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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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Endocrinology 149, 2306–12. The gastrointestinal hormone ghrelin modulates inhibitory neurotransmission in deep laminae of mouse spinal cord dorsal horn. 2008

Vergnano, A.M., Ferrini, F., Salio, C., Lossi, L., Baratta, M. and Merighi, A.

Notes: The authors investigated the expression pattern of type 1a growth hormone secretagogue receptor (type 1a GHSR), a receptor for ghrelin. In situ RT-PCR was performed on paraformaldehyde-fixed, paraffin-embedded mouse spinal cord tissue. Prior to in situ RT-PCR, tissue sections were treated with proteinase K and triethanolamine, then dewaxed. Reverse transcription was performed using the Reverse Transcription System and oligo (dT)15 primers; followed by amplification using the PCR Master Mix in the presence of 11-digoxygenin-dUTP (1mM). Amplification products were detected using an anti-digoxygenin, alkaline phosphatase-conjugated goat antibody and nitro blue tetrazolium/5-bromo-3-indolylphosphate-p-toluidine salt (NBT/BCIP). (3968)

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Appl. Environ. Microbiol. 74, 2288–97. The genomes of the non-clearing-zone-forming and natural-rubber-degrading species Gordonia polyisoprenivorans and Gordonia westfalica harbor genes expressing Lcp activity in Streptomyces strains. 2008

Bröker, D., Dietz, D., Arenskötter, M. and Steinbüchel, A.

Notes: Natural rubber-degrading bacteria fall into two categories: those forming clearing zones on latex overlay plates and those that do not. To investigate this degradation process, the authors amplified latex-clearing protein (lcp) homologs from non-clearing-zone-forming bacteria using degenerate PCR primers based on lcp sequences from clearing-zone forming species. The 3´ region of the lcp gene in G. westfalica was amplified by nested PCR using biotinylated primers, and the amplified products were cloned in the pGEM®-T Easy Vector and sequenced using universal M13 forward and reverse primers. (3907)

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J. Biol. Chem. 283, 16868–75. The hairless phenotype of the Hirosaki hairless rat is due to the deletion of an 80-kb genomic DNA containing five basic keratin genes. 2008

Nanashima, N., Akita, M., Yamada, T., Shimizu, T., Nakano, H., Fan, Y. and Tsuchida, S.

Notes: The mutation responsible for the hairless phenotype was linked to a 80kb deletion of chromosome 7q36. Because many basic keratin genes are located at 7q36, the authors examined keratin gene expression in the Hirosaki hairless rat using RT-PCR. Expression of kb21, kb23 and kb26 was not detected, whereas other basic keratin genes were expressed. RT-PCR was performed using the AccessQuick™ RT-PCR System and 0.5µg of total RNA isolated from rat skin for 21–30 cycles. (3887)

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Genetics 179, 177-192. The small ubiquitin-like modifier (SUMO) and SUMO-conjugating system of Chlamydomonas reinhardtii. 2008

Wang, Y., Ladunga, I., Miller, A.R., Horken, K.M., Plucinak, T., Weeks, D.P. and Bailey, C.P.

Notes: These authors used computational biology to screen the genome of the alga Chlamydomonas reinhardtii for SUMO (small ubiquitin-like modifier) homologs. They identified several SUMO and SUMO-like sequences. One of these proteins, crSUMO96, which was recognized by the A. thaliana anti-SUMO antibody, was studied in detail. During their studies, the authors used the PureYield™ RNA Midiprep System to isolate total RNA from C. reinhardtii cells. This RNA was used in real-time RT-RCR assays to detect mRNA transcripts for the various SUMO-like proteins. The Plexor® Two-Step qRT-PCR System was used for the real-time assays. For expression studies, cDNA encoding the various proteins was amplified and subcloned into the pGEM®-T Easy Vector before transfer into an expression vector. (3875)

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Eukaryot. Cell 7, 1965–1979. Transcriptome for photobiological hydrogen production induced by sulfur deprivation in the green alga Chlamydomonas reinhardtii. 2008

Nguyen, A.V., Thomas-Hall, S.R., Malnoë, A., Timmins, M., Mussgnug, J.H., Rupprecht, J., Kruse, O., Hankamer, B. and Schenk, P.M.

Notes: The authors analyzed the transcriptional activity of wild-type Chlamydomonas reinhardtii cultures sampled at different time points during the aerobic and anaerobic phase of the photobiological hydrogen production process under sulfur-depleted conditions. C. reinhardtii were grown in photobioreactors, carefully extracted, centrifuged and flash-frozen in liquid nitrogen. RNA was purified using the SV Total RNA Isolation System following the plant centrifugation protocol without sample grinding. The eluted RNA was quantitated and integrity checked by gel electrophoresis and qRT-PCR. Total RNA was used to synthesize labeled cDNA using the ChipShot™ Indirect Labeling and Clean-Up System. The labeled cDNA was used for probing microarrays. (4022)

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Appl. Environ. Microbiol. 74, 1886–91. Use of Drosophila S2 cells as a model for studying Ehrlichia chaffeensis infections. 2008

Luce-Fedrow. A., Von Ohlen, T., Boyle, D., Ganta, R.R. and Chapes, S.K.

Notes: The authors infected Drosophila S2 cells with Ehrlichia chaffeensis to determine if Drosophila is a model system to study E. chaffeensis pathogenesis. E. chaffeensis was also grown in canine macrophage-like DH82 cells, the most common host cell line. Infections were assessed by RT-PCR using the Access RT-PCR System, 0.5–1.0µg of RNA and primers specific to the E. chaffeensis 16S rRNA gene. Housekeeping genes for ribosomal protein 49 and canine glyceraldehyde-3-phosphate dehydrogenase were amplified as control targets for Drosophila and DH82 cells, respectively. Negative controls without reverse transcriptase were performed to be sure that DNA was absent from the RNA samples. (3888)

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Exp. Biol. Med. 232, 1195–1203. 13-cis-Retinoic acid alters intracellular serotonin, increases 5-HT1A receptor, and serotonin reuptake transporter levels in vitro. 2007

O'Reilly, K.C., Trent, S., Bailey, S.J. and Lane, M.A.

Notes: The authors examined the regulatory effect of 13-cis-retanoic acid (13-cis-RA) on genes that encode proteins involved in serotonergic neurotransmission in the RN46A-B14 cell line, which was derived from rat embryonic raphe nuclei. Northern blot analysis was performed to quantitate mRNA levels of these genes in 13-cis-RA-treated and untreated cells. cDNA templates for generating Northern blot probes were synthesized by reverse transcription using the Reverse Transcription System followed by PCR. The Reverse Transcription System was also used in RT-PCR to check for the expression of retinoic acid and retinoid X receptors (RAR and RXR, respectively) in RN46A-B14 cells. Briefly, 1µg of total RNA was treated with DNase, reverse transcribed using oligo (dT) primers, then amplified by PCR using RARα, RARβ, RXRα, RXRβ/γ primers. (3790)

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Anticancer Res. 27, 3843–8. Pamidronate down-regulates urokinase-type plasminogen activator expression in PC-3 prostate cancer cells. 2007

Iguchi, K. et al.

Notes: In this paper, the authors hypothesized that bisphosphonates, which are used to prevent tumor metastasis, affect expression of urokinasetype plasminogen activator (uPA), which seems to be critical for prostate cancer metastasis. The authors examined the effect of several bisphosphonates on uPA expression in PC-3 cells. Pamidronate treatment resulted in lower uPA mRNA levels. To investigate the cause, the authors created a uPA reporter construct (pGL3-uPA) by cloning the 5′-flanking region of the human uPA gene upstream of a firefly luciferase reporter gene in the pGL3-Basic Vector. PC-3 cells were seeded at a density of 3 × 104 cells/well in 24-well culture plates and transfected with 0.5µg of pGL3-uPA and 1ng of the Renilla luciferase phRL-TK Vector using FuGENE® 6 Transfection Reagent. At 48 hours post-transfection, the authors measured reporter activity using the Dual-Luciferase® Reporter Assay System to learn that treatment with 100µM pamidronate inhibited transcription of the uPA gene. (4384)

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Assay Drug Dev. Technol. 5, 237–245. A bioluminescent-based, HTS-compatible assay to monitor G-protein-coupled receptor modulation of cellular cyclic AMP 2007

Kumar, M., Hsiao, K., Vidugiriene, J. and Goueli, S.A.

Notes: The authors of this paper introduce a luminescent assay to monitor changes in cellular cAMP concentration. The assay can be used to study the activity of G-protein coupled receptors that modulate adenylate cyclase activity. The assay is compatible with high-throughput screening in 96-, 384- and 1536-well formats. (3928)

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Mol. Biol. Cell 18, 2795–804. A developmentally regulated chaperone complex for the endoplasmic reticulum of male haploid germ cells. 2007

van Lith, M., Karala, A.R., Bown, D., Gatehouse, J.A., Ruddock, L.W., Saunders, P.T. and Benham, A.M.

Notes: PDILT is a protein disulfide isomerase (PDI) homolog under developmental control and is induced during puberty. To determine whether PDILT expression coincides with the first wave of spermatogenesis, the authors examined expression of PDILT in 2-, 15-, 30-, 45- and 58-day old rats using RT-PCR. Total RNA was isolated from rat testis, and 50ng was amplified using the AccessQuick™ RT-PCR System and primer pairs specific for PDILT, PDI and calmegin, a testis-specific chaperone that is expressed upon the appearance of spermatocytes. Primer pairs were designed to span introns to avoid amplification of genomic DNA. PCR products were analyzed on a 1% agarose gel. Results showed that PDILT mRNA was first detected during the onset of spermatogenesis. (3765)

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Development 134, 2889–2894. A discrete period of FGF-induced Erk1/2 signalling is required for vertebrate neural specification. 2007

Stavridis, M.P., Lunn, J.S., Collins, B.J. and Storey, K.G.

Notes: The authors studied the role of the Erk1/2 signaling pathway during neural specification in mouse embryonic stem (ES) cells. Undifferentiated ES cells express high levels of the pluripotent marker Nanog but do not express fibroblast growth factor (Fgf5), an early marker of differentiation of ES cells, or Sox1, an early neural transcription factor gene. Using quantiative PCR, levels of Nanog, Fgf5 and Sox1 mRNA were quantitated during ES differentiation in the presence and absence of a MEK inhibitor. Prior to quantitative PCR, 1µg of total RNA was reverse transcribed using ImProm-II™ Reverse Transcriptase. By measuring these mRNA levels, the authors determined that inhibition of the Erk1/2 pathway blocked ES differentiation. (3726)

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J. Biol. Chem. 282, 29211–21. A novel CaV1.2 N terminus expressed in smooth muscle cells of resistance size arteries modifies channel regulation by auxiliary subunits. 2007

Cheng, X., Liu, J., Asuncion-Chin, M., Blaskova, E., Bannister, J.P., Dopico, A.M. and Jaggar, J.H.

Notes: The authors identified a novel subunit of the voltage-dependent L-type Ca2+ channel (CaV1.2) with a cysteine-rich N-terminus using rapid amplification of cDNA ends (5´ RACE). The 5´ RACE products were amplified using nested PCR, then cloned into the pGEM®-T Easy Vector and sequenced using the T7 Promoter Primer. (3801)

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J. Virol. Methods 144, 86–90. A rapid DNA hybridization assay for the evaluation of antiviral compounds against Epstein-Barr virus. 2007

Prichard, M.N., Daily, S.L., Jefferson, G.M., Perry, A.L. and Kern, E.R.

Notes: The authors developed an assay to evaluate antiviral compounds for Epstein-Barr virus (EBV) infections. EBV DNA was isolated using the Wizard® SV 96 Genomic DNA Purification System, and 5µl was used for real-time PCR to quantify the viral DNA. Cytotoxicity of the antiviral compounds was assessed using treated, uninfected Akata cells compared to those with no treatment or infection. After three days when the viral DNA was harvested, cell viability was measured using the CellTiter-Glo® Luminescent Cell Viability Assay. (3761)

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Am. J. Pathol. 171, 19–31. A sertoli cell-specific knockout of connexin43 prevents initiation of spermatogenesis. 2007

Brehm, R., Zeiler, M., Rüttinger, C., Herde, K., Kibschull, M., Winterhager, E., Willecke, K., Guillou, F., Lécureuil, C., Steger, K., Konrad, L., Biermann, K., Failing, K. and Bergmann, M.

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)

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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. Immunol. 179, 4829–4839. Aging up-regulates expression of inflammatory mediators in mouse adipose tissue. 2007

Wu, D., Ren, Z., Pae, M., Guo, W., Cui, X., Merrill, A.H. and Meydani, S.N.

Notes: The authors examined the role of ceramide and NF-κB in age-related adipose tissue inflammation in mice. Levels of inflammation-associated molecules, IL-1β, IL-6, TNF-α, COX-2 and peroxisome proliferator-activated receptor (PPAR)-γ mRNA, were quantified by quantitive PCR (qPCR). RNA was isolated from adipose tissues, and first-strand cDNA was synthesized using the Reverse Transcription System prior to qPCR. mRNA levels were also quantified in peritoneal macrophages grown in the presence of young adipocyte-conditioned medium and old adipocyte-conditioned medium. Viability of the primary adipocytes used in these experiments was confirmed using the CellTiter 96® AQueous One Solution Cell Proliferation Assay and CytoTox 96® Non-Radioactive Cytotoxicity Assay. (3777)

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Forensic Sci. Int. 170, 68–72. Allele frequency distribution for 21 autosomal STR loci in Bhutan. 2007

Kraaijenbrink, T., van Driem, G.L., Tshering of Gaselô, K. and de Knijff, P.

Notes: The authors determined the allele frequencies for 21 autosomal STR loci in 936 individuals from the Royal Kingdom of Bhutan using the AmpFlSTR® Identifiler® kit, PowerPlex® 16 System and the F13A01, FESFPS, F13B, LPL (FFFL) Multiplex. DNA was extracted from whole blood samples using the Autopure LS® kit and amplified as directed by the manufacturers. Amplified products were detected using an ABI PRISM® 3100 Genetic Analyzer. (3822)

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Forensic Sci. Int. 168, 227–231. Allele frequency distribution for 21 autosomal STR loci in Nepal. 2007

Kraaijenbrink, T., van Driem, G.L., Opgenort, J.R., Tuladhar, N.M. and de Knijff, P.

Notes: Allele frequency data for 21 autosomal loci was studied in 953 unrelated individuals belonging to 12 major Nepalese groups. DNA was extracted from whole blood using the Autopure LS® kit, then amplified using the PowerPlex® 16 System, AmpFlSTR® Identifiler® kit or F13A01, FESFPS, F13B, LPL Multiplex (FFFL) Multiplex. Amplification products were analyzed using the ABI PRISM® 3100 Genetic Analyzer. Several new alleles not reported in the NIST short tandem repeat database were detected in this population. (3811)

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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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J. Forensic Sci. 52, 1073–6. Characterization of the variant allele 9.2 of Penta D locus. 2007

Miozzo, M.C., Maxzud, M.K., Pacharoni, C.M., Mutal, S.A. and Modesti, N.M.

Notes: During casework analysis using the PowerPlex® 16 System, the authors identified an off-ladder allele at the Penta D locus as the microvariant allele 9.2. DNA from individuals with the 9.2 allele was amplified using a single primer pair specific for Penta D, and the amplification products were purified and sequenced to characterize the microvariant allele. PCR products were purified using the Wizard® PCR Preps DNA Purification System. Sequence analysis revealed that the 9.2 allele has 10 STR repeats and a TAA deletion in the 3´ flanking region. (3771)

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Proc. Natl. Acad. Sci. USA 104, 10637–10642. Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization. 2007

Rendón, M.A., Saldaña, Z., Erdem, A.L., Monteiro-Neto, V., Vázquez, A., Kaper, J.B., Puente, J.L. and Girón, J.A.

Notes: The authors identified an adherence factor of enterohemorrhagic E. coli that is involved in colonization of cultured epithelial cells. This factor, named E. coli common pilus (ECP), is encoded by the ecpA gene, which is present 96% of E. coli strains tested, as determined by PCR. The remaining 4% of the strains were found to be deficient in the ECP operon, as determined by multiplex PCR amplification of ecpR, ecpA, epcB and ecpC sequences. PCR were performed using GoTaq® Green Master Mix. An ecpA deletion mutant exhibited impaired adherence compared to the wildtype E. coli strain. Complementation of the mutant strain with the plasmid pMR13, the pGEM®-T Vector cotaining the ecpA gene, restored the strain's ability to adhere to epithelial cells. (3719)

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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, 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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