Notes: The NanoBiT Protein-Protein Interaction System was utilized to investigate the interaction of human A₃ adenosine receptor, a G protein-coupled receptor, and β-arrestin2. Specifically, the importance of specific C-terminal phosphorylation sites for A₃AR and β-arrestin2 coupling was determined using protein truncations. To determine the optimal fusion tags for this assay, multiple constructs were tested in the presence of an A₃AR selective agonist. Unlike previous studies in rat models, C-terminal human A₃AR truncations displayed no major impact on β-arrestin2 coupling. (5077)

Notes: The authors present an overall strategy for using the CRISPR/Cas9 system for reporter tagging of endogenous loci. As examples, NanoLuc^® and TurboGFP-tagged reporter cell lines were generated. PCR was performed using GoTaq^® Polymerase. To detect the integration event, we combined a primer binding in the cassette (NanoLuc^® or TurboGFP) with a gene-specific primer. Cells were analysed using the NanoGlo^® Luciferase Assay. (4755)

Notes: Taq Polymerase was used in this experiment to amplify eight microsatellite loci, according to the tagged primer method.  (4893)

Notes: These authors investigated the role of matrix metalloproteinase (MMP-9) in a reversible model of cancer that is initiated by infection with intracellular Theileria parasites. They found that gene induction by parasite infection was associated with trimethylation of histone H3K4 (H3K4me3) at the MMP-9 promoter. The H3K4 methyltransferase SMYD3 was the only histone methyltransferase upregulated upon infection. They therefore investigated the role of SMYD3 overexpression on MMP-9 expression and cell migration, identifying SMYD3 as an important new regulator of MMP-9 transcription. During the study they used the GloMax® Multi Luminometer to measure luminescence and absorbance in reporter and cell viability assays. They also used the Dual-Luciferase® Reporter Assay to measure SMYD3 activity in cells transfected with a SMYD3 reporter, and the pGL4-hRluc/TK plasmid for normalization of the experimental reporter activity. GoTaq® DNA polymerase was used in semi-quantitative RT-PCR. (4189)

Notes: The authors used GoTaq® DNA Polymerase to amplify cDNA generated from total RNA (RT-PCR) extracted from murine erythroid leukemia (MEL) cells and mouse erythroid burst-forming units (BFU-Es). These cells were used to study the molecular mechanism of 5-aza-2'-deoxycytidine (5-aza-CdR)-induced erythroid differentiation, a process involved in azanucleotides for treating myelodysplastic syndromes (MDS) that reduces the risk of transformation to acute myeloid leukemia (AML). Treatment of these cells with 5-aza-CdR, a hypomethylation reagent, upregulated genes responsible for heme production and iron uptake. The pGL3 basic vector and promoter were used to create plasmid constructs of different E-box regulatory sequences with a luciferase reporter. The plasmids were cotransfected with c-Myc, Max or both transcription factors into human hepatocytes (HepG2). The Dual-Luciferase® Reporter Assay System was used to identify that the –6kb E-box of the transferrin receptor 1 (TfR1) promoter was a strong enhancer for inducing TfR1 expression when c-Myc and Max formed functional complexes that bound to it. Bisulfite sequencing was performed to study methylation patterns after 5-aza-2’-CdR treatment using the pGEM-T® Easy Vector system to ligate the isolated DNA fragments for TfR1 and Fech (ferrochetalase), which were transformed into E coli. for final sequencing. (4176)

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: Snail is a transcriptional repressor of E-cadherin that enhances both cell attachment and cell detachment in Madin Darby canine kidney (MDCK) and A4231 cells. To investigate this effect, the authors used Western blot analysis and RT-PCR to monitor protein and mRNA levels of the major adhesive proteins expressed in epithelial cells: laminin, heparin sulfate proteoglycan and collagens. For RT-PCR, total RNA was isolated from transiently transfected snail-expressing MDCK and A431 cells and untransfected cells, then reverse transcribed. The resulting cDNA was amplified by PCR using GoTaq^® DNA Polymerase; glyceraldehyde-3-phosphate dehydrogenase was amplified as an internal control. The ability of Snail to regulate the integrin αV promoter was also examined by cloning the promoter and several promoter deletions upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. Each of these constructs (1µg) and 20ng of pRL-CMV Vector were transfected into MDCK and MDCK/snail cells, and luminescence was measured using the Dual Luciferase Assay System. (3882)

Notes: To study the role of connexin42 (cx43) in testis development, the authors generated a conditional cx43 knockout mouse, which lacked the cx43 gene in Sertoli cells. To confirm that the cx43 gene was deleted in these mice, PCR was performed using primers specific to cx43, 1X Colorless GoTaq^® Flexi Reaction Buffer, 2mM MgCl₂, dNTPs and 0.15µl of GoTaq^® DNA Polymerase. Tissue-specific deletion of cx43 was confirmed by amplifying RNA isolated from mouse testis, heart and tail was also confirmed using the same PCR components. (3713)

Notes: The authors studied the defective prophage KplE1 in E. coli K12 to map the binding sites of proteins required for recombination. Prior to in vivo excision assays in two E. coli K12 strains, the presence of three DNA sequences required for recombination was confirmed by PCR using GoTaq^® DNA Polymerase. In vitro excision assays were also performed using linear and supercoiled DNA substrates that were purified using the Wizard^® PCR Clean-Up System. Finally the phage-encoded integraseS (IntS) mRNA was quantitated by real-time RT-PCR. The RNA template was purified from E. coli K12 using the PureYield™ RNA Midiprep System. (3722)

Notes: The authors characterized expression of extracellular proteins by white-rot fungus, Phanerochaete chrysosporium, grown on wood. Temporal expression of these proteins was monitored by relative quantitative RT-PCR. Two micrograms of total RNA was reversed transcribed using 1µg of Random Primers at 37°C for 1 hour. PCRs with one set of PCR primers were performed using 0.5 units of GoTaq^® DNA Polymerase, 1X reaction buffer, 250µM each dNTP, 0.5µM each primer and 1µl of cDNA. PCRs with two sets of PCR primers were performed using 2.5 units of GoTaq^® DNA Polymerase, 1.6X reaction buffer, 500µM each dNTP, 0.5µM each primer and 1µl of cDNA. (3708)

Notes: The authors developed a multilocus sequence typing scheme (MLST) to examine sequence variation and discriminate between Aspergillus fumigatus strains. They also examined the distribution of MAT1-1 and MAT1-2 sexual idiomorphs in 100 clinical and environmental isolates. Sexual idiomorphs were determined using PCR and a reverse primer to both idiomorphs and a forward primer specific to either MAT-1 or MAT-2. PCRs consisted of 2mM MgCl₂, 200µM DNTPs and 2.5 units of GoTaq^® DNA Polymerase. (3714)

Notes: GoTaq^® DNA Polymerase was used in PCR to test dog blood for the presence of Babesia canis. Genomic DNA isolated from dog blood was analyzed with primers to the variable 5’ region of the Babesia canis 18S rRNA gene. PCR was performed in 50µl reactions containing 1.25 units of GoTaq^® DNA Polymerase and 10µl of GoTaq^® Reaction Buffer. (3367)

Notes: In the process of demonstrating the role of IN in HIV-1 integration in yeast, the authors purified all DNA vectors and PCR products with the Wizard® Plus SV Miniprep System and Wizard® SV Gel System. PCR products were generated using Taq DNA Polymerase. The pGEM®-T Vector was used to clone amplification products. Sequencing was performed using BamHI, religated with T4 DNA Ligase. (3704)

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: 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: 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: 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: In this study, GoTaq^® DNA Polymerase was used in two-step RT-PCR. The ImProm-II™ Reverse Transcription System was first used to produce cDNA using an oligo d(T)₁₅ primer. PCR was then performed using GoTaq^® DNA Polymerase. Each reaction contained 2μl cDNA, 10μl GoTaq^® Reaction Buffer, 1μl dNTP (10mM), 0.2μl GoTaq^® DNA Polymerase, 1μl each primer (10pmol) and 34.8μl nuclease-free water. PCR was performed at 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 60 seconds for 35 cycles, and 72°C for 10 minutes.PCR products were visualized by agarose gel electrophoresis containing ethidium bromide and then sequenced.
(3368)

Notes: Mouse bone marrow cells and calvarial osteoblasts were cocultured for 6 days with or without 50 μM of IBMX. Total RNA was then isolated from the cells and cDNA templates prepared. cDNAs were subjected to PCR amplification with GoTaq^® DNA Polymerase. Primers for mouse PDE4s, TRANCE, CTR, cathepsin K and GAPDH genes were used in this study. The PCR program was as follows: 32 (all mouse PDE4s, TRANCE, CTR, and cathepsin K) or 28 (GAPDH) cycles, after an initial denaturation step at 94°C for 3 minutes, then denaturation at 94°C for 30 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 60 seconds, with a final extension at 72°C for 10 minutes. (3356)

Notes: In this study, GoTaq^® DNA Polymerase was used in amplification reactions to test for the recruitment of DNA damage checkpoint proteins RPA, Rad9, and ATR in ChIP assays. DNA was isolated from cells treated with UV irradiation or acetoxyacetylaminofluorene. The DNA and DNA damage checkpoint proteins RPA, Rad9, and ATR were crosslinked together and the complexes immunoprecipitated with antibodies toward RPA, Rad9, and ATR. Amplification reactions contained primers designed to amplify regions of the p53 and β-globin genes. (3366)

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: 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: 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: 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)