Notes: The authors identified a bacterial strain that can use the toxin 3-nitropropionic acid (3NPA) as its sole carbon and nitrogen sources and cloned the genes that encode the enzymes responsible for the initial steps in the 3NPA degradation pathway. The Pseudomonas library used to clone these genes was created using genomic DNA isolated using the Wizard^® SV Genomic DNA Purification System. Closely related genes were amplified from other bacterial species for phylogenetic analysis. PCRs were performed using the GoTaq^® Hot Start Polymerase. (4164)

Notes: To better examine the molecular mechanisms of small bowel adenocarcinomas, DNA was extracted from paraffin-embedded tissue and tested for microsatellite instability (MSI) using the MSI Analysis System, Version 1.2. The PCR products were run on the Applied Biosystems 3130 Genetic Analyzer and analyzed using GeneScan® software. If two or more of the BAT-25, BAT-26, NR-21, NR-24 or MONO-27 monomorphic markers had altered lengths, the tumors were designated as MSI unstable. (4112)

Notes: Certain bacteriophages can promote host cell sporulation under unfavorable conditions to increase survival of the host and prophage. These types of phages, known as spore-converting phages, have been found in terrestrial Bacillus species. In this article the authors examined the effect of prophages on sporulation of 11 Bacillus isolates from the Gulf of Mexico. Potential prophages in the Bacillus isolates were detected by PCR using unique PCR primer sets for each prophage genome and GoTaq® Green Master Mix. One of these isolates, B14905, was examined in more detail; the genome of this isolate was isolated using the Wizard® Genomic DNA Purification Kit, then sequenced. (4091)

Notes: The vasculoproliferative disorder peliosis hepatis has been linked to Bartonella henselae infection in humans and dogs. The authors sought to determine if this is true in cats, the natural reservoir for B. henselae, using immunohistochemistry (IHC) and PCR. Tissue sections from 26 cats with peliosis hepatis were formalin-fixed and paraffin-embedded and subjected to IHC using a B. henselae-specific antibody. To detect B. henselae DNA, PCR was performed using GoTaq® Flexi DNA Polymerase, genus-specific primers for the pap31 gene of Bartonella, 1mM MgCl₂ and total DNA isolated from 8µm paraffin-embedded tissue sections. The presence of Bartonella DNA was confirmed by PCR using species-specific primers that target the B. henselae heat-shock protein (htrA). These studies found no link between B. henselae infection and peliosis heptis in cats. (4094)

Notes: The authors compared seedling growth rates, photosynthesis rates and expression levels of heat-responsive genes in the heat-resistant wild rice Oryza meridionalis and the domesticated rice O. sativa when grown at optimal and elevated temperatures. Proteins that were up- or downregulated in response to heat were identified by two-dimensional gel electrophoresis coupled with nano liquid chromatography on line with tandem mass spectrometry (nanoLC-MS/MS). Trypsin was used to cleave proteins prior to nanoLC-MS/MS. Semi-quantitative RT-PCR was performed using the GoTaq® Green Master Mix to determine if the heat-responsive proteins were transcriptionally regulated. (4092)

Notes: The authors investigated the expression of genes encoding histone-like (H-NS) proteins from the self-transmissible pCAR1 plasmid and Pseudomonas putida KT2440 genome, as well as the interaction of H-NS family members in vitro. Gene expression was quantified using quantitative RT-PCR and RNA templates that were treated with RQ1 RNase-Free DNase to degrade contaminating DNA. Interactions between Pmr and other H-NS proteins were monitored using pull-down assays. His-tagged Pmr was expressed in E. coli, purified and used as bait for FLAG-tagged H-NS family proteins. Protein purification of His-tagged proteins was performed using the MagneHis™ Protein Purification System. (4119)

Notes: The authors investigated the mechanism of microRNA (miRNA)-mediated regulation of both endogenous mRNAs and artificial reporter constructs. To determine whether an m⁷G cap and poly(A) tail are required for repression, the authors created plasmids containing eight synthetic, tandem miR-30 recognition sequences in the 3´ untranslated region (UTR) of a Renilla luciferase gene under the control of a 5´UTR that confers either cap-dependent or cap-independent translation. They used these plasmids as a template for in vitro transcription, then transfected in vitro transcripts with and without an m⁷G cap and poly(A) tail into 293T cells, along with miR-30 miRNA (and miR-21 as a negative control). Similar experiments were performed by transfecting 293T cell with the Renilla luciferase constructs and miR-30 and miR-21 expression vectors. Finally, the authors exchanged the artificial 3´ UTR of the Renilla luciferase construct with the 3´ UTR of BACH1, a transcription factor that is regulated by miR-155, and cotransfected 293T cells with the BACH1 3´ UTR construct and an miR-115 expression vector to examine regulation via an endogenous 3´ UTR. The Promega Primer Extension System was used to compare miR-21, miR-30 and miR-155 RNA levels in transfected and untransfected 293T cells to determine endogenous levels of these miRNAs. Renilla luciferase activity was determined using the Renilla Luciferase Assay System. The authors used Northern blot analysis and quantitative RT-PCR to determine Renilla luciferase RNA levels (and GAPDH levels for normalization purposes). The Plexor® One-Step qRT-PCR System was used for qRT-PCR. (4048)

Notes: Listeria monocytogenes uses the internalin A protein (InlA) to cross the intestinal barrier and cause foodborne illness. Mutations in inlA can introduce a premature stop codon, producing a truncated InlA protein that is secreted rather than associated with the bacterial cell wall. Strains with these inlA mutations have reduced virulence. The authors describe an inlA single nucleotide polymorphism (SNP)-based genotyping assay to distinguish isolates with the inlA mutations and use this assay to screen >1,000 L. monocytogenes isolates from ready-to-eat foods and human listeriosis cases. The assay involves amplification of the full-length inlA gene using GoTaq® Colorless Master Mix, purification of amplified products, then single-base-pair extension reactions. (4099)

Notes: The authors characterized mouse neocortical interneurons that express 5-HT_3A, a ligand-gated cation channel activated by 5-hydroxytryptamine (serotonin), during embryonic development. Transgenic mice that expressed green fluorescent protein (GFP) under the control of the 5-HT_3A promoter were created. Single 5-HT_3A-expressing neurons within 300µm brain sections of transgenic mice at various stages of embryonic development were subjected to whole-cell path-clamp recordings to examine their electrophysiological properties. To confirm activation of the 5-HT_3A promoter in these cells, GFP expression was visualized by fluorescence microscopy without breaking the patch clamp seal. The contents of these single neurons then were aspirated and expelled into a 10µl reverse transcription reaction. After the reverse transcription, PCR was performed to simultaneously detect mRNAs encoding two isoforms of glutamic acid decarboxylase, three calcium-binding proteins, three neuropeptides, two transcription factors and reelin, a protein thought to be involved in neuronal migration and morphology. Two rounds of PCR using nested primers were required to detect these mRNAs. PCRs were performed using GoTaq® DNA Polymerase. Amplified products were visualized by agarose gel electrophoresis, using the 100bp DNA Ladder as a size standard. (4096)

Notes: Human Genomic DNA:Male was used as a negative control in standard PCR and Sanger Sequencing to confirm fusion genomic breakpoints identified by NGS experiments. (4534)

Notes: The Wizard® SV Gel and PCR Clean-Up System was used to purify PCR products prior to pyrosequencing. (4546)

Notes: These authors studied the formation of centrioles in Drosophila spermatids. Genomic DNA was extracted from whole flies using the Wizard^® SV Genomic DNA Purification System. The DNA was then subjected to PCR, purified and sequenced. RNA was purified from whole flies using the SV Total RNA Isolation System. After RNA extraction, the samples were used in RT-PCR. (4064)

Notes: Mineral accumulation was studied in Arabidopsis thaliana comparing loci involved with growing in soil versus hydroponics. An F2 population derived from a cross between Landsberg erecta (Ler; maternal parent) and Eringsboda-1 (Eri-1; paternal parent) was propagated by single seed descent for nine successive generations in soil.
The flower buds of three plants per line were collected, and the DNA extracted using the Wizard® Magnetic 96 DNA Plant System and used for genotyping with 90 amplified fragment length polymorphism PCR (AFLP) and 39 single sequence length polymorphisms (SSLP) markers to build a genetic map of quantitative trait loci (QTL). (4136)

Notes: The authors studied how the basal rate of protein synthesis in primary cortical neurons was affected by chronic treatment of with a variety of neurotrophic factors and cytokines. Rat eukaryotic elongation factor 2 (eEF2) was cloned by PCR then subcloned into the pCI Mammalian Expression Vector and electroporated into neurons. After 72 hours, the neurons were harvested and used in various translation assays including ribosomal transit time. (4070)

Notes: To gain a better understanding of how Xylella fastidiosa causes diseases in grapes, the authors mutated conserved regulatory genes, including gacA, that affect expression of virulence-related factors in other species. The relative expression levels of gacA in wildtype and mutated strains were examined using RT-PCR. The authors also identified and quantified a number of genes that were regulated by GacA by microarray analysis. Microarray results were confirmed using RT-PCR. RT-PCR was performed using the AccessQuick™ RT-PCR System. (4052)

Notes: This study examined the genotype-phenotype correlation of the two major UGT isoforms, UGT1A1 and UGT1A6, involved in resveratrol metabolism. Genomic DNA was isolated from 30mg human liver tissue samples (normal and metastatic) using the Wizard^® SV Genomic DNA Purification System. The purified DNA was eluted with 65°C water and 200–400ng of eluted DNA was used in a PCR-RFLP UGT1A6 genotyping assay. Amplification was carried out using PCR Master Mix in a final volume of 50µl, and the amplimers digested with appropriate restriction enzymes. (4018)

Notes: The authors describe a new form of PCR, co-amplification at lower denaturation temperature PCR (COLD-PCR), to detect low-level somatic mutations. This technique is based on the facts that a) each DNA sequence has a critical denaturation temperature (T_c), which is lower than the melting temperature (T_m) and below which PCR efficiency decreases dramatically and b) Tc depends on DNA sequence. The authors used GoTaq^® Flexi DNA Polymerase and mutation-specific TaqMan^® probes for tumor protein 53 (TP53) and epidermal growth factor receptor (EGFR) to detect low-level somatic mutations in a mixture of wildtype and mutant DNAs. Conventional TaqMan^_® technology can detect mutant alleles at an abundance of 10–20% of that of the wildtype allele; using COLD-PCR the authors were able to increase selectivity 15- to 30-fold, detecting as little as 0.8% mutuant alleles. (4038)

Notes: This paper explored the effect of methyl-CpGmodified single-stranded oligonucleotides (ssODN) on gene repair. The Wizard^® Genomic DNA Purification Kit was used to isolate gDNA from methylCpG-ssODN-treated myoblasts derived from limb muscle of neonatal C57 mice that stably expressed the mismatch repair site. One microgram of purified gDNA was digested overnight with 5U of restriction enzyme, purified, resuspended in 20µl of water and 5µl used in real-time PCR to determine if the target had been repaired. (4062)

Notes: Glutathione levels were assessed in human coronary artery endothelial cells (HCAECs) as a measure of oxidative stress. HCAECs were treated with either 100ng/ml eotaxin, a newly discovered chemokine, or pretreated with 2µmol/l MnTBAP for 30 minutes followed by eotaxin treatment for 45 minutes. Positive controls were treatment with 10µg/ml antimycin A and 2ng/ml TNF-α. Cellular glutathione was measured using the GSH-Glo™ Glutathione Assay. (4011)

Notes: The authors examined the role of two promoters in the regulation of fabA, an enzyme involved in unsaturated fatty acid synthesis. fabA transcript levels were quantified using real-time quantitative RT-PCR using ImProm-II™ Reverse Transcriptase, followed by a SYBR^® Green method. (4053)

Notes: In this study, the authors wanted to examine the ability to trace the origin of tomato goods from fresh to processed. They tested several DNA extraction procedures for fresh tomato, tomato sauce, tomato puree, tomato pulp, whole peeled S. Marzano PDO (Protected Designation of Origin) tomato, whole peeled tomato, tomato concentrate and ‘‘Arrabbiata sauce”. Homogenized material (200mg) was extracted in three replicates using seven different methods including the Wizard^® DNA Clean-Up System. The DNA extracted was then analyzed by agarose gel electrophoresis, quantified and tested in PCR using SSR loci. The authors concluded that the Wizard^® DNA Clean-Up System was the most effective of the DNA extraction methods tested and yielded the greatest number of successful amplification reactions with lowest investment of personnel time and money. (4003)

Notes: This study examined the effects of folic acid supplementation on the offspring of pregnant rats fed a protein-restricted diet. Genomic DNA was extracted from rat adipose tissue using the Wizard^® SV Genomic DNA Purification System. The purified DNA was then incubated with methylation-sensitive restriction enzymes and then used in real-time PCR. (4066)

Notes: The authors use the Flexi^® Cloning System to convert their cDNA clones to expression-ready clones. They wanted clones that could be used for comprehensive analysis with the HaloTag^® Technology. They also describe a method of transferring ORFs between Flexi^® Vectors in a 96-well plate format. They also used Wizard^® SV 96 Plasmid DNA Purification, Wizard^® SV PCR Clean-Up, and Wizard^® SV Gel and PCR Clean-Up Systems. (4056)

Notes: The authors examined whether HMGB1 and HMGB2 proteins could affect promoter activity of the topoisomerase IIα gene. Portions of the topoisomerase IIα gene promoter were cloned into the pGL3 Basic Vector, and Saos-2 cells were cotransfected with the resulting constructs, an HMGB1- or HMGB2-expressing plasmid and the pRL-tk Vector as a control for normalization. Firefly and Renilla luciferase activities were determined using the Dual-Luciferase Reporter Assay. To determine whether HMGB1 and HMGB2 promoted binding of the transcription factor nuclear factor-Y (NF-Y) to the topoisomerase IIα promoter, the authors used a chromatin immunoprecipitation (ChIP) assay. Two populations of Saos-2 cells, one of which expressed HMGB1 or HMGB2 and one that had expression inhibited, were fixed with formaldehyde, then treated to shear chromatin. Immunoprecipitation was performed using an anti-NF-Y antibody, and the amount of DNA bound to the NF-Y was quantified by semi-quantitative PCR using GoTaq^® Hot Start DNA Polymerase and Green GoTaq^® Flexi Reaction Buffer. (4037)

Notes: These authors used short heteronuclear RNAs (shRNA) in multiple cancer cell lines to silence hypoxia-inducing factor-2α (HIF-2α), a gene that many cancers exploit to increase angiogenesis and activate fundamental receptor tyrosine kinase signaling pathways to gain growth signal autonomy. Western blotting and RT-PCR were used to monitor the level of HIF-2α silencing. RT-PCR was performed using the AccessQuick™ RT-PCR System. The authors also examined the level of tyrosine kinase activation in shRNA-treated cells by Western blot analysis. To examine levels of ERK1/2 phopshorylation, the authors used the Anti-ERK 1/2 pAb to compare levels of activated and unactivated ERK1 and 2. (4050)