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: The authors used ISSR PCR to quantitate genomic instability using matched tumor (OSCC) and normal oral squamous cell samples. The inter-repeat region bands of similar molecular size that were altered in more than one case of OSCC were reamplified, gel purified and cloned into the pGEM T Vector for sequencing. (2635)

Notes: Researchers performed a 5´ RACE analysis on the human CCR8 and mouse CX3CR1 transcripts.  PCR-amplified products from the reactions were cloned in to the pGEM^®-T Vector for further analysis.  Also, the human CCR8 and mouse CX3CR1 promoters were cloned into the pGL3-Basic Vector and transfected into THP1 and Jurkat cells. For transfections, 15 x 10⁶ cells in 750μl were used with 15μg of construct in electroporation reactions. Transfectants were grown for 48 hours and analyzed with the Bright-Glo™ Luciferase Assay System. A fourfold increase in activity was observed compared to the pGL3-Basic Vector alone. (2729)

Notes: cDNA fragments from the Nogo gene were amplified from genomic DNA and cloned into the pGEM^®-T vector. The Wizard^® Plus Mini- and Midiprep kits were used to purify the plasmids from bacterial cells. (2658)

Notes: In this paper, the SV Total RNA Isolation System was used to isolate total RNA from Gigaspora margarita. The authors reported isolating total RNA from 100 spores or 100mg of mycorrhizal roots. One microliter of the isolated RNA was used in RT-PCR to amplify Gigaspora margarita metallothionein (MT)-like polypeptide (GmarMT1) RNA. The researchers also cloned the GmarMT1 coding region using the pGEM^®-T Vector. (3077)

Notes: PAI-1 is an inhibitor of tissue-type plasminogen activator (t-PA) and has been shown to have neuroprotective activity through the TGF-β pathway. The authors performed RTPCR experiments to study PAI-1 expression in cultured neurons and astrocytes. To confirm specificity of PAI-1 amplified products, the products were cloned into pGEMT vectors and sequenced. Wildtype and PAI-1 deficient astrocytes were transfected  (using the cationic lipid reagent, Transfast Transfection Reagent) with a luciferase reporter gene driven by the TGF-β1 response element (CAGA-luc) to determine if PAI-1^-/- cells could transduce TGF-β1 signal. Luciferase activity was quantitated using the Luciferase Assay Kit. (2608)

Notes: M-MLV Reverse Transcriptase and RNasin® Ribonuclease Inhibitor were used in a reaction to generate first strand cDNA.  The generated cDNA was used as a template for quantitative PCR in a Taqman® Assay.  The pGEM®-T Vector System was used to clone copies of each target gene so that standards could be generated.  The Wizard® Genomic DNA Purification Kit was used to isolated genomic DNA from the S. epidermidis to generate a standard curve for quantitation of genomic DNA contamination of samples. (2291)

Notes: The interleukin (IL)-1ß stimulated release of granulocyte macrophage colony stimulating factor (GM-CSF) from lung epithelial cells was explored in this study. To test the promoter activity of GM-CSF, the promoter and enhancer regions were amplified by PCR from human peripheral blood mononuclear cells and cloned into the pGEM^®-T vector. After verification by sequencing, the promoter and enhancer were cloned individually and together into the pGL3-Basic Vector. Additionally, an Xho I/Sal I fragment containing the HSV tk promoter, a gene conferring neomycin resistance, and a poly-A tail were cloned into the Sal I site of pGL3 to allow the production of stable transfectants. To perform stable transfections, 20µl of the Tfx™-50 transfection reagent was incubated with 8µg of plasmid in serum-free medium for 15 minutes at room temperature.  Preconfluent human A549 type II alveolar carcinoma cells were incubated with the transfection mix for 2 hours after washing with serum-free medium. The cells were then cultured in fresh medium for 16 hours before the addition of  0.5mg/mL G-418. After 21 days, foci of cells developed and were harvested for use in luciferase assays. The cells were plated into 24-well plates, grown to confluency and incubated in serum-free medium. Stimulation by 1ng/ml IL-1ß or 1µM PMA for 12 hours was followed by lysate production and measurement of luciferase activity using the Luciferase Assay System and a Turner luminometer. Luciferase readings were standardized against total protein measurements. The PolyATract^® System IV was also used prior to a Northern Blot to obtain purified mRNA. (2742)

Notes: Poly(A)⁺ RNA was purified from total RNA by the PolyATtract^® mRNA Isolation System. PCR products were purified by agarose gel electrophoresis and were cloned into pGEM^®-T and pGEM^®-T Easy Vector. Genes were obtained by RT-PCR using the Access RT-PCR System. (1097)

Notes: Total RNA was isolated from a virulent strain of Salmonella enterica, SL1344 using the SV Total RNA Isolation System. The Access RT-PCR System was used to characterize the transcriptional organization of the prg operon. The pgrH gene was amplified by RT-PCR and cloned into the pGEM^®-T vector (2305)

Notes: The Access RT-PCR System was used to amplify Vα₁₀Jα₂₄ mRNA of the TCR alpha chain and the resulting product was cloned with the pGEM^®-T Vector System. (0734)

Notes: Authors use the Wizard^® Genomic DNA Purification Kit to isolate DNA from newborn mice livers. They performed nested PCR on V(H)-D-J(H) genes and cloned these into the pGEM^®-T Vector and sequenced the rearrangements using the fmol^® DNA Cycle Sequencing System. (0908)

Notes: The pSP73 Vector was used for routine subcloning. The pGEM®-T Vector System was used for subcloning of PCR products. (1320)

Notes: Adult mice were perfused with 4% paraformaldehyde and 7% picric acid in PBS. The brains were removed and post-fixed for 4hr in the same buffer and stored overnight at 4°C in 30% sucrose. Twenty micron frozen sections were prepared and probed with a 1:200 dilution of the Anti-CNP mAb overnight and the antibody was recognized with a rhodamine-conjugated secondary antibody. The pGEM®-T Vector System was also used to clone RACE products. (0568)

Notes: In this paper, DNA visualized on a sequencing gel is stained with the SILVER SEQUENCE™ DNA Sequencing Systems, and PCR products are cloned into the pGEM^®-T Vector. (0187)

Notes: The p53 cDNAs expressed from thymic lymphoma cells were amplified with the Access RT-PCR System and the resulting products were cloned with the pGEM^®-T Vector System. No details of the reaction or whether or not total RNA was used were provided. (0812)

Notes: The complete cDNA for the CD1.1 gene was amplified and subcloned into the pTARGET™ Mammalian Expression Vector. The 1020bp, 339 amino acid protein was stably expressed in CHO cells following selection with G-418 sulfate. Expression was confirmed by Northern blot and FACS analysis with an mAb to the CD1.1 protein. The pGEM®-T Vector System was used for routine cloning of both PCR and RT-PCR products. The Prime-a-gene® Labeling System was used create probes for cosmid library screening. (1303)

Notes: Poly A+ RNA was isolated from Arabidopsis total RNA using the PolyATtract^® mRNA Isolation System. The isolated RNA was used for Northern analysis and both 3' and 5' RACE. The products of the RACE reactions were subcloned into the pGEM^®-T Vector. (1189)

Notes: The authors used the pGEM^®-T Vector System to clone the 4.3kb dTTP II gene from a Drosophila melanogaster embryonic cDNA library. (0512)

Notes: In this paper, Tfl DNA polymerase was used for two-step RT-PCR. The amplified fragments were cloned into the pGEM®-T Vector. Promega's Terminal Deoxynucleotidyl Transferase (TdT) was used to end label specific oligos for screening a frog brain cDNA library. T7 and T3 RNA polymerases were used to make digoxigenin labeled riboprobes for in situ hybridization. (1281)

Notes: Streptavidin MagneSphere^® Paramagnetic Particles (SA-PMPs) were used to capture biotinylated primer probe in a random access retrieval of genetic information by PCR (rargip) screening [ABE, K., (1992) Rapid isolation of desired sequences from lone linker PCR amplified cDNA mixtures: application to identification and recovery of expressed sequences in cloned genomic DNA. Mamm. Genome 2:252-259]. The pGEM^®-T Vector System and PolyATract^® mRNA Isolation System were also used. (0513)

Notes: Poly-A+ RNA was isolated from rat cerebellar total RNA with the PolyATtract^® mRNA Isolation System and used for semi-quantitative RT-PCR. The targets of the RT-PCR were cloned with the pGEM^®-T Vector System and sequenced to show they quantitated the correct targets. (0671)

Notes: Taq DNA Polymerase was used for PCR. The PCR products were purified with the Wizard^® PCR Preps DNA Purification System and cloned with the aid of the pGEM^®-T Vector System. (0624)

Notes: RT-PCR was performed with degenerate primers and the resulting 110bp product was subcloned with the pGEM®-T Vector System. The 110bp fragment was used to screen a cDNA library and four positive plaques were identified. (0650)

Notes: The RNasin^® Ribonuclease Inhibitor and the pGEM^®-T Vector System were used in this study. The inhibitor was used to protect RNA during the T4 RNA ligase step for 5' RACE analysis. (1643)