Notes: The authors characterize an Arabidopsis cytokinin-receptor mutant. RNA was isolated from leaf tissue from wildtype and transgenic Arabidopsis plants expressing various components of the cytokinin signaling pathway. The RNA was reverse transcribed using the ImProm-II™ Reverse Transcription System, and real-time PCR was performed to quantitate response regulator proteins in the signaling pathway. (3450)

Notes: Researchers used the GoTaq^® DNA Polymerase in PCR screens for excisions around a CYP6d4 gene in the HA-1829 strain of Drosophila. PCR was performed in a 20μl reaction volume using 0.4 Units of GoTaq^® DNA Polymerase, 2.75mM MgCl₂ and 1μl of DNA (equivalent to the DNA in approximately 1/5 to 1/10 of a fly). (3363)

Notes: GoTaq^® DNA Polymerase was used in PCR to detect Lactococcus lactis phage DNA strains in whey samples. Phage DNA templates were amplified directly from DNase treated and boiled whey samples. For these reactions, the researchers use 0.25µl (1.25 units) of GoTaq^® DNA Polymerase for each 50μl reaction. Primers were designed to distinguish various strains of Lactococcus lactis phage receptor-binding protein genes. (3362)

Notes: The authors linked outbreaks of acute gastroenteritis in Italy and France with consumption of oysters contaminated with Norovirus. In Italy Norovirus RNA was detected in fecal samples from infected individuals using the Access RT-PCR System and primers for the Norovirus polymerase region. (3792)

Notes: Researchers used GoTaq^® DNA Polymerase to test sheep and goat blood samples for the presence of Babesia DNA. Primers were designed around the 18S rRNA sequence of Babesia sp. PCR was performed in a 50µl reaction volume using 1 unit of GoTaq^® DNA Polymerase. Ten microliters of each amplification reaction were loaded on gels and subjected to electrophoresis. (3380)

Notes: In this study, a real-time PCR assay coupled with a DNA extraction method were used to detect staphylococcal enterotoxin (SE)-encoding genes in milk, a source of staphylococcal food poisoning. Pasteurized milk prepared with known concentrations of Staphylococcus aureus was used to generate a standard curve; experimental samples were from a staphylococcal food-poisoning outbreak that occurred in Japan in June 2000. PCR inhibition was overcome by using the following DNA purification method: a sample of milk (100µl) was added to an equal volume of 0.2M sodium hydroxide and incubated at 37°C for 20 minutes. The alkaline-treated sample was neutralized using 10µl of 3M sodium acetate (pH 5.4), extracted with 1ml of petroleum ether, and centrifuged at 13,000 × g for 10 minutes at 25°C. The aqueous phase was transferred to a fresh tube and bacterial DNA was purified from the aqueous solution using the Wizard^® SV Genomic DNA Purification System. (3677)

Notes: Variation in five microsatellite loci and five SNPs covering all Arabidopsis thaliana chromosomes was examined using samples from four individuals from each of 37 local populations, including North America, England, Eastern and Western Europe, Asia, and a selection of ecotypes. Genomic DNA was extracted from leaf tissue using the Wizard^® Magnetic 96 DNA Plant System and a GenoGrinder^® instrument then used for amplification with the 10 loci chosen. (3427)

Notes: To examine genetic variation in Anopheles funestus, genomic DNA was extracted from single mosquito carcasses (minus the abdomen) using the Wizard^® SV 96 Genomic DNA Purification System on a Beckman Coulter Biomek^® FX workstation. The purified genomic DNA was diluted 1:10 in water to ~5ng/µl for PCR analysis. (3415)

Notes: To test the feasibility of the Mycobacterium bovis Ag85B gene as a DNA vaccine, the gene was amplified by PCR, introducing EcoRI and XbaI restriction endonucleases onto the primers, digested with the restriction enzymes and ligated into the pCI Mammalian Expression Vector. After transformation, the constructs were isolated using the Wizard^® Plus Minipreps DNA Purification System and analyzed using endonuclease digestion and DNA sequencing. To confirm production of a maltose binding protein (MBP)-Ag85B fusion protein in E. coli, the cell lysate was analyzed by Western blot, stained with a rabbit anti-MBP serum and secondary antibody Anti-Rabbit IgG (Fc), AP Conjugate (1:10,000 dilution) and developed with BCIP/NBT. BALB/c mice pretreated with 10mM cardiotoxin prior to intramuscular DNA immunization using construct pCI-Ag85B, administered at days 0, 15, 30, and 45. The immunization course was 50µl of DNA at a concentration of 1µg/µl in PBS with each animal receiving a total of 100µg of plasmid DNA. Antibody response was assessed at 15 days with an ELISA assay and cytokine presence tested two weeks after immunization using splenocytes for ELISA assays and ICC. (3552)

Notes: The authors developed a PCR method to detect the presence of meat and bone meal (MBM) in animal feed even if the MBM had been heat treated, and discern whether the animal component is bovine or porcine in origin. The genomic DNA from 100mg of various feedstuffs with known and unknown amounts of MBM, fishmeal or poultry feed or a combination of these compounds was isolated using the Wizard^® Magnetic DNA Purification System for Food with the KingFisher System. Real-time PCR was performed using 5μl of extracted DNA. (3750)

Notes: The authors examined the locations and levels of apoptosis-related Fas, Fas ligand, Bcl-2, Bax and caspase-3 proteins in ovarian tissue throughout the rat estrus cycle. Protein levels were determined using Western blotting, and proteins were localized by immunhistochemistry. The presence of Fas, Fas ligand, Bcl-2, Bax and caspase-3 mRNA in various ovarian tissues was monitored by RT-PCR. Reverse transcription was performed using the ImProm-II™ Reverse Transcription System, 1µg of RQ1 RNase-free DNase-treated RNA and an oligo(dT) primer. One microliter of the reverse transcription reaction was amplified by PCR, and 10µl of the amplification products were analyzed by agarose gel electrophoresis. (3724)

Notes: In this study, genomic DNA was extracted from 99 frozen thymic epithelial tumor samples using the Wizard^® SV Genomic DNA Purification System. Purified DNA was used in TaqMan SNP genotyping assays for 13 epidermal growth factor receptor (EGFR) gene mutations. (3585)

Notes: To further understand the transcription factor paired-box 2 (PAX2) gene, 2µg of total RNA isolated from Kaposi’s sarcoma cells were reverse transcribed, and 5µl of the RT reaction was amplified. The PCR product was then cloned into the pTARGET™ Mammalian Expression Vector and two clones identified: PAX2 in the sense orientation and a second in the antisense orientation. The clones were then transfected into two different cell lines: the antisense PAX2 into renal tumor-derived endothelial cells where PAX2 is expressed, and the sense into normal human microvascular endothelial cells where PAX2 is not normally expressed. The effects of PAX2 expression on the cell lines were then examined. (3496)

Notes: PMSRA expression was examined in 4-week old plants exposed to 10μM methyl viologen, 100μM cercosporin, photo-oxidative stress or ozone. Samples were ground in liquid nitrogen and total RNA isolated. Total RNA (2.5μg) was reverse transcribed into cDNA with random primers d(N)₁₀, then amplified using gene-specific primers for PMSRA1—PMSRA5 and an antibody-mediated hot start containing a mixture of GoTaq^® DNA Polymerase and Taq antibody (BD Biosciences, Mountain View CA). In a total volume of 25μl, the RT-PCR reaction mixture contained 2.5 units of GoTaq^® DNA Polymerase, 10mM Tris–HCl (pH 8.5), 60mM KCl, 2.4mM MgCl₂ and 300μM of each dNTP. For each RT-PCR, a plant 18S universal internal standard (Ambion, Austin TX) was included as a loading control. Amplification reaction conditions were as follows: 27 cycles at 94°C for 45 seconds, 55°C for 45 seconds and 72°C for 1 minute. For each analysis, three rounds of RT-PCR were conducted with three independently isolated total RNA samples. Twenty microliters from each PCR were fractionated by 1.5% (w/v) agarose gel in Tris–borate EDTA buffer and stained with 0.5% (w/v) ethidium bromide. (3370)

Notes: The authors devised an mRNA display system to generate a peptide library with multiple nonnatural amino acids incorporated into the proteins, an important feature of peptide libraries for successful drug discovery. An mRNA with 3 four-base codons at a random position was used as a template in an in vitro translation system in the presence of charged tRNAs carrying four-base codons. In vitro translations were performed using 3.6 × 10¹³ molecules of mRNA template and the E. coli S30 Extract System. The mRNA template contained a T7 tag sequence, so the translation products could be detected using an anti-T7 tag antibody and the Anti-Mouse IgG (H+L), AP Conjugate. The mRNA-displayed peptides also incorporated a polyhistidine tag so that they could be purified using the MagneHis™ Ni-Particles. After selecting for the desired protein characteristic, the mRNA portion of the mRNA-displayed peptides was reverse transcribed and quantitated by real-time PCR. PCR products were cloned into the pGEM^®-T Vector prior to sequencing. (3651)

Notes: In this study, the effects of amino acid substitutions in porcine corticosteroid-binding globulin gene (Cbg) were tested on CBG binding and affinity. Genomic DNA was isolated from whole blood of 92 female pigs studied. Cbg cDNA was obtained by reverse transcribing pig liver total RNA using M-MLV Reverse Transcriptase followed by PCR. The 1257pb cDNA PCR product was ligated into the pTARGET™ Mammalian Expression Vector. The GeneEditor™ in vitro Site-Directed Mutagenesis System was used to introduce four different codon substitutions in the Cbg cDNA. Once created, the mutated and unmodified Cbg cDNA constructs were transfected into HEK-293T (human embryonic kidney) cells. After 48 hours, the supernatant was collected to analyze secreted CBG. (3498)

Notes: The authors used the PowerPlex^® 16 System to gather population data from 429 unrelated individuals in northern and northeastern Argentina. Blood or buccal swabs were collected, and DNA was extracted organic extraction. Fifteen autosomal STR loci were amplified using the PowerPlex^® 16 System and a GeneAmp^® PCR System 9600 or 9700, and amplified products were detected using an ABI PRISM^® 310 or 3100-Avant Genetic Analyzer. (3820)

Notes: The authors generated population data from 913 individuals living in two patagonian provinces in Argentina using the PowerPlex^® 16 System. DNA was collected as a blood or buccal swab sample, isolated using by organic extraction and amplified using the GeneAmp^® PCR System 9600 or 9700. Amplification products were detected using an ABI PRISM^® 310 or 3100-Avant Genetic Analyzer. (3814)

Notes: The authors determined allele frequency data in the Tujia population in China using the PowerPlex® 16 System. DNA was extracted from 98 whole blood samples, and 1ng was amplified using the GeneAmp® PCR System 9600. Amplified products were analyzed using the ABI PRISM® 310 Genetic Analyzer. (3774)

Notes: The authors selected repetitive elements in the human genome using a novel technique: GREM. T4 DNA Ligase was used to ligate adapters to digested genomic DNA prior to PCR, and exonuclease III was used to generate the necessary 5´ termini. After the final amplification, the PCR products were cloned into the pGEM^®-T Vector, then sequenced. (3550)

Notes: This paper highlights the first use of the HaloTag™ Interchangeable Protein Labeling Technology in plant cells. The cDNA for the HaloTag™ protein was amplified by PCR from the pHT2 Vector and cloned into the pGEM^®-T Easy^® Vector, from which it was excised and transferred to a second vector where its expression was under the control of the cauliflower mosaic virus (CaMV)-³⁵S promoter. The construct was transformed into tobacco protoplasts and bioballistically transformed into tobacco leaf cells. Localization was followed using the HaloTag™ TMR and diAcFAM Ligands. (3503)

Notes: In this study, the Flexi^® Vector Systems was compared with the Gateway^® Cloning System to determine its utility in high-throughput expression cloning by subcloning 96 human target genes. A direct comparison between pVP16, the Gateway^® vector and the equivalent Flexi^® Vector, pVP33A or K, was achieved by modifying pVP16 with the barnase gene and PmeI/SgfI restriction sites, duplicating the design available in the commercial Flexi^® Vectors. Capture of genes by PCR amplification of the cDNAs was similar for both systems, but the timeline for the Flexi^® Vector system was shorter at 6–8 days compared to 12 days for the Gateway^® system. They also found the Flexi^® Vector System was lower cost and more accurate due to the shorter primers required for the Flexi^® Vector cloning. The authors found nearly twofold fewer missense errors due to the shorter amplification primers. Ninty-six cDNAs were amplified simultaneously in their protocol and PCR products were cleaned up using either the MagneSil^® PCR Clean-Up System or Wizard^® SV 96 PCR Clean-Up, ligated into the Flexi^® Vector, and transformed into Select96™ Competent Cells. The study also compared transfer of cDNA inserts between different Flexi^® Vectors and transfer of cDNA inserts between different Gateway^® vectors and found similar performance in the two systems. For the Flexi^® Vector test set, the authors sequenced the clones, validating the high fidelity transfer of cDNA inserts between Flexi^® Vectors. (3533)

Notes: Fifteen microliter amplification reactions performed using 0.5 Units of GoTaq^® DNA Polymerase and 25 ng BAC or genomic DNA were used as templates for sequencing portions of the QTL region of porcine chromosome 7. Amplification products were verified by gel electrophoresis followed by ethidium bromide staining. (3361)

Notes: Total RNA was extracted from human astrocytes and control A549 cells. First strand cDNA was synthesized from 3μg of total RNA using random hexamers. PCR was performed on the cDNA samples using primers for DR4, DR5, and GAPDH with GoTaq^® Green Master Mix. The PCR was performed with an initial denaturation at 94°C for 2 minutes, followed by cycles of 30 seconds at 95°C, 30 seconds at 55°C, and 45 seconds at 72°C. The PCR products were analyzed on a 1.5% agarose gel and stained with ethidium bromide. (3372)

Notes: The gene expression profile of human umbilical cord matrix stem (UCMS) cells was determined by microarray analysis and confirmed by RT-PCR. The 50 most highly expressed genes in UCMS cells tended to fall into several categories: genes associated with neurotrophic effects and morphogenesis, the three germ layers, the undifferentiated state of embryonic stem cells and extracellular adhesion molecules. The biotin dUTP-labeled cDNA probes used to query the UCMS cell microarray were synthesized using 200 units of M-MLV Reverse Transcriptase and 4µg of total RNA. (3469)