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J. Biomol. Scr. 15, 418–26. Epitope mapping of antibodies using a cell array-based polypeptide library. 2010

Maier, R.H., Maier, C.J., Rid, R., Hintner, H., Bauer, J.W. and Onder, K.

Notes: The authors developed a high-density protein array using a recombinant peptide library to map the epitope recognized by a commercially available anti-vitamin D receptor (VDR) monoclonal antibody. By screening 2304 overlapping VDR peptides, they were able to identify the 37-amino-acid epitope. The library was created by amplifying the 1.2kb VDR coding region, cleaning the PCR product with the Wizard® SV Gel and PCR Clean-Up System, sonicating the PCR product, then cloning the VDR fragments into a bacterial expression vector that confers a glutathione-S-transferase (GST) tag. The epitope was verified by showing that the 37-amino-acid sequence was recognized in Western blot analysis and enzyme-linked immunosorbent assay (ELISA); the full-length VDR, also expressed as GST fusion protein, was used as a positive control. These GST fusion proteins were purified using the MagneGST™ Protein Purification System. (4154)

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Methods in Mol. Biol. 577, 25-39. High-Throughput Construction of ORF Clones for Production of the Recombinant Proteins 2009

Yamakawa, Hisashi

Notes: The authors use the Flexi® Cloning System to convert their cDNA clones to expression-ready clones. They wanted clones that could be used for comprehensive analysis with the HaloTag® Technology. They also describe a method of transfereing ORFs between Flexi® Vectors in a 96-well plate format. They also used Wizard® SV 96 Plasmid DNA Purification, Wizard® SV PCR Clean-Up, and Wizard® SV Gel and PCR Clean-Up Systems. (4056)

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Proc. Natl. Acad. Sci. USA 105, 8914-8919. An epoxide hydrolase involved in the biosynthesis of an insect sex attractant and its use to localize the production site. 2008

Abdel-Latief, M., Garbe, L.A., Koch, M., and Ruther, J.

Notes: These authors amplified and characterized a putative epoxide hydrolase gene from the jewel wasp Nasonia vitripennis. PCR fragments were amplified from genomic DNA, purified from gels using the Wizard® SV Gel and PCR Clean Up System and then subcloned into the pGEM®-T Easy Vector. The plasmid DNA was purified using the PureYield™ Midiprep System. Linearized plasmids were used for in vitro transcription of RNA for use in RNA interference experiments. (3903)

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BMC Genomics 9, 315. The complete mitochondrial genome of the Antarctic springtail Cryptopygus antarcticus (Hexapoda: Collembola). 2008

Carapelli, A., Comandi, S., Convey, P., Nardi, F. and Frati, F.

Notes: To sequence the mitochondrial genome one of the most widespread and common collembolan species of Antarctica, springtail Cryptopygus antarcticus. Specimens were collected from Killingbeck I during a 2002 polar expedition and frozen in liquid nitrogen. The Wizard® SV Genomic Purification System was used to extract total DNA from the samples and the complete mitochondrial genome was amplified twice, first with universal primers and sequenced, and then using long PCR with specific primers. The long PCR products were mechanically sheared, blunt end repaired and purified using the Wizard® SV Gel and PCR Clean-Up System. The fragments were then cloned, transformed and sequenced. (3976)

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J. Biol. Chem. 282, 21798–21809. Control and regulation of KplE1 prophage site-specific recombination: a new recombination module analyzed. 2007

Panis, G., Méjean, V. and Ansaldi, M.

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)

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Plant Physiol. 145, 547–558. Diversity of acetyl-coenzyme A carboxylase mutations in resistant Lolium populations: Evaluation using clethodim. 2007

Yu, Q., Collavo, A., Zheng, M.Q., Owen, M., Sattin, M. and Powles, S.B.

Notes: The authors characterized mutations in the acetyl-coenzyme A carboxylase (ACCase) gene that confer resistance to the herbicide clethodim in the grass weed Lolium rigidum. The ACCase gene was amplified from clethidem-resistant and susceptible plants, then sequenced to identify previously unknown mutations. Amplifications of ACCase were performed using 300ng of genomic DNA and GoTaq® Green Master Mix. The Wizard® SV Gel and PCR Clean-Up System was used to purify PCR products directly or from agarose gels. (3721)

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Emerging Infect. Dis. 13, 1756-1758. Human Bocavirus infection in children with gastroenteritis, Brazil. 2007

Albuquerque, M.C., Rocha, L.N., Benati, F.J., Soares, C.C., Maranhão, A.G., Ramírez, M.L, Erdman, D., and Santos, N.

Notes: In this study, the Wizard® Genomic DNA Purification Kit was used to extract DNA from diluted fecal samples. The extracted DNA was used in PCR with specific primers to detect viral sequences. PCR fragments were gel-purified using the SV Gel and PCR Clean-Up System prior to sequencing to confirm the Bocavirus DNA identity. (4221)

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Brain Res. 1127, 66–75. Molecular characterization and gene expression of the pituitary adenylate cyclase-activating polypeptide (PACAP) in the lizard brain. 2007

Valiante, S., Prisco, M., Capaldo, A., Zambrano, I., De Falco, M., Andreuccetti, P., Laforgia, V., and Varano, L.

Notes: The authors cloned pituitary adenylate cyclase-activating polypeptide (PACAP) from lizard (Podarcis sicula) brain. They then isolated total RNA from lizard brain using the SV Total RNA Isolation System and used 4µg of total RNA in a reverse transcription with ImProm-II™ Reverse Transcriptase and oligo(dT)15 primers at 37°C for 1.5 hours. The PACAP cDNA was amplified by PCR, and the resulting PCR products were cleaned up using the Wizard® SV Gel and PCR Clean-Up System prior to sequencing. (3666)

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J. Endocrinol. 197, 201–12. Neonatal exposure to bisphenol A modifies the abundance of estrogen receptor alpha transcripts with alternative 5'-untranslated regions in the female rat preoptic area. 2007

Monje, L., Varayoud, J., Luque, E.H. and Ramos, J.G.

Notes: The authors investigated the effect of neonatal bisphenol A (BPA) exposure in rats on expression of estrogen receptor α (ERα) transcripts. Alternative ERα transcripts in preoptic area of treated and untreated rats were quantified using real-time RT-PCR. Reverse transcription was performed using 4µg of total RNA, 200pmol random primers and 300 units M-MLV Reverse Transcriptase. Real-time PCR was performed using SYBR® Green I to quantify amplified products. To determine if the changes in BPA-induced ERα transcript expression were caused by DNA methylation, the methylation status of the five ERα promoters was examined by bisulfite modification. Genomic DNA was isolated from rat tissue using the Wizard® Genomic DNA Purification Kit, denatured with NaOH, then treated with hydroquinone and sodium bisulfite. Prior to methylation-specific PCR, DNA was cleaned up using the Wizard® DNA Purification Resin as directed by the manufacturer. PCR products were cleaned up again using the Wizard® SV Gel and PCR Clean-Up System, then subjected to restriction enzyme digestion and agarose gel electrophoresis to reveal methylation-dependent sequence differences. (3911)

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J. Biol. Chem. 282, 14194-14204. Regulation of the interleukin-7 receptor α-promoter by the Ets transcription factors PU.1 and GA-binding protein in developing B cells. 2007

Dekoter, R.P., Schweitzer, B.L., Kamath, M.B., Jones, D., Tagoh, H., Bonifer, C., Hildeman, D.A., and Huang, K.J.

Notes: The interleukin-7 receptor is composed of γ and α subunits, encoded by the genes il7rg and il7r, respectively. The α subunit is expressed in developing B cells and is downregulated upon maturation. These authors investigated the mechanisms of transcriptional regulation of the il7r gene using 5´ RACE, EMSA, RNA interference and chromatin immunoprecipitation analyses. Potential promoter regions identified by 5´ RACE analysis were cloned into the pGL3-Basic luciferase reporter vector for further study. The promoter constructs were transiently transfected into the 38B9 pro-B cell line along with the control pRL-TK Vector, which expresses Renilla luciferase, and the Dual-Luciferase® Reporter Assay System was used to assess luciferase activity from the various promoter constructs. The promoter construct having the highest activity was chosen, and site directed mutagenesis was used to identify specific regions within the promoter fragment that may be important for activity. Sequence analysis was then used to identify a conserved Ets transcription factor binding site within the putative il7r promoter region. To determine whether the ETS transcription factor GABP binds to this Ets region, the authors performed chromatin immunoprecipitation analysis with an anti-GABP antibody. Immunoprecipitated DNA was then PCR-amplified with primers specific for the Ets region or control primers. The Wizard® SV Gel and PCR Clean-Up System was used to purify the amplified fragments prior to semiquantitative PCR analysis. (3626)

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Genetics 175, 1047-1058. Single-gene detection and karyotyping using small-target fluorescence in situ hybridization on maize somatic chromosomes. 2007

Lamb, J.C., Danilova, T., Bauer, M.J., Meyer, J.M., Holland, J.J., Jensen, M.D., and Birchler, J.A.

Notes: These authors generated a set of probes that could be used in fluorescence in situ hybridization (FISH) analyses for karyotyping studies on maize chromosomes. Specific target regions composed of genes or gene clusters and free from repetative elements were identified for each chromosome. Target regions were amplified by PCR, gel purified using the Wizard® SV Gel and PCR Clean-Up System, and tested in a FISH assay. Probes showing low background were selected, subcloned into the pGEM® -T Vector and sequenced to confirm identity. (3627)

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Appl. Environ. Microbiol. 73, 2860-2870. The microbial community structure in petroleum-contaminated sediments corresponds to geophysical signatures. 2007

Allen, J.P., Atekwana, E.A., Atekwana, E.A., Duris, J.W., Werkema, D.D., and Rossbach, S.

Notes: These authors studied microbial community structure at various locations in an aged underground petroleum plume. DNA was purified from soil samples collected from different sites within a contaminated area. 16S rRNA genes were then amplified from the isolated DNA, and the PCR products were run on a gel and purified using the Wizard® SV Gel and PCR Clean-Up System. After subcloning into a TA vector, the 16S RNA genes were sequenced and used to identify the various Phyla represented and characterize the microbial populations present throughout the site. (3625)

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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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Nucl. Acids Res. 34, 6215-6224. Chromosomal integration of LTR-flanked DNA in yeast expressing HIV-1 integrase: down regulation by RAD51 2006

Desfarages, S., San Filippo, J., Fournier, M. Calmels, C., Caumont-Sarcos, A., Litvak, S., Sung, P., Parissi, V.

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)

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Infect. Immun. 74, 3825-3833. Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of insertional mutants. 2006

Shin, S.J., Wu, C-W., Steinberg, H. and Talaat, A.M.

Notes: In this study, insertional mutagenesis with the transposon TN5367 was used to generate a library of M. paratuberculosis mutants. Sequences containing transposons were then amplified, gel purified using the Wizard® SV Gel and PCR Clean-Up System, and cloned into the pGEM®-T Easy Vector prior to sequencing. Bioinformatic screening was then used to identify potential virulence determinants for further study in a mouse model of M. paratuberculosis infection. (3534)

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J. Biol. Chem. 281, 13915–13921. NDR2 acts as the upstream kinase of ARK5 during insulin-like growth factor-1 signaling. 2006

Suzuki, A., Ogura, T. and Esumi, H.

Notes: A deletion mutation of the serine/threonine protein kinase NDR2 was created by PCR using two mutagenesis primers and two plasmid-based primers. After amplification, the two products were run on a 1% agarose gel and extracted using the Wizard® SV Gel and PCR Clean-Up System. The purified fragments were mixed, annealed, re-amplified and then digested prior to cloning into an expression vector. The human colorectal cancer cell lines HCT-116, DLD-1, and SW480 used in the study were seeded into a 24-well plate at 5 × 104/well, and transfected using the TransFast™ Transfection Reagent. The transfection was assessed with a green fluorescent protein expression vector. (3438)

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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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Nucl. Acids Res. 34, 2109–2116. The Drosophila termination factor DmTTF regulates in vivo mitochondrial transcription. 2006

Roberti, M., Bruni, F., Polosa, P.L., Gadaleta, M.N. and Cantatore, P.

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)

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J. Bacteriol. 187, 8026-8038. Comparative Analysis of Two Classes of Quorum-Sensing Signaling Systems that Control production of Extracellular Proteins and Secondary Metabolites in Erwinia Carotovora Subspecies 2005

Chatterjee, A., Cui, Y. Hasegawa, H., Leigh, N., Dixit, V., Chatterjee, A.K.

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)

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Appl. Environ. Microbiol. 70, 2779–2785. Acquisition of an Agrobacterium Ri plasmid and pathogenicity by other α-Proteobacteria in cucumber and tomato crops affected by root mat. 2004

Weller, S.A., Stead, D.E. and Young, J.P.W.

Notes: Researchers used the Wizard® Genomic DNA Purification Kit to isolate genomic DNA from non-Agrobacterium field bacteria samples. The isolated genomic DNA was used in PCR to amplify regions of the 16S rRNA gene. The PCR products were cleaned up using the Wizard® SV Gel and PCR Clean-Up System and used in sequencing reactions. The paper also mentions the use of the Wizard® Magnetic DNA Purification System for Food to isolate Agrobacterium radiobacter from cucumber root mats grown in the lab. (3190)

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Biochem. Biophys. Res. Commun. 321(1), 259-265. Cloning of hOST-PTP: the only example of a protein-tyrosine-phosphatase the function of which has been lost between rodent and human 2004

Cousin, W., Courseaux, A., Ladoux, A., Dani, C., and Peraldi, P.

Notes: Researchers used GoTaq® DNA polymerase to amplify 139bp and 815bp regions of hOST-PTP cDNA for detection and probe synthesis. The full-length 4006bp cDNA was amplified with Pfu DNA Polymerase. Fragments were gel purified using the Wizard® SV Gel and PCR Clean-Up System prior to cloning into the pGEM®-T Easy Vector.  The Prime-a-Gene® Labeling System was used to make 32P-dCTP labeled probes, which were used to screen cDNA clones. (3111)

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J. Biol. Chem. 278 (39), 37937–37947. Cellular prostaglandin E2 production by membrane-bound prostaglandin E synthase-2 via both cyclooxygenases-1 and -2. 2003

Murakami, M., Nakashima, K., Kamei, D., Masuda, S., Ishikawa, Y., Ishii, T., Ohmiya, Y., Watanabe, K. and Kudo, I.

Notes: The Wizard® SV Gel and PCR Clean-Up System was used to purify a mutant ~870bp prostaglandin E synthase (PGES) PCR product. The purified fragment was then subcloned into the pLenti6/V4-D-TOPO vector and sequenced. (2749)

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J. Nanobiotechnology 1:2. mRNA analysis of single living cells. 2003

Osada, T., Uehara, H., Kim, H. and Ikai, A.

Notes: Researchers used the Wizard® SV Gel & PCR Clean-Up System to purify rat c-fos RT-PCR products that were then quantified by A260 absorbance. These products were used as standards for quantitative PCR reactions. The researchers used an Applied Biosystems Prism 7000 sequence detection system to perform the 50μl quantitative PCR reactions. (2678)

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Ann. Clin. Microbiol. Antimicrob. 2, 11. Mutations in the 23S rRNA gene are associated with clarithromycin resistance in Helicobacter pylori isolates in Brazil. 2003

Ribeiro, M.L., Vitiello, L., Miranda, M.C., Benvengo, Y.H., Godoy, A.P., Mendonca, S. and Pedrazzoli Jr., J.

Notes: The Wizard® SV Gel and PCR Clean-Up System was used to purify a 280 base-pair PCR product, which was then directly sequenced. (2791)

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