Notes: The authors of this study investigated the role of apoptosis signal-regulating kinase 1 (ASK1) in neural cell apoptosis during retinal development and ischemic injury. Nucleotides 283 to 713 of the ASK1 cDNA were amplified by PCR and cloned into the pGEM^®-T Easy Vector, and sense and antisense probes for in situ hybridization experiments were generated. Anti-ACTIVE^® p38 polyclonal antibody was used for immunohistochemistry analyses to investigate the localization of phosphorylated p38 in mouse retina. (3530)

Notes: After bioinformatics analysis of the Bacillus anthracis genome for potential vaccine targets, the 197 ORFs were selected for amplification. Using the genomic DNA of B. anthracis strain Vollum, all PCR products were generated using a 5’ primer incorporating the T7 promoter, the Kozak sequence, unique restriction enzyme sites and a start codon and a 3’ primer with a stop codon and three more unique restriction sites. The amplicons were then in vitro transcribed and translated using the TNT^® T7 Quick for PCR DNA system and radiolabeled with [³⁵S]methionine. The products were analyzed by SDS-PAGE and autoradiography. To assess the immunoreactivity of the translated products, the proteins were immunoprecipitated using anti-B. anthracis antisera. The seropositive candidate amplified ORFs were cloned into the pCI Mammalian Expression Vector using compatible RE sites. These vectors were introduced into ICR mice using a Helios gene gun system (0.5µg plasmid DNA on 1µm gold particles) and the mice immunized in 2 week intervals. (3515)

Notes: The authors wanted to develop a more sensitive assay to detect codon 816 pathogenic variations in people diagnosed with systemic mastocytosis. The Wizard^® Genomic DNA Purification Kit was used to isolate DNA from peripheral blood and bone marrow aspirate samples. To extract DNA from 2–5 micron, paraffin-embedded samples of bone marrow trephine, skin, spleen or liver, the tissues were digested with Proteinase K for four days at 56°C prior to DNA purification using the Magnesil^® Genomic Fixed Tissue System. The isolated DNA was subjected to two assays: enriched sequencing of mutant alleles (ESMA) after BsmAI restriction enzyme digestion, and allele-specific competitive blocker PCR (ACB-PCR). (3575)

Notes: To purify stolbur phytoplasma DNA from total DNA of infected periwinkle plants, two rounds of suppression subtractive hybridization (SSH) were performed, followed by amplification with Taq DNA polymerase. The resultant PCR products (1µl) were ligated into 50ng of pGEM^®-T Easy Vector using 3 units T4 DNA Ligase. After transformation of DH10B cells, ampicillin-resistant colonies were grown and the plasmids purified using the Wizard^® Plus SV Minipreps DNA Purification System. The insert lengths were estimated after EcoR I digestion and agarose gel electrophoresis prior to amplification and labeling with digoxigenin. These probes were used for dot hybridization with denatured healthy or infected plant DNA (10µg) and the corresponding plasmid as a positive control (100 ng). (3436)

Notes: To examine the effect of Substance P (SP) on COX-2 expression, the COX-2 promoter region spanning –2069 to –66 bp was cloned by PCR and subcloned into the pGL3-Basic Vector (pGL3-Cox-2). Nontransformed human colonic epithelial NCM460 cells overexpressing neurokinin-1 receptor (NK-1R; NCM460-NK-1R) were seeded in 12-well plates and transiently transfected with pGL3-Cox-2 with either a transfection control pRL-TK Vector or siRNA or both. The siRNA molecules used were for AK1, JAK2 (Upstate Biotechnology), STAT3, STAT5, STAT6 or a control siRNA. The transfected cells were serum starved for 24 hours, treated with SP for 4 hours and then lysed. The cell extracts were measured for firefly and Renilla luciferase activities using the Dual-Luciferase^® Reporter Assay System. Relative luciferase activity was a ratio of COX-2 promoter firefly activity to Renilla activity; results were expressed as percentage of control group without SP stimulation. To mutate the STAT binding elements, the pGL3-Cox-2 construct was modified using the GeneEditor™ in vitro Site-Directed Mutagenesis System. The resulting mutant constructs were tested in the same system as the wildtype COX-2 promoter. (3520)

Notes: The COX-2 promoter was cloned by PCR into the pGL3 Vector. NCM460-NK-1R cells were transiently transfected with the cloned promoter and the pRL-TK vector as an internal control or siRNA targeted against various JAK/STAT genes. The Dual-Luciferase^® Assay was used to measure promoter activity. The wildtype COX-2 promoter was mutagenized using the GeneEditor™ in vitro Site-Directed Mutagenesis System. (3384)

Notes: The authors developed a new set of miniplex primers for DNA typing of degraded DNA from human skeletal remains. The miniplex primers produced smaller amplicons (50–280 base pairs) than standard STR systems. The DNA-typing results obtained with the miniplex primers were compared to results obtained with the PowerPlex^® 16 System. The authors determined that larger loci failed to amplify when using degraded DNA and that the degradation cut-off length of template fragments occurred predominantly at 200bp and is not kit-dependent. (3808)

Notes: The authors identify PARTING DANCERS as a gene involved in male meiosis in Arabidopsis using a microarray generated from meiotic-stage anthers. To confirm the sequence obtained by microarray analysis, RT-PCR was performed and the resulting cDNA was cloned into the pGEM^®-T Vector and sequenced. To generate probes for in situ hybridization, fragments of ptd were amplified, cloned into the pGEM^®-T Vector, then labeled with digoxygenin through in vitro transcription. (3468)

Notes: To examine if the depletion of Drosophila transcription termination factor (DmTTF) after RNAi treatment could reduce the gene copy number, genomic DNA was isolated from RNAi-treated and untreated Drosophila embryonic D.Mel-2 cells using the Wizard^® Genomic DNA Purification Kit. The mitochondrial ND3 gene and the nuclear H2B histone gene were used as probes for the Xho I-digested, Southern-blotted genomic DNA to compare the treatment groups. The Wizard^® SV Gel and PCR Clean-Up System was used to clean up the PCR-amplified probes. (3418)

Notes: To examine possible Ig gene rearrangement of B-cell neoplasias after Kaposi sarcoma–associated herpesvirus infection, transgenic mice expressing KSHV latency-associated nuclear antigen (LANA) were created. Genomic DNA was isolated from murine spleens using the Wizard^® SV Genomic DNA Purification System, and 1.7ng of the purified genomic DNA was used in PCR analysis. (3414)

Notes: Monocarboxylate transporters (MCT) transport lactic acid across the cell membrane. The promoters of 4 MCT family members (MCT1, MCT2, MCT3 and MCT4), were amplified by PCR and cloned into the pGEM^®-T Easy Vector. The sequences were confirmed, and the promoters were cloned into the pGL3-Basic Vector. The Dual-Glo™ Luciferase Assay System was used to quantitate promoter activity under basal and hypoxic conditions in HeLa cells. The pRL-SV40 Vector was used to normalize for differences in transfection efficiency. (3463)

Notes: To characterize sequence variability of the catalytic center of the hepatitis delta virus (HDV) ribozyme, which includes a self-cleaving motif, the authors developed an in vitro selection process that allowed them to select self-cleaving sequence variants from a pool of HDV ribozymes. The selection process started with a library of DNA oligonucleotides corresponding to the HDV ribozyme sequence that had been randomized at a 25-nucleotide sequence. The oligos contained known sequences at the 5´ and 3´ ends to allow amplification. The library was amplified by PCR, then transcribed. The resulting RNA was separated by polyacylamide gel electrophoresis, and the self-cleaved transcripts were excised, purified and reverse transcribed. A 3´ extension using terminal deoxynucleotidyl transferase added a known sequence to the 3´ end so that the resulting cDNA could by amplified by PCR. Thus another cycle of selection could begin. The PCR products generated at the start of the process and at each cycle were cloned into the pGEM^®-T Vector. (3467)

Notes: GoTaq^® DNA Polymerase was used in RT-PCR. First-strand cDNA was synthesized from 3μg of total RNA. PCR was performed using the first-strand cDNA as a template, the primers TM4SF2, and GoTaq^® DNA Polymerase, with 42 cycles of 94°C for 30 seconds, 50°C for 30 seconds, and 72°C for 60 seconds. (3354)

Notes: In this study, coding sequence abnormalities in desert hedgehog (DHH), a gene involved in male sex differentiation, were investigated. Genomic DNA was isolated from paraffin-embedded gonadal tissue from individuals with mixed gonadal dysgenesis and from three controls using the MagneSil^® Genomic, Fixed Tissue System. The purified DNA was then analyzed by exon-specific PCR, single-stranded conformation polymorphism (SSCP) analysis, and sequencing. (3447)

Notes: The authors used 5´ and 3´RACE to amplify the gene mimitin, and the resulting cDNA was cloned into the pGEM^®-T Vector. A genomic DNA fragment containing the mimitin promoter sequence was amplified by PCR and cloned into the pGEM^®-T Vector. The promoter was then cloned into the pGL3-Basic Vector. The activity of the wildtype and mutated promoters was determined using a luciferase assay. The pRL CMV Vector was used to normalize for differences in transfection efficiency. (3466)

Notes: Mouse adenovirus type 1 (MAV-1) was detected in DNA extracted from the lungs of mice after PCR amplification of the E1A region of MAV-1. For these assays, 80ng of total DNA was added to a 20µl PCR reaction containing 0.5 units of GoTaq^® DNA Polymerase, 4µl of 5X GoTaq^® Buffer, dNTPs and primers for MAV-1 E1A. The amplified products were separated on a 1.8% agarose gel and stained with ethidium bromide. (3381)

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)