^® Protein Purification System. Lem3 was cleaved from the HaloLink™ Resin using TEV protease.

  (4350)

Notes: The authors of this study sought to determine how a diet enriched with low bush blueberries would affect the metabolic potential of gut microbes. For this study, they fed rats either a control diet or the control diet plus 8% weight/volume powder derived from low bush blueberries. After six weeks, rats were sacrificed and DNA was extracted from stool. Genomic DNA concentration was determined using the Quantus™ Fluorometer prior to being subjected to next generation sequencing (NGS). (4502)

Notes: The authors characterized the gut microbiota in two populations of Sprague Dawley rats—one that was fed the AIN93 diet and the other was fed the AIN93 diet with lowbush wild blueberry powder. After 6 weeks, rats were sacrificed and DNA was purified from the contents of the proximal colon and subjected to whole genome sequencing to identify the resident bacterial species. The DNA concentration was determined using the QuantiFluor™-ST Fluorometer prior to Illumina next-generation sequencing. (4239)

Notes: These authors describe use of a microfluidic device based on mechanically induced trapping of molecular interactions (k-MITOMI) for kinetic characterization of 768 biomolecular interactions in parallel. Using this approach, they measured the association and dissociation kinetics of numerous transcription factors with their associated consensus sequences. The TNT® T7 Quick Coupled Transcription/Translation System was used to express the transcription factors from linear templates, either on-chip or in bulk reactions. (4247)

Notes: Genomic rearrangements detected by NGS were mapped and confirmed by PCR. Human Genomic DNA: Male was used as a control for the confirmatory experiments. (4539)

Notes: The E3 ubiquitin ligase murine double minute 2 (MDM2) regulates transcriptional activity and expression levels of the p53 tumor suppressor protein. In this article, the authors show that the cell fate determinant NUMB interacts with MDM2, disrupting the interaction of MDM2 and p53 and preventing the ubiquitination of p53. To study protein-protein interactions between NUMB and MDM2, the authors used the pFN2A (GST) Flexi® Vector to express full-length and partial NUMB sequences as GST fusion proteins for protein pull-down experiments. To examine their hypothesis that the interaction between NUMB and MDM2 increases p53 levels and thus apoptosis in cells, they treated ZR75 human breast carcinoma cells and A375 human melanoma cells with NUMB-derived peptides and small molecule ligands that block the interaction between NUMB and MDM2. These treatments resulted in increased levels of p53 protein, Annexin V staining as well as caspase-3/7 activity, as determined using the Caspase-Glo® 3/7 Assay System. (4351)

Notes: In this study the Wizard® Genomic DNA Purification Kit was used to isolate DNA from Anabaena sp. (4299)

Notes: This study identified that miR-21 is significantly overexpressed in human cervical squamous cancer tissues and cell lines, and showed that the level of miR-21 correlates with tumor differentiation, and regulates proliferation, apoptosis, and migration of HPV16-positive cells. The authors used gene expression profiling and luciferase reporter assay to identify candidate target genes for miR-21. Wildtype and mutant 3′-UTR sequences from the target gene CCL20 containing putative binding sites for miR-21 were subcloned into the psiCHECK-2 Vector and used to transfect HEK293 cells. The Dual-Luciferase® Reporter Assay System and GloMax® Multi Luminometer were used to measure luciferase activity from both constructs. (4192)

Notes: This study describes the clinical observations and laboratory investigations performed on 170 of the 2,000 suspected West Niles Virus (WNV) cases. These cases were admitted to Aravind Eye Hospital, Madurai, Tamil Nadu with ocular complications. Conventional reverse transcription PCR (RT-PCR) and real-time RT-PCR assays were used to detect WNV infection. In addition, reverse transcription loop-mediated isothermal gene amplification (RT-LAMP) was performed to determine the feasibility of using this method as an alternative cost-effective tool to the real-time RT-PCR.

After proving negative for DENV- and CHIKV, samples were tested for the presence of WNV-specific RNA by RT-PCR, real-time RT-PCR and RT-LAMP assays. RNA was extracted from the patient serum, plasma and infected culture supernatant. The RNA was then eluted in 50µl of nuclease-free water and used as template in the AccessQuick™ RT-PCR System, with primer pairs targeting the env gene.

Amplification was performed in a 50µl total reaction volume with the AccessQuick™ RT-PCR System, using 50pmol of each forward and reverse primer and 2µl of extracted viral RNA. (4331)

Notes: Mice RNA samples were converted to cDNA using an oligo-dT primer with the Reverse Transcription System, ethanol precipitated, vacuum dried and transferred to another lab. There they were reconstituted in 20μl of molecular biology grade water.

Detection of caliciviruses in the wild mice samples was attempted using generic calicivirus primers targeting sequences encoding conserved amino acid motifs in the RNA-dependent RNA polymerase (RdRp) region of ORF1. Two microliters of cDNA was used in 25μl PCR reactions using the GoTaq® Green Master Mix. Laboratory mouse RNA samples were tested only with MNV-specific primers in the AccessQuick™ RT-PCR System using 2μl RNA as template.

PCR products were cloned into pGEM-T® Vector and sequenced using M13 forward and reverse primers on an ABI PRISM® 3730 DNA Analyzer. (4330)