Notes: These authors used the PowerPlex^® Y System to characterize Y-STR haplotype frequencies in southern populations in Korea. Blood and buccal swabs were collected from 259 Korean males, and DNA was isolated with the QIAamp DNA Mini Kit (Qiagen). The authors amplified 5–10ng of each sample (much more than the recommended 0.5–1.0ng) and detected the amplification products using an ABI PRISM^® 310 genetic analyzer. (3637)

Notes: The authors generated population data for 16 autosomal STR loci using the PowerPlex^® 16 System and PowerPlex^® ES Monoplex System, SE33 (JOE), for 200 unrelated individuals in northern Portugal. DNA was collected as blood stains, extracted using Chelex^® resin and amplified using a GeneAmp^® PCR System 9700 as directed by the manufacturer. Amplified products were detected using an ABI PRISM^® 3100 Genetic Analyzer. (3823)

Notes: A transposase protein (Tnp) open reading frame (amino acids 1 to 345) was amplified from the Drosophila mauritiana Mos1 mariner transposable element using GoTaq^® DNA polymerase. The Tnp open reading frame was then cloned with into the pGEM^®-T Easy Vector and sequenced before further subloning. Tnp protein was purified and used in gel-shift assays to demonstrate Tnp binding regions in the Mos1 transposable element. (3344)

Notes: The Wizard^® SV 96 Plasmid Purification System was used to purify plasmid DNA on the Beckman Coulter Biomek^® NX Laboratory Automation Workstation. Plasmid DNA quantity and quality were assessed by spectrophotometry, restriction digestion and PCR. The Wizard^® SV 96 Plasmid DNA Purification System was also used to purify plasmid DNA from bacterial clones isolated in a bacterial two-hybrid screening. (3300)

Notes: Total RNA was isolated from HeLa cells (wild-type and stably transformed) and 2μg of the total RNA was reverse transcribed into cDNA. PCR was performed using 1 or 2μl of the cDNA, 0.5μM of each gene-specific primer (GAPDH, Bcl-2, or Bcl-xl), 0.2mM dNTP mix and GoTaq^® DNA Polymerase (5U/μl) with the corresponding 5X GoTaq^® Reaction Buffer in a final volume of 20μl. The initial denaturation step at 95°C for 2 minutes was followed by 25 cycles of denaturation at 94°C for 30 seconds (15 seconds for GAPDH), annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, and a final extension step at 72°C for 10 minutes. The PCR products were separated on 2% agarose gels. (3369)

Notes: These authors describe map-based cloning tools for Chlamydomonas reinhardtii, (green yeast). In this case-based study of existing and projected resources for C. reinhardtii mapping, mcd4 and mcd5 mutants were mapped by crossing to the infertile strain known as Chlamydomonas grossii, S1-D2, with several known marker sites. To reduce the time and expense of mapping, bulked segregant analysis (BSA) and marker duplexing were evaluated. In BSA, DNAs from multiple segregating progeny (up to six in this study) are combined, and results from PCR-based markers are examined for significant bias from a roughly equal contribution from each parent. This case study used 57–72 markers to span the 1,107-cM genome using approximately 3,350 PCR amplifications. The researchers picked single colonies from a plate and placed each colony in a hypotonic EDTA solution. After boiling for 5 minutes, the supernatant was collected by centrifugation and the DNA quantitated. Twenty nanograms of DNA thus isolated was used as the template for PCR. The reaction included GoTaq^® DNA polymerase with the enhancers 8.5%glycerol and 0.83% formamide in a 30µl reaction. Forty cycles of amplification were performed and the results were analyzed by agarose gel electrophoresis. The various amplification reactions included the use of BSA and both monoplex and duplex reactions with amplicons of 90bp–625bp. (3279)

Notes: The host gene E3L is required for vaccinia virus infection and has anti-apoptosis activity. The authors examine the ability of E3L to regulate transcription of several genes related to apoptosis, immune response and viral pathogenesis. IL-6, NF-AT, p53, NF-κB, Ap-1 and cAMP response elements were cloned upstream of a TATA box and the firefly luciferase reporter gene. Renilla luciferase (pRL-null Vector) was used to normalize for transfection efficiency. Luciferase activities were measured using the Dual Luciferase^® Reporter Assay System. The authors also show that the Z-DNA binding region of E3L is important for transcriptional regulation. HeLa cells were transfected with an expression vectors expressing full-length E3L, E3L with a deletion of the Z-DNA binding domain or E3L with point mutations in residues important for Z-DNA binding. The AccessQuick™ RT-PCR System was used to quantitate IL-6, NF-AT and p53 mRNAs; β-actin was used as a control for RNA input. (3452)

Notes: An open reading frame encoding a putative DNA polymerase, SpPol4, was amplified from S. pombe genomic DNA by PCR. The resulting PCR product was cloned into the pGEM^®-T Easy Vector, and the sequence was verified. Based on sequence analysis, the authors hypothesize that SpPol4 has deoxyribose phosphate (dRP) lyase activity, suggesting that the enzyme plays a role in base excision repair. The authors perform a dRP lyase activity assay with an oligonucleotide substrate labeled using [³²P]ddATP and terminal deoxynucleotidyl transferase. (3465)

Notes: Production of extracellular proteins in E. Carotovora subspecies that are critical for the development of soft-rotting disease of plants, is controlled by quorum-sensing signals, plant signals and assorted transcriptional factors and post-transcriptional regulators. Of these, post-transcriptional regulation by the RsmA-RsmB RNA pair is critical. RsmA is a small RNA-binding protein that promotes decay of RNA. RsmB specifies an untranslated regulatory RNA that binds RsmA and neutralizes its regulatory effect. Many of the transcription factors and QS signals known to regulate extracellular protein production actually act via these post-transcriptional regulators. ExpR, the putative AHL (N-acetyl homoserine lactone) receptor of E. carotovora subspecies carotovora, activates transcription of rmsA and AHL prevents this activation. The authors generated PCR fragments using primers for rsmA71 and rsmA153 and then used the Wizard(R) SV Gel and PCR Clean-Up System (Cat.# A9281) to purify PCR-generated DNA fragments used in gel mobility shift assays. Band shift assays revealed that ExpR is a DNA-binding protein and that its DNA-binding property is modified by AHL. In addition they showed that RsmA overproduction is responsible for inhibition of extracellular protein. (3572)

Notes: To prepare single rat NK cells for reverse transcription, the cells were sorted by flow cytometry and lysed using 0.5% NP-40, 2.2µl of 5X M-MLV Reverse Transcriptase Buffer, 0.3µl of 0.1mol/l DTT, 5.5µl DEPC-treated water and 1U of RNasin^® Plus RNase Inhibitor. After a 30-minute incubation on ice, the lysates were heated to 65°C for 90 seconds, cooled to 22°C for 3 minutes and placed back on ice. For the RT reaction, 1U RNasin^® Plus RNase Inhibitor, 60 U of M-MLV Reverse Transcriptase, 2.5mmol/l dNTPs and 100µM primers were added to these single-cell lysates. (3390)

Notes: The authors examined interindividual differences in cytochrome P450 CYP1A1 and CYP1B1 expression levels in human lymphocytes before and after exposure to dioxins and dioxin-like compounds. CYP1A1 and 1B1 mRNA levels were assessed with semi-quantitative RT-PCR using the Access RT-PCR System. (3432)

Notes: The presence or absence of intercellular adhesion genes (icaA and icaD) in various strains of Staphylococcus epidermidis isolated in medical facilities was investigated using PCR amplification and GoTaq^® DNA Polymerase. Each reaction contained 150ng of template. GoTaq^® Green Reaction Buffer was used in the PCR. (3359)

Notes: The authors investigated common variants of the APOA5 gene that have been associated with differences in plasma triglyceride (TG) levels. PCR fragments containing either the –1131T --> C promoter variant or containing both the –1131T --> C and –3G --> A promoter variants were cloned into the pGEM^®-T Vector System. The fragments were subsequently cloned into the pGL3 Basic Vector and transiently transfected into Huh7 and HepG2 cells along with the luciferase control vector, pRL-TK. The cells were lysed 48 hours after transfection and Luciferase activity was measured with the Dual-Luciferase^® Reporter Assay System. The function of the 1891T --> C variant in the 3´ UTR was tested the same way; with the exception that site-directed mutagenesis was performed to introduce the T --> C at position 1891 before the fragment was cloned into the pGL3 Basic Vector. The functionality of the Kozak sequence –3A --> G variant was determined by cloning cDNAs into the pGEM^®-7Zf Vector. Transcription/translation experiments were performed using the TNT^® Quick Coupled Transcription/Translation System and the proteins were labeled using the FluorTect™ Green_Lys System. In addition, a primer extension inhibition assay was performed using capped mRNAs generated with the Riboprobe^® System –T7 and the Ribo m⁷G Cap Analog. Ribosome binding reactions were performed using the Rabbit Reticulocyte Lysate System, Nuclease Treated. (3460)

Notes: On November 20, 2002, an explosion in a munitions facility left 7 people dead, 100 injured and 5 missing. The authors used DNA typing to identify 2 tissue samples and 19 bone samples. These samples, as well as reference samples from relatives of the missing persons, were analyzed using the PowerPlex^® 16 System. DNA was extracted using phenol:chloroform:isoamyl alcohol extraction and concentrated, then amplified using 28–30 cycles. For bone samples, the cycle number was increased to 35. The success rate was 90% (19 of 21 samples identified; 2 of 21 samples had a high degree of contamination and could not be identified). (3819)

Notes: These authors studied the effect of transgenic Bacillus thuringiensis (Bt) corn feed containing the Cry1A gene on broiler chicken performance. They also analyzed the degradation of the Cry1A(b) transgene in the digestive tract of the chickens. Blood and samples from cecum, jejunum, gizzard, and crop were collected from ten broilers per treatment (fed Bt corn versus near isogenic corn). Genomic DNA was isolated from blood samples and the intestinal contents of the crop and gizzard using the Wizard^® Genomic DNA Purification Kit. Fifty nanograms of purified DNA were then used in PCR analysis. (3678)

Notes: To rapidly characterize somatic mutations in the EGF receptor in lung cancer tissues, genomic DNA was extracted from lung biopsies using the Wizard^® SV Genomic DNA Purification System. The extracted DNA was used in a LightCycler SNP genotyping assay to determine the genotype of common loci associated with improved patient response to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor. (3588)

Notes: To confirm that the rat IL-10 transgene under the control of a liver-specific promoter was expressed in mice, mouse tails were subjected to Proteinase K digestion and genomic DNA isolation using the Wizard^® SV 96 Genomic DNA Purification System. PCR analysis was used to distinguish between mouse and rat IL-10. (3555)

Notes: Immunohistochemistry, Western blotting, and real-time PCR were used to determine levels of COX-2 in papilloma and normal laryngeal tissue. Explant cultures of human normal laryngeal and papilloma cells were used to define the signaling pathways that regulate COX-2 expression and investigate the potential of targeting COX-2 as a strategy to suppress papilloma growth. To measure the effects of prostagladin E₂ (PGE₂) on cell number, papilloma cells were cultured in keratinocyte growth media containing 250 or 500 nmol/L PGE₂ or an equal volume of DMSO for 24 hours. The relative measure of viable cells was determined by the CellTiter 96™ Non-Radioactive Cell Proliferation Assay.
(3306)

Notes: To simulate isolating Salmonella typhimurium genomic DNA from various sample types, 2 to 2 × 10⁶ genome equivalents and 200µl lysis buffer were added to pelleted material from fishmeal, pig feces and chicken neck skin. After vortexing the samples, the genomic DNA was isolated using the MagneSil^® KF, Genomic System on a KingFisher^® workstation. To analyze the purified DNA, 5µl was used in real-time PCR with four replicates. (3425)

Notes: In this study, the researchers used the MagneHis™ Protein Purification System to purify recombinant, histidine-tagged Tribolium castaneum (red grain beetle) esterase from Pichia pastoris. The T. castaneum esterase gene, termed TCE, was cloned into pGAPZα A, pGAPZ B, and pPICZ B vectors and P. pastoris cultures transformed with each vector were analyzed for esterase activity. TCE yields varying from 7-80mg/L were obtained from P. pastoris cultures containing the above constructs using the MagneHis™ Protein Purification System. Specific activities of histidine-tagged TCE ranged from 4.5 to 5.7 U/mg. (3297)

Notes: To learn more about the molecular mechanism of thermostability of sucrose phosphate synthase (SPS) from Halothermothrix orenii, the gene was cloned in the pTrcHisA expression vector by PCR, transformed into E. coli and induced for 4 hours with 1mM IPTG. The cells were lysed by sonication and freeze/thaw cycles, and the bacterial lysate was cleared by centrifugation. The HisLink™ Protein Purification Resin was added to the cleared lysate and incubated for 1 hour with gentle agitation. The mixture was transferred to a chromatography column and washed with at least 50 column volumes of 20mM Tris-HCl (pH 7.5), 500mM NaCl, 10mM imidazole. Recombinant SPS was eluted in 20mM Tris-HCl (pH 7.5), 100mM NaCl, 500mM imidazole and the imidazole removed by diafiltration. The purified protein was then allowed to crystallize and used for X-ray diffraction to determine structure. (3281)

Notes: Suppression subtractive hybridization (SSH) was performed on six breast cancer tumors. The PCR products were T/A cloned, grown and isolated using the Wizard^® MagneSil^® Plasmid Purification System. The purified plasmid DNA was then sequenced using BigDye^® 3.0 Sequencing Mix and the ABI 3700 Genetic Analyzer. (3446)

Notes: Having observed that blunt 27mers had increased potency in RNAi compared to 21mers or 27mers with 3' or 5' overhangs, these authors investigated what differences may account for these changes in gene silencing activity using the same target sequence in enhanced green fluorescent protein (EGFP). For one experiment, a PCR-generated fragment of the EGFP coding region spanning sites EGFPS1 and EGFPS2 was cloned into psiCHECK™-2 Vector in both sense and antisense orientations. Also, a PCR-generated fragment of the human heterogeneous nuclear ribonucleoprotein H (hnRNPH) coding region spanning sites H1 and H3 was similarly cloned in sense and antisense orientations. HEK293 cells were transfected with 150ng EGFP sense and antisense vectors plus EGFPS2 or control duplex RNAs. HCT116 cells were transfected with 100ng sense and antisense hnRNPH vectors with H3 or control duplex RNAs. The Dual-Luciferase^® assay was used to evaluate luciferase expression 24 hours post-transfection. In a separate EGFP RNAi experiment, the Steady-Glo^® Luciferase Assay System was used to monitor firefly luciferase activity to normalize transfection of HEK 293 cells. A further RNAi experiment targeted the firefly luciferase gene in the pGL3-Control Vector cotransfected with 20, 2 or 0.4 nM siRNA duplexes into HeLa cells. After 48 hours, the cells were lysed and 10µl tested using the Luciferase Assay System. To test the level of expression of human La antigen targeted for gene silencing, total RNA was harvested from HeLa cells using the SV 96 Total RNA Isolation System, reverse transcribed and used in real-time PCR. (3289)

Notes: In vitro cultures of P. alba were incubated in growth chambers under various conditions. The axenic plants of P. alba were sprayed with either methyl jasmonate (100 lM), methyl salicylate (1mM), or ethanol as a control, and then grown under illumination at 18 hours/day for 24 hours at 25°C. Total RNA was subjected to semi-quantitative RT-PCR using GoTaq^® DNA polymerase and primers for hybrid poplar isoprene synthase. For normalization, actin was used as an external standard. (3355)

Notes: To examine the role of Rac3 in nervous system development, knockout mice were created. Total RNA from postnatal day 0 (P0) and day 9 (P9) brains from wild-type (+/+), heterozygous (+/-), and knockout (-/-) mice was reverse transcribed and a ~260bp product amplified by PCR using GoTaq^® DNA Polymerase and primers for either Rac3 or Rac1 (a Rac3 homolog). No amplification product for Rac3 was observed in the knockout mice. (3328)