Notes: Nuclear transfer (NT) embryos (macaque fibroblasts introduced into matured metaphase II stage rabbit oocytes) were placed into PCR tubes containing 10µl of lysis solution, ReadyAmp™ Genomic DNA Purification System resin and 200µg/ml proteinase K. To break open the cells and access the genomic DNA, the embryos were incubated for 30 minutes at 55°C, boiled for 10 minutes, centrifuged for 1 minute then used for PCR. The amplified DNA was then cloned into the pGEM^®-T Easy Vector and sequenced to determine if the product was macaque or rabbit. (3423)

Notes: The authors examine the role of brain-derived neurotrophic factor (BDNF) in alcohol addition. RNA was isolated from primary rat hippocampal neurons cultured in the absence or presence of ethanol. The RNA was reverse transcribed using the Reverse Transcription System, and BDNF and GPDH RNAs were quantitated by fluorescent real-time PCR. BDNF and nerve growth factor (NGF) protein levels were monitored using the BNDF and NGF Emax^® ImmunoAssay Systems. (3441)

Notes: Genomic DNA was isolated from murine plasma samples using the ReadyAmp™ Genomic DNA Purification System. The purified genomic DNA was then used for PCR genotype analysis of the CD14 and TLR2-KO loci. (3422)

Notes: The Wizard® SV Genomic DNA Purification System was used to isolate genomic DNA from the E. coli K-12 derivative, MC4100.  The isolated genomic DNA was used as a template to PCR clone the Escherichia coli 2-phosphoglycolate phosphatase (gph) gene.  The PCR plasmid constructs were also purified with the Wizard® Plus SV Minipreps DNA Purification System. (3218)

Notes: Genomic DNA was isolated from mouse primary granulocytes, macrophages, B lymphocytes and T lymphocytes using the Wizard^® SV Genomic DNA Purification System. The isolated genomic DNA was used to identify blood cells from progenitor transgenic mice by PCR with specific primers to a β-galactosidase transgene.  (3236)

Notes: In this study, the Wizard^® Genomic DNA Purification Kit was used to isolate chromosomal DNA from Salmonella enterica serovar Typhi. The isolated DNA was used in multiplex PCR to determine the presence or absence of certain DNA regions in various clinical isolates.  (3127)

Notes: The Wizard^® SV Genomic DNA Purification System was used to purify genomic DNA from blood fluke Schistosoma mansoni worm cercariae shed from Biomphalaria glabrata snails. The genomic DNA was used in PCR to type each individual S. mansoni worm in a cercariae. (3230)

Notes: GoTaq^® DNA Polymerase was used in a multiplexed genotyping reaction with three primers to differentiate wild-type, heterozygous, and mutant transgenic mouse alleles simultaneously. The wild-type target was 670bp and the mutant or knockout target was 1,030bp. Various other amplimers (1.4kb, 471bp, & 311bp) were subcloned with the aid of the pGEM^®-T Easy Vector System. (3360)

Notes: The Wizard® Genomic DNA Purification Kit was used to purify genomic DNA from Bacillus cereus.  The isolated DNA was used in PCR to create probes for Northern blotting Bacillus cereus total RNA to detect rsbV and orf4 gene transcripts.  (3100)

Notes: In this study, the SV Total RNA Isolation System was used to isolate total RNA from venom sacs from Naja Atra (Asian cobra). The Access RT-PCR System was used to reverse transcribe atratoxin and atratoxin-b cDNA from the isolated RNA. The amplified cDNAs were then cloned into the pGEM®-T vector. (3126)

Notes: These authors performed gel shift (EMSA) assays to determine whether purified VirA binds to the vapA promoter. Radiolabeled DNA fragments for the EMSA assays were prepared using DNA Polymerase I Large (Klenow) Fragment. Primer extension using ImProm-II™ Reverse Transcriptase localized the transcription start site within the vapA promoter. To characterize the transcriptional organization of the virR gene cluster, the authors performed reverse transcription using ImProm-II™ Reverse Transcriptase and Random Primers, followed by PCR using a combinations of primers in opposite orientations throughout the gene cluster. The plasmids used in this study were purified by alkaline lysis method or using the Wizard^® Plus SV Minipreps DNA Purification System.
(3563)

Notes: The authors of this study investigated the function of the daf-15 gene in C. elegans. The daf-15 gene encodes the C. elegans ortholog of raptor, a protein involved in the TOR signaling pathway. In C. elegans, daf-15 transcription is regulated by DAF-16, a FOXO transcription factor which is itself regulated by the insulin/IGF-1 signaling pathway. This work links regulation of the TOR pathway, which controls cell growth, to the insulin/IGF-1 pathway, known to affect lifespan, development and metabolism. Three candidate daf-15 genes were amplified by PCR and cloned into the pGEM^®-T Vector. The Riboprobe^® SP6/T7 transcription system was used to transcribe RNA from the candidate clones. Semiquantitative RT-PCR was performed to compare daf-15 mRNA levels in daf-2 and daf-16; daf-2 mutant animals. The mRNA was purified from total worm RNA using the PolyATtract^® mRNA Isolation System. (3633)

Notes: The authors examined the ybxI gene in Bacillus subtilis 168 for beta-lactamase activity and penicillin recognition as the primary structure of YbxI was similar to that of beta lactamases. The SV Total RNA Isolation System was used to isolate RNA from an exponential growth phase B. subtilis 168 culture. To determine if yxbI was present in the harvested total RNA, the Access RT-PCR System was used to amplify the yxbI cDNA and the positive control B. subtilis yjbJ mRNA. The products were run on a 1% agarose gel and stained with ethidium bromide. (3073)

Notes: In this study, siRNA was used to reduce the level of the PU.1 transcription factor.  The siRNA was generated using the T7 RiboMAX™ Express RNAi System with design assistance from the siRNA Target Designer (http://www.promega.com/siRNADesigner/). Annealed siRNA was purified by isopropanol precipitation. Forty-eight hours after transfecting 2.5ug of siRNA into RAW264.7 cells, RNA and protein were isolated from the cells and the siRNA effect was analyzed by RT-PCR and Western blot. (3062)

Notes: The authors investigate a potential internal ribosome entry site (IRES) in the 5´ untranslated region (UTR) of cellular inhibitor of apoptosis protein 1 (c-IAP1). The c-IAP1 5´ UTR was amplified, cloned into pGEM®-T Vector, sequenced, then inserted into a dicistronic reporter vector between Renilla and firefly luciferase sequences. Using the Dual-Luciferase® Reporter Assay System, IRES activity was evaluated in Rabbit Reticulocyte Lysate and transiently transfected cells. The pSV-β-Galactosidase Control Vector was used as a control for transfection efficiency. Because splicing events were removing part of the Renilla luciferase coding region, the authors chose to use RNA transfection of cells. The ImProm-II™ Reverse Transcription System was used for the reverse transcription step of RT-PCRs to amplify intercistronic regions of the dicistronic RNA to examine mRNA splicing. (3429)

Notes: The SV 96 Total RNA Isolation System was used to isolate total RNA from human umbilical vein endothelial (HUVEC) cells, primary keratinocytes (NHEK-adults) and human rheumatoid arthritis synovial fibroblasts (RASF) infected with adenovirus expressing siRNAs targeted at a variety of messages. The isolated RNA was used in real-time SYBR Green RT-PCR reactions to investigate the amount of message present after infection.  For reverse transcriptase reactions, the researchers used 5-100ng of purified total RNA.  Results were normalized to GAPDH message levels. (2752)

Notes: The acetylation status of histones was explored as an indicator of functional gene expression. The acetylation status of core histones H3 and H4 were assessed in treated and untreated cells by crosslinking chromatin, immunoprecipitating the chromatin with acetyl-H3 and acetyl-H4 specific antibodies. The crosslinking was reversed and the immunoprecipitated were phenol chloroform extracted then amplified for the mouse histone H4 gene. Amplifications were also set up with untreated genomic DNA to insure the amplification was within the linear range and to supply standards for gel-based quantitation.  All amplifications were performed with the PCR Master Mix. (2611)

Notes: The Wizard^® SV 96 Plasmid DNA Purification System was used to purify random subtractive PCR clones from DH5α cells.  The subtractive PCR clones were made from porcine spherical and tubular conceptuses.  Purified clones were denatured and screened with a DIG High Prime DNA Labeling and Detection Starter Kit (Roche Applied Science). (2748)

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)

Notes: Improm-II™ Reverse Transcriptase was used to clone U7T snRNA isolated from Drosophila nuclear extracts. The authors used 1ng of extracted U7T snRNA, 30ng of a U7T primer, and 1μl of the Improm-II™ Reverse Transcriptase. cDNAs from the reaction were PCR-amplified and cloned. The resultant clones were used to make probes for Northern blot analysis. (2723)

Notes: The authors of this paper describe testing and comparing five genomic DNA purification kits for their ability to isolate bacterial DNA from Neisseria meningitidis-, Streptococcus pneumoniae-, and Haemophilus influenzae-inoculated whole blood samples. The Wizard^® SV 96 Genomic DNA Purification System was considered to be the best kit for its ease of use and ability to be automated on a Roboseq 4200 PE liquid-handling robot. The Wizard^® SV 96 Genomic DNA Purification System produced good yields of high quality DNA that displayed sensitivity levels down to 2 genome copies when used as template in fluorescence-based PCR  (3209)

Notes: The authors examined the putative Pseudomonas aeruginosa homolog to the E. coli small multidrug resistance gene EmrE and its possible role in P. aeruginosa intrinsic drug resistance. Total RNA was isolated from 1-2ml log-phase or overnight stationary-phase P. aeruginosa PAO1 cultures grown in LB using the SV Total RNA Isolation System. After treating the RNA with DNase, 0.1µg RNA was used in the Access RT-PCR System to measure emrE_Pae expression and the constitutive rpsL gene. The two amplification products were then analyzed on a 1.7% agarose gel. (3074)

Notes: The Wizard^® Genomic DNA Purification Kit was used to purify Bacillus anthracis chromosomal DNA, which was then used in PCR to amplify a luxN ortholog (named open reading frame BA5047). Amplified DNA containing the luxN ortholog region was then cloned into the pGEM^®-T Easy Vector. This construct, when transformed into Vibrio harveyi (AI-1^-, and AI-2⁺), allowed the detection of an upregulated signaling system by a bioluminescent assay. (3092)

Notes: Researchers used the pGEM®-T Vector System to clone the entire 1.4kb Shiga toxin type 2 gene (Stx2) from E. coli O157-H7 C600 (933W). The resultant construct, named pGEMTStx2, was used as a template in PCR to amplify each region of the gene corresponding to Shiga toxin type 2 subunits A and B. Each PCR product was digested with BamHI and EcoRI before ligation into pCDNA 3.1+ (Invitrogen) to create pStx2ΔA and pStx2B. Mice were then immunized with either one or both of these constructs and another construct expressing murine granulocyte-macrophage colony-stimulating factor. Expression of each subunit in mouse tissue was verified by RT-PCR with specific primers and the AccessQuick™ RT-PCR System. (2701)

Notes: In this paper, the authors describe use of ImProm-II™ Reverse Transcriptase to transcribe cDNAs for Quantitative RT-PCR. Reverse transcription (RT) reactions were performed with 1μg total RNA from treated rat PC12 cells. Light Cycler reactions were setup with 1-3μl cDNA from the RT reactions. The researchers incubated cells with EGF and bFGF and analyzed HIF-2α mRNA levels. For these experiments, PC12 cells were incubated with 30ng/ml EGF or 50μM bFGF for 8 hours. Erk1&2 phosphorylation was examined by Western blot using the Anti-ACTIVE® MAPK pAb. Western results were visualized by chemiluminesent detection and analyzed with a digital luminescent image analyzer. The Dual-Luciferase® Reporter Assay System was used to analyze PC12 cell-expressed luciferase from pEpoEm1-luc and pEpoE-luc promoter constructs that were normalized with the pRL-TK vector. Cells were harvested 17-18 hours post transfection and treatment. (2725)