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Mol. Cell. Endocrinol. 264, 50-60. Novel estrogen receptor beta transcript variants identified in human breast cancer cells affect cell growth and apoptosis of COS-1 cells. 2007

Treeck, O., Pfeiler ,G., Horn, F., Federhofer, B., Houlihan, H., Vollmer, A., and Ortmann, O.

Notes: This study identified two novel transcript variants of the estrogen receptor ERβ that were expressed in the ERα-negative breast cancer cell line MDA-MD-231. These variants were identified after amplification of ERβ transcripts from the breast cancer cell line by RT-PCR. The amplification products were then excised from gels and subcloned into the pTARGET™ Mammalian Expression Vector prior to sequencing. COS1 cells, which do not express the estrogen receptor, were then stably transfected with full-length ERβ or one of the splice variants and the effects on cell proliferation, apoptosis, and estrogen response were evaluated. In COS1 cells expressing either ERβ or the transcript variants cell proliferation decreased and basal apoptosis (caspase 3/7 activity) increased, compared to cells transfected with vector alone. Exposure to therapeutic doses of tamoxifen induced apoptosis in cells expressing the full-length ERβ but not in cells expressing either of the variant isoforms. (3618)

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Clinica Chimica Acta 381, 171-175. Microsatellite mutation in the maternally/paternally transmitted D18S51 locus: two cases of allele mismatch in the child. 2007

Narkuti, V., Vellanki, R.N., Gandhi, K.P., Doddapaneni, K.K., Yelavarthi, P.D. and Mangamoori, L.N.

Notes: The authors describe two cases of paternity dispute, one with a maternally mismatched allele at the D18S51 locus and a second with a paternally mismatched D18S51 allele. Seventeen autosomal STR loci were analyzed using the PowerPlex® 16 System and AmpFlSTR® Identifiler® kit. Amplifications were performed using a GeneAmp® PCR System 9700, and amplification products were detected using an ABI PRISM® 310 Genetic Analyzer. Y-STR loci and mitochondrial DNA hypervariable regions HV1 and HV2 were also examined. Sequence analysis of the D18S51 locus revealed an expansion of the maternal allele by one repeat unit in one case and an expansion of the paternal allele by two repeat units in the second case. (3809)

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J. Forensic Sci. 52, 870–3. Concordance study between the AmpFlSTR MiniFiler PCR amplification kit and conventional STR typing kits. 2007

Hill, C.R., Kline, M.C., Mulero, J.J., Lagacé, R.E., Chang, C.W., Hennessy, L.K. and Butler, J.M.

Notes: The authors analyzed 1,308 samples for concordance between the Identifiler® kit, AmpFlSTR® Minifiler™ kit and PowerPlex® 16 System. DNA was isolated from liquid blood using the manual DNA IQ™ System protocol, and STR amplifications were performed as per the manufacturer's recommendations except that reaction volumes were decreased by half. Amplified products were analyzed using an Applied Biosystems 3130xl and POP™-4 or POP™-6 polymer. Twenty seven disconcordant phenotypes were identified between the Minifiler™ and Identifiler® kits, 14 between Minifiler™ and PowerPlex® 16 kits, and 4 between PowerPlex® 16 and Identifiler® kits. (3770)

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J. Biol. Chem. 282, 29847–29854. Differential regulation of vitamin D receptor (VDR) by the p53 family: p73-dependent induction of VDR upon DNA damage. 2007

Kommagani, R., Payal, V. and Kadakia, M.P.

Notes: The authors examined transcriptional regulation of the vitamin D receptor (VDR) by p53 and p63, a member of the p53 family, under stressed and unstressed conditions. Reporter constructs with the full-length and minimal VDR promoters controlling expression of firefly luciferase were cotransfected with p53 or p63 expression constructs, and transcriptional activation of the VDR promoter was monitored using the Dual-Luciferase® Reporter 100 Assay System. Results were normalized to Renilla luciferase activity. Interaction between p73, another member of the p53 family, and the VDR promoter was examined using chromatin immunoprecipitation. The imunnopreciptated chromatin was reverse crosslinked, DNA was eluted and VDR and p21 sequences were detected by PCR using GoTaq® Green Master Mix. (3715)

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J. Biomol. Scr. 12, 546–559. Optimization procedure for small interfering RNA transfection in a 384-well format. 2007

Borawski, J., Lindeman, A., Buxton, F., Labow, M. and Gaither, L.A.

Notes: A lentiviral expression vector containing the firefly luciferase gene from a pGL3 Vector was transduced into SKOV3 cells in 384-well plates, transfected with various siRNAs and analyzed 72 hours later. The luciferase expression was determined using the Bright-Glo™ Luciferase Assay System and cell viability assessed using the CellTiter-Glo® Luminescent Cell Viability Assay. (3729)

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Assay Drug Dev. Technol. 5, 127–136. Bioluminescent assays for high-throughput screening 2007

Fan, F. and Wood, K.V.

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

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J. Biol. Chem. 282, 9883–94. Cell confluence-induced activation of signal transducer and activator of transcription-3 (Stat3) triggers epithelial dome formation via augmentation of sodium hydrogen exchanger-3 (NHE3) expression. 2007

Su, H.W., Yeh, H.H., Wang, S.W., Shen, M.R., Chen, T.L., Kiela, P.R., Ghishan, F.K. and Tang, M.J

Notes: The authors tested their hypothesis that Na+-H+ exchangers (NHE) are involved in the formation of multicullar dome structures in confluent Madin-Darby canine kidney (MDCK) cells and that the Stat3 pathway is involved in regulation of NHEs. The authors performed semi-quantitative RT-PCR to monitor NHE3 mRNA levels in MDCK cells expressing a constitutive Stat3 mutant or a dominant-negative Stat3 mutant. The reverse transcription step was performed using Promega M-MLV Reverse Transcriptase. RAlso, Stat3 activities in low-density cultures and high-density cultures were compared using a reporter gene assay. Four copies of the Stat3-binding site were cloned upstream of a firefly luciferase reporter gene, and the resulting vector, along with the pRL-TK Vector for normalization, were transfected into MDCK cells. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System. (3910)

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Nucl. Acids Res. 35, 1245-1256. Dual role of DNA methylation inside and outside of CTCF-binding regions in the transcriptional regulation of the telomerase hTERT gene 2007

Renaud, S., Loukinov, D., Abdullaev, Z., Guilleret, I., Bosman, F.T., Lobanenkov, V. and Benhattar, J.

Notes: Telomeres shorten by 50–100 bases with each cell division, making the telomere a "mitotic counter" that can limit cellular lifespan. Telomerase is a two-component protein consisting of a reverse transcriptase (hTERT) bound to its own RNA template that can act to maintain telomere length in dividing cells. Telomerase is highly active in dividing cells such as germ cells, stem cells and many cancers. This paper investigated the role of methylation of the hTERT promoter and the transcription factor CTCF in regulation of telomerase activity. LacZ reporter plasmids driven by the hTERT minimal promoter were transiently transfected into HeLa cells, and reporter assays were performed on lysate generated using Passive Lysis Buffer. The hTERT minimal promoter did not show activity if all of the CpG sites were methylated. The promoter and first exon of hTERT were amplified using PCR Master Mix from sodium bisulfite-treated genomic DNA isolated from telomerase-positive cell lines and tissues. The resulting fragments were cloned using the pGEM®-T Vector System II. For the methylation cassette assay, methylated and unmethylated fragments were cloned into a methylated or unmethylated vector using the LigaFast™ Rapid DNA Ligation System. The authors conclude that methylation plays a dual role in regulating hTERT expression. CTCF will bind to the first exon of hTERT when the hTERT CpG island is not methylated, resulting in downregulation of hTERT expression. Although CTCF cannot bind the hTERT promoter when the DNA is completely methylated, the methylation itself completely represses transcription. In situations where there is partial methylation of the promoter, such as in tumor cells, CTCF cannot bind to the promoter, but the partial methylation is not enough to repress transcription, and hTERT is expressed. (3641)

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J. Biol. Chem. 282, 10953–10962. Evidence for 1,25-dihydroxyvitamin D3-independent transactivation by the vitamin D receptor: uncoupling the receptor and ligand in keratinocytes. 2007

Ellison, T.I., Eckert, R.L. and MacDonald, P.N.

Notes: While the absence of the Vitamin D receptor (VDR) has profound effects in skin cells, mutation of 25-hydroxyvitamin D 1α-hydroxylase (24OHase), the enzyme required for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone biosynthesis, has little effect on the skin. To determine how VDR may transactivate independent of the 1,25(OH)2D3 ligand, the human 24-hydroxylase promoter was amplified from MCF-7 genomic DNA, digested with XhoI and HindIII and inserted into the pGL3-Basic Vector. Mutations in the proximal and distal vitamin D response elements in the human 24-hydroxylase promoter were introduced using the GeneEditor™ Site-Directed Mutagenesis System. HaCaT cells, primary human fibroblasts or primary human keratinocytes were seeded at a density of 3.2 × 104 cells/well in 12-well plates and transiently transfected with reporter constructs. After 18 hours, the cells were exposed to 1,25(OH)2D3, 9-cis-retinoic acid, ethanol vehicle, or no additive and harvested 24 hours later. The luciferase activity of the cell lysates was measured using the Dual-Luciferase® Reporter Assay System. Five micrograms of RNA purified from mouse keratinocyte and fibroblast cultures was reverse transcribed and amplified for the 24OHase transcripts using the PCR Master Mix. The products were analyzed on ethidium bromide-stained 2% agarose gels. (3695)

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Nucl. Acids Res. 35, 2390–2402. Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance. 2007

Wu, J., Apontes, P., Song, L., Liang, P., Yang, L. and Li, F.

Notes: To study how Hoechst33342 upregulates the expression and promoter activity of survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, nested deletions of the survivin promoter driving a firefly luciferase reporter gene (pLuc-1430c ) were created. The vector was digested with SalI, the ends filled in using α-phosphorothioate dNTPs, digested a second time with BamHI and subjected to Exonuclease III digestion at 25°C. Aliquots of the 5’ end deletions were removed every 15–30 seconds, religated, transformed and analyzed by PCR and sequencing. Transient transfection experiments were carried out using HeLa cells seeded in 24-well plates and cotransfected 490ng of a pLuc-survivin construct and 10ng of pRL-TK Vector or in U937 cells using 2µg of survivin promoter constructs. After 24 hours, the HeLa cells were treated with Hoechst33342 and harvested 8–24 hours later. For U937 cells, the medium was changed with or without added drugs and the cells lysed after 36 hours. Reporter expression was assessed using the Dual-Luciferase® Reporter Assay System. (3697)

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Cancer Res. 67, 1472-1486. Adaptation of energy metabolism in breast cancer brain metastases. 2007

Chen, E.I., Hewel, J., Kreuger, J.S., Tiraby, C., Weber, M.R., Kralli, A., Becker, K., Yates, J.R., and Felding-Habermann, B.

Notes: This study investigated protein expression profiles in tumors from breast cancer brain metastases. Circulating tumor cells were isolated from a patient with stage IV breast cancer and injected into SCID mice. Tumor cells were then recovered from brain and bone lesions that subsequently developed in these mice. The protein expression profiles of the parent cell line were then compared with those from brain and bone tumors. More than 300 proteins that were up- or down-regulated in the brain tumor cells were identified. Classification of these proteins by function revealed that the majority were associated with cellular metabolism. Sixty-three differentially expressed proteins, including mainly cellular redox-active proteins and proteins involved in glucose and fatty acid oxidation, were selected for further study. Based on the expression data, a metabolic profile of brain metastatic cells was generated. Up-regulation of genes involved in oxidative energy metabolism as indicated by the proteomic analysis was confirmed by quantitative real-time PCR analysis. Consistent with the observation of increased glycolysis and oxidative phosphorylation, the authors also found that levels of cellular ATP were increased in cells from brain metastases. The CellTiter-Glo® Luminescent Cell Viability Assay was used to measure ATP levels in the primary, bone, and brain-derived tumor cells. The authors suggest that adaptation of the tumor cell energy metabolism is a key development in breast cancer brain metastasis. (3613)

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J. Appl. Microbiol. 98, 1001-1009. Detection of lactococcal 936-species bacteriophages in whey by magnetic capture hybridization PCR targeting a variable region of receptor-binding protein genes. 2006

Dupont, K., Vogensen, F.K., and Josephsen, J.

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)

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Appl. Environ. Microbiol. 72, 2539-2546. Cloning and sequencing of the ompA gene of Enterobacter sakazakii and development of an ompA-targeted PCR for rapid detection of Enterobacter sakazakii in infant formula. 2006

Mohan Nair, M.K. and Venkitanarayanan, K.S.

Notes: The outer membrane protein A (ompA) gene of Enterobacter sakazakii was amplified using PCR primers based on E. coli ompA sequences. The resulting PCR product was ligated into the pGEM®-T Easy Vector, and the sequence was confirmed. The ompA sequence was used to develop a PCR for detection of Enterobacter sakazakii in reconstituted infant formula. (3464)

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Exp. Parasitol. 112, 63-65. Babesia canis vogeli: A novel PCR for its detection in dogs in Australia. 2006

Martin, A.R., Dunstan, R.H., Roberts, T.K., and Brown, G.K.

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)

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Appl. Environ. Microbiol. 72, 5463–5468. Comparative, collaborative, and on-site validation of a TaqMan® PCR method as a tool for certified production of fresh, campylobacter-free chickens. 2006

Krause, M., Josefsen, M.H., Lund, M., Jacobsen, N.R., Brorsen, L., Moos, M., Stockmarr, A. and Hoorfar, J.

Notes: To test if a real-time PCR assay would be able to detect Campylobacter spp. in various chicken samples, several laboratories were involved in a collaborative trial. Each lab was given 18 1ml samples, including 6 chicken neck skin samples, 6 shoe cover fecal samples and 6 cloacal swab samples. DNA was extracted from these samples using the MagneSil® KF, Genomic System, first tested using 20µl of paramagnetic particles (PMPs) and then using 75µl PMPs. Five microliters of purified DNA was used in real-time PCR analysis. (3558)

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J. Clin. Microbiol. 44, 3285-3291. Rapid and sensitive detection of single Cryptosporidium oocysts form archived glass slides. 2006

Sunnotel, O., Snelling, W.J., Xiao, L., Moule, K., Moore, J.E., Millar, B.C., Dooley, J.S.G. and Lowery, C.J.

Notes: These researchers used laser-capture microscopy followed by real-time PCR to detect and identify Cryptosporidium oocysts in stained fecal smears and water samples on glass slides. After microdissection of single oocysts or groups of oocysts from the stained slides, DNA was extracted and real-time PCR performed using primers specific for the cryptosporidial 18s rRNA gene. To confirm primer specificity and the identity of the real-time PCR products, the amplimers were recovered from the LightCycler® capillaries at the end of each each real-time experiment. Products were then separated on agarose gels and purified using the Wizard® SV Gel and PCR System prior to sequencing using a BigDye® terminator cycle sequencing kit from Applied Biosystems. (3532)

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J. Clin. Microbiol. 44, 3878–82. Detection of multiple noroviruses associated with an international gastroenteritis outbreak linked to oyster consumption. 2006

Le Guyader, F.S., Bon, F., DeMedici, D., Parnaudeau, S., Bertone, A., Crudeli, S., Doyle, A., Zidane, M., Suffredini, E., Kohli, E., Maddalo, F., Monini, M., Gallay, A., Pommepuy, M., Pothier, P. and Ruggeri, F.M.

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)

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Clin. Chem. 52, 2250–2257. Sensitive detection of KIT D816V in patients with mastocytosis. 2006

Tan, A., Westerman, D., McArthur, G.A., Lynch, K., Waring, P. and Dobrovic, A.

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)

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Anal. Bioanal. Chem. 385, 1045-1054. Effective PCR detection of animal species in highly processed animal byproducts and compound feeds. 2006

Fumière, O., Dubois, M., Baeten, V., von Holst, C. and Berben, G.

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)

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Pesticide Biochem. Physiol. 84, 236-242. Deletion of Cyp6d4 does not alter toxicity of insecticides to Drosophila melanogaster. 2006

Hardstone, M.C., Baker, S.A., Gao, J., Ewer, J., and Scott, J.G.

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)

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Appl. Environ. Microbiol. 72, 6070-6078. An oxidoreductase is involved in cercosporin degradation by the bacterium Xanthomonas campestris pv. zinniae. 2006

Taylor, T.V., Mitchell, T.K. and Daub, M.E.

Notes: Fungi of the genus Cercospora are plant pathogens that cause leaf spot and blight diseases, and produce the polyketide toxin cercosporin. The bacterium Xanthomonas campestris is able to rapidly degrade cercosporin. In this study, X. campestris mutants unable to degrade cercosporin were created by chemical mutagenesis. Complementation studies with a plasmid-based library of X. campestris DNA showed that the ability to degrade cercosporin was restored upon transformation with plasmids containing an oxidoreductase gene and a putative transcriptional regulator. These genes were then amplified from the mutant strains by high-fidelity PCR. The PCR products were separated by agarose gel electrophoresis, purified using the Wizard® SV Gel and PCR Clean-Up System, and subcloned into the pGEM®-T Easy Vector. The mutant genes were then sequenced to identify the nature of the mutations. (3531)

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J. Clin. Microbiol. 44, 2750–2759. Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification. 2006

Zucol, F., Ammann, R.A., Berger, C., Aebi, C., Altwegg, M., Niggli, F.K., and Nadal, D.

Notes: A panel of 11 Gram-negative and 11 Gram-positive bacterial species was used to develop a real-time PCR detection method. Initially DNA was purified from 1ml of various dilutions of bacteria resuspended in a saline solution using either the QIAamp DNA blood mini kit or the Wizard® SV Genomic DNA Purification System. The results suggested that the Wizard® SV Genomic DNA Purification System extraction protocol was superior in disrupting the bacterial cell wall (especially of Gram-positive bacteria), to allow release of bacterial DNA. Using DNA purified with the Wizard® SV Genomic DNA Purification System, detection of S. aureus and E. coli at concentrations as low as 101 CFU per PCR was achieved. The Wizard® SV Genomic DNA Purification System purification protocol provided in eNotes online (www.promega.com/enotes/applications/ap0051_tabs.htm) was used with the following modifications: The bacterial pellet was resuspended in 400µl of enzymatic lysis solution (47mM EDTA, 25mg/ml lysozyme, 20µg/ml lysostaphin) and incubated for 2 hours at 37°C. Next, 19.2mg/ml proteinase K was added (final concentration 0.4mg/ml), and the mixture was incubated for 1 hour at 55°C. Finally, the Nuclei Lysis Solution and RNase Solution were added, mixed and incubated for 10 minutes at 80°C. For real-time PCR analysis, 2µl of genomic DNA was used. (3676)

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Protein Expr. Purif. 47, 562–570. High efficiency single step production of expression plasmids from cDNA clones using the Flexi Vector cloning system. 2006

Blommel, P.G., Martin, P.A., Wrobel, R.L., Steffen, E. and Fox, B.G.

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)

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Plant Sci. 170, 705-14. Expression profile analysis and biochemical properties of the peptide methionine sulfoxide reductase A (PMSRA) gene family in Arabidopsis. 2006

Romero, H.M., Pell, E.J. and Tien, M.

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)

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J. Med. Microbiol. 55, 273–277. Development of a routine laboratory direct detection system of staphylococcal enterotoxin genes. 2006

Nakayama, A., Okayama, A., Hashida, M., Yamamoto, Y., Takebe, H., Ohnaka, T., Tanaka, T. and Imai, S.

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)

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