Notes: The authors initiated translation on a poly(U) mRNA using biotin-labeled phenylalanine tRNA with isolated ribosomes. The reaction was allowed to bind to Streptavidin MagneSphere^® Paramagnetic Particles, washed four times and then shaken in buffer to remove the ribosomes.  The isolated rRNA was then extracted with phenol/chloroform, ethanol precipitated and used in RT-PCR. (3082)

Notes: Researchers cloned the Photinus and Renilla luciferase ORFs into the pSP64 Poly(A) Vector to create a dual-reporter vector named SP6P. A similar vector, SP6R.4G(-508/-3).P, was created in which a 5´ untranslated region from the Saccharomyces cerevisiae TIF4631 gene was cloned between the two reporter genes. These two vectors were used to transform yeast strains. The resultant transformants were lysed using Passive Lysis Buffer and a modified lysis procedure.   Lysates were analyzed for luciferase activities using the Dual-Luciferase^® Reporter Assay System and a TD20/20 luminometer. The researchers also cloned and sequenced the 5´  untranslated region of TIF4631 by using a RACE-PCR technique followed by cloning the PCR amplimers into the pGEM^®-T Vector. (2845)

Notes: In this article, a 2.5- and a 1.7-kb fragment of the myostatin promoter were amplified by PCR and cloned into the pGEM®-T Easy vector.  These clones were then subcloned into the pGL3-Basic Vector.  Deletion mutants of the constructs were made and used to transient transfect mouse muscle C₂C₁₂ cells.  The Luciferase Assay System was then used to analyze transfectants.  The researchers also injected luciferase-reporter constructs into mouse muscle.  Luciferase activity in the mouse muscle was examined by grinding isolated muscles in liquid nitrogen and resuspending them in Cell Culture Lysis Buffer.  Ten micron cryosections of the muscles were also used in immunohistochmical staining experiments.  For these experiments, the researchers used a 1:50 dilution of Promega’s polyclonal anti-luciferase antibody, a secondary antibody and tertiary fluorescein-labeled conjugate.  The slides were counter stained with DAPI. (3147)

Notes: The authors cloned and sequenced the dsRNA from Penicillium chrysogenum virus (PcV) and analyzed the gene products of the segmented virus. After reverse transcription followed by PCR to confirm the 5’ and 3’ ends of the dsRNA segments, RT-PCR was used to amplify the complete cDNAs for the four viral segments. The products were cloned into pGEM®-T Vector after A-tailing. To confirm the ORFs predicted for each of the four segments, the cDNAs were added to TNT® T7 Quick Coupled Transcription/Translation System reactions that included radiolabeled methionine. The resulting proteins ranged in size from 94-128 kDa and were separated on an 8% SDS-PAGE gel and analyzed by autoradiography. Gradient-purified PcV virions were digested with Sequencing Grade Modified Trypsin at 37°C for 18 hours and the digestion products were separated by reverse-phase HPLC on a C18 column. Two highly resolved peptides were selected for amino acid sequencing by automated Edman degradation. (3089)

Notes: The entire sti gene (~7kb) of Drosophila was PCR cloned into the pGEM^®-T Vector. The cloned sequences were then sequenced and analyzed for mutations. The researchers also used the T7 RiboMAX™ Large Scale RNA Production System to create dsRNAs from PCR-amplified regions of the sti and GFP coding regions. TransFast™ Transfection Reagent was used to transfect 2 x 10⁶ Schneider S2 cells with 10 μg of dsRNA in a 35mm Petri dish. The cells were fixed after transfection and examined for multinucleate cells, or immunocytochemically stained for actin and anillin. (3259)

Notes: This paper describes use of the MagneSil® Genomic, Fixed Tissue System for purification of genomic DNA from paraffin-embedded gonadal tissues. The isolated genomic DNA was used in PCR to amplify a sequence from Desert hedgehog (DHH) Gene. The PCR products were used in sequencing reactions. (3179)

Notes: COS-7 cells were harvested and total RNA isolated 24 hours after transfection with plasmids expressing trans-activating proteins. Five micrograms of RNA was reverse transcribed with oligo(dT) primer using the M-MLV Reverse transcriptase. One tenth of the reaction volume (2μl) was then used as template in PCR to amplify a gene presumed to be controlled by the trans-activators (pS2; 210bp) or a control gene (36B4 ribosomal phosphoprotein; 408bp). The PCR products were amplified using GoTaq^® DNA polymerase. (3203)

Notes: HeLa and Saos-2 were transiently transfected with luciferase reporter vectors made from the pGL3-Promoter vector containing various intron sequences from the human integrinα3 gene ITGA3. The first intron sequence was originally cloned by PCR cloning into the pGEM®-T Easy vector. Cell cultures were assessed for luciferase activity by making lysates with the Glo Lysis Buffer and assaying with the Steady-Glo® Assay kit. (3225)

Notes: Total RNA was isolated from leaf tissue from Arabidopsis plants treated with rhizobacteria Serattia marcescens strain 90-166, 300μM benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH), 100μM jasmonic acid (JA), or water for 2 days. Reverse transcription reactions were performed on 1–5μg of total RNA using 200 units of reverse transcriptase, 500ng oligo d(T)_12–16mer primer and 500μM dNTPs in a final volume of 20μl. Semi-quantitative RT-PCR was performed in a final volume of 75μl using 1.5μl of cDNA, 1X PCR buffer (with 1.5 mM MgCl₂), 200μM dNTP, 200nM of each gene specific primer (primers were designed from the Arabidopsis PR-1 gene) and 1.5 units of GoTaq^® DNA Polymerase. To ensure that only host genes and not the viral RNA transcripts were amplified, the RT reactions were performed using oligo d(T) primers. As a loading control, parallel reactions using elongation factor primers were performed. PCR conditions for all genes were as follows: One cycle of 4 minutes at 94°C, 30 seconds at 55°C and 30 seconds at 72°C was followed by 34 cycles of 30 seconds at 92°C, 30 seconds at 59°C and 30 seconds at 72°C. A 5μl aliquot was removed from each reaction after 25, 30, and 35 cycles. These aliquots were analyzed on a 1% agarose gel stained with ethidium bromide. (3371)

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)