Notes: The Wizard^® Genomic DNA Purification Kit was used to purify genomic DNA from Burkholderia thailandensis and Burkholderia pseudomallei.  The genomic DNA was used in PCR to amplify the arabinose assimilation operon.  (3104)

Notes: Two genes (CzcR and CzcS) encoding the CzcCBA (cobalt/zinc/cadmium) heavy metal efflux pump were cloned using  Pfu DNA Polymerase and genomic DNA from PT5 and PT1105 strains of Pseudomonas aeruginosa. The products were 900 and 1,600 bp, respectively. RNA from 5 different strains of Pseudomonas aeruginosa was isolated and treated with RQ1 RNase-Free DNase.  The RNA was used in reverse transcription reactions with the ImProm-II™ Reverse Transcriptase and random primers.  Copy-DNAs produced from the reaction were stored at -20°C until use in real-time PCR.  (3044)

Notes: cDNA was synthesized from 5μg total RNA from patient skin fibroblasts using the specific ALG1 β1,4 mannosyltransferase primer in the presence of 5% DMSO and 1 unit of ImProm-II™ Reverse Transcriptase. Reverse transcription reactions were performed at 42°C for one hour.  PCR amplification of the cDNA produced ~1,200bp amplimers.  (3180)

Notes: Using the SV Total RNA Isolation System before RT-PCR, expression levels of AtGGT-IB were compared in flowers, leaves, stems, and root tissues of wildtype and era1-2 Arabidopsis plants. First-strand synthesis was performed with M-MLV Reverse Transcriptase using 500ng of RNA and an equal amount of oligo(dT). A 1:10 dilution of this product was used for amplification. (2855)

Notes: This paper describes the use of RQ1 RNase-Free DNase to remove DNA from TRIZOL- purified total RNA. The researchers describe stopping RQ1 RNase-Free DNase reactions with RQ1 RNase-Free DNase Stop Solution and heating the reactions at 64°C before using the samples in RT-PCR.  (3187)

Notes: DNA was purified from blood stains and buccal swabs with DNA IQ™ System and two other comparative methods. A high throughput AluQuant^® assay on the BioMek^® 2000 was compared to a quantiblot method for quantifying human genomic DNA. DNA samples extracted with the DNA IQ™ System had less variability than the quantiblot method. The AluQuant^® System showed a similar level of sensitivity, reproducibility and precision compared to the quantiblot method.  (3007)

Notes: MCF-7 cells were incubated for 24 hours in PRF-SFM and then detached and plated on uncoated dishes or dishes coated with 2μg/cm² poly-L-Lysine (P-Lys) in PBS, 30μg/ml fibronectin (Fn) in PBS or 30μg/ml type IV Collagen (Col) in 10mM acetic acid. The cells plated on uncoated dishes were treated both with and without 10nM estradiol (E2). After 24 hours, total cellular RNA was extracted and reverse transcribed using M-MLV reverse transcriptase. Briefly, reverse transcription was performed on 1μg of total RNA in a final volume of 10μl by incubation at 37°C for 30 min with 200U of M-MLV reverse transcriptase, 0.4μg oligo-dT, 0.5μM dNTP and 24U RNasin^® Ribonuclease Inhibitor, followed by heat denaturation for 5 minutes at 95°C. Subsequent PCR analysis was performed on 1μl of the RT product in a final volume of 25μl. Primers were used to amplify the 210bp fragment of PS2 and the 304 bp fragment of cathepsin D. Amplification of 408bp of ribosomal RNA 36B4 was performed as control. The PCR mixture consisted of 1.25U GoTaq^® DNA Polymerase, 1X PCR buffer (10mM Tris-HCl, 50mM KCl), 2.5mM MgCl₂, 0.2mM each dNTP, 0.6μM of each PS2 primer or cathepsin D primer and 0.2μM of each 36B4 primer. PCR was performed for 20 cycles at 95°C for 1 minute, 59°C for 2 minutes and 72°C for 1 minute. Ten microliters of the PCR products were separated on a 1.2% agarose gel. (3377)

Notes: The Wizard^® Genomic DNA Purification Kit was used to purify genomic DNA from Yersinia pestis Kimberley53 strain.  The isolated genomic DNA was digested with restriction endonucleases and then used in PCR amplifications. The amplified products were sequenced to verify the presence of a transposon insertion site.  (3103)

Notes: The researchers used pGEM®-T Easy Vectors (Cat.# A1360) to clone Zika virus PCR products. (4651)

Notes: GoTaq^® DNA Polymerase was used to amplify a portion of Human Papillomavirus 16 (HPV 16) from cervical carcinoma-derived genomic DNA. The amplified region of HPV 16 was then purified and used in sequencing reactions. (3358)

Notes: The AccessQuick™ RT-PCR System was used to analyze mRNA expression of Hypoxia Inducible Factor-1α (HIF-1α) in HCT116 cells. RT-PCR was performed using 0.5μg of Trizol-isolated total RNA and HIF-1α specific primers. (3060)

Notes: GoTaq^® DNA Polymerase was used to amplify Green Fluorescent Protein and β-actin cDNA. This paper describes use of a “Touchdown” PCR procedure for amplification of the β-actin cDNA. Gels are presented displaying the amplification products from the reporter constructs and cells used in the experiments. (3129)

Notes: The authors used real-time quantitative PCR to characterize cytokine response after HIV infection of human lymphoid tissues. To synthesize first-strand cDNA, total RNA was reverse transcribed using the ImProm-II™ Reverse Transcription System: 200U of ImProm-II Reverse Transcriptase, 2µg of total RNA, 500ng of oligo(dT) primer, 500µM dNTPs 3mM MgCl₂ and 24U of RNase inhibitor in 1x ImProm-II™ reaction buffer. To obtain viral stocks for infection, 293T cells were transfected with the proviral plasmids pNL4-3 and pYU-2 using the ProFection^® Mammalian Transfection System—Calcium Phosphate. (3455)

Notes: A fusion protein construct was made using the Monster Green™ Fluorescent Protein phMGFP Vector and the PCR-amplified Open Reading Frame E (ORF-E) from Bovine herpesvirus 1. The construct was transfected into mouse neuroblastoma (Neuro-2A) cells (ATCC CCL131), human neuroblastoma (SK-N-SH) cells, rabbit skin cells and bovine kidney cells. Transfected cells were visualized using an Olympus FW500/BX60 confocal microscope with 488nm excitation laser and 522nm emission filter set. The ORF-E-MGFP protein was localized in discreet domains within the nucleus of Neuro-2A and SK-N-SH cells. In rabbit skin cells and bovine kidney cells the ORF-E-MGFP protein was detected in the cytoplasm and nucleus. (3029)

Notes: The SV 96 Total RNA Isolation System was use to purify total RNA from stably transfected MCF-7 cell clones expressing deletion mutants of SEL1L, a protein implicated in breast tumor formation. One microgram of total RNA purified with the SV 96 Total RNA Isolation System was then used in RT-PCR. (2846)

Notes: The authors identified a TERMINAL FLOWER homolog, CsTFL, in Washington navel oranges (Citrus sinensis) and investigated its role and the role of other genes in juvenility and flower production. The CsTFL gene was amplified from genomic DNA using PCR and degenerate primers, and amplification products were cloned into the pGEM^®-T Easy Vector. The resulting clones were sequenced using the fmol^® DNA Cycle Sequencing System. The CsTFL cDNA was amplified by RT-PCR, using 4 µg of total RNA from whole flowers and ImProm-II™ Reverse Transcriptase. The amplified cDNA was then cloned into the pGEM^®-T Easy Vector. To evaluate CsTFL gene copy number and allele origins, the authors performed a Southern blot with 10 µg of genomic DNA and a CsTFL probe labeled with the Prime-a-Gene^® Labeling System. To characterize CsTFL expression in various citrus tissues, RT-PCR was performed with 2 µg of total RNA and ImProm-II™ Reverse Transcriptase. The levels of CsTFL RNA and other RNAs were determined by cDNA synthesis using ImProm-II™ Reverse Transcriptase, followed by real-time PCR. Amplification products were quantitated by SYBR^® Green fluorescence. Standard curves for each real-time PCR target were generated using known amounts of in vitro transcribed RNA. Prior to reverse transcription and real-time PCR, total RNA samples were treated with RQ1 RNase-Free DNase to remove DNA contaminants. (3650)

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)