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Proc. Natl. Acad. Sci. USA 100, 9422-9427. Cloning and characterization of the Drosophila U7 small nuclear RNA. 2003

Dominski, Z., Yang, X.C., Purdy, M. and Marzluff, W.F.

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)

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J. Biol. Chem. 278 (10), 8018-8027. Nuclear factor-κB and mitogen-activated protein kinases mediate nitric oxide-enhanced transcriptional expression of interferon-kβ. 2003

Jacobs, A.T. and Ignarro, L.J.

Notes: Promega T4 Polynucleotide Kinase was used to end-label an oligo representing a NF-kB binding sequence element with [γ-32P] ATP (7,000 Ci/mmol).  Specific primers to IFN-β and IκB-α messenger RNA were used in RT-PCR to generate products for cloning into the pGEM®-T Easy Vector. The resulting plasmids were linearized with Nco I and Spe I, respectively, and used as templates for in vitro transcription using the Riboprobe® System to generate probes for use in an RNase protection assay.  The antisense probes were labeled with [α-32P]CTP (800 Ci/mmol) in the Riboprobe® reactions.  RNase ONE™ Ribonuclease was also used in this study.  (3197)

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J. Clin. Invest. 109, 923-930. The melanin-concentrating hormone receptor 1, a novel target of autoantibody responses in vitiligo 2002

Kemp, E.H., Waterman, E.A., Hawes, B.E., O'Neill, K., Gottumukkala, R.V.S.R.K., Gawkrodger, D.J., Weetman, A.P., and Watson, P.F.

Notes: T4 Polynucleotide Kinase was used to phosphorylate primers before ligating them into a phagemid vector to allow cloning of Sfi I-restricted DNA fragments. Phagemid was prepared from XL1-Blue cells using the Wizard® Minipreps DNA Purification System. Inserts were PCR amplified from the phagemid and purified using the Wizard® PCR Preps DNA Purification System. In separate experiments, the cDNA for the melanin concentrating hormone receptor 1 cDNA was cloned from total melanocyte RNA using MMLV Reverse Transcriptase. In vitro translation of the cDNA was performed using the TnT T7 Coupled Reticulocyte Lysate System and Canine Pancreatic Microsomal Membranes. (2602)

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Genetics 160, 225-234. Cloning and characterization of the Tribolium cataneum eye-color genes encoding tryptophan oxygenase and kynurenine 3-monooxygenase 2002

Lorenzen, M.D., Brown, S.J., Dennell, R.E., and Beeman, R.W.

Notes: The Wizard® Genomic DNA Purification Kit was used to isolate genomic DNA from individual Tribolium cataneum flour beetles. The isolated genomic DNA was used for PCR amplification for recombinational mapping related to eye color, PCR-based deletion breakpoint analysis, and was EcoR I-digested for Southern blotting analysis. (2504)

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Oncogene 20, 260-269. Modulation of apoptosis by procaspase-2 short isoform: selective inhibition of chromatin condensation, apoptotic body formation and phosphatidylserine externalization 2001

Droin, N., Rebe, C., Bichat, F., Hammann, A., Bertrand, R., and Solary, E.

Notes: The cDNA for the short isoform of the caspase-2 mRNA was amplified and cloned directly into the pTARGET™ Mammalian Expression Vector. The protein was stably expressed in U937 human leukemic cells by selection with the antibiotic G-418.  Stable clones were identified by PCR of the genomic DNA of selected colonies with primers to the T7 promoter of the pTARGET™ Vector and the downstream primer of the caspase-2 message. Overexpression of the protein interfered with etoposide-mediated apoptosis. (2597)

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J. Biol. Chem. 276, 37520–37528. Identification of a novel kinesin-related protein, KRMP1, as a target for mitotic peptidyl-prolyl isomerase Pin1. 2001

Kamimoto, T., Takeru Zama, Aoki, R., Muro, Y. and Hagiwara, M.

Notes: The pGEM®-T Easy Vector was used to clone the 5.4kb open reading frame of the kinesin-related protein (KRMP1). The gene was later subcloned into fusion tagged expression vectors and used in point mutation analysis of the protein’s active site.  For some of these studies, the researchers used Promega Recombinant Human cdc2 Kinase as a substrate in in vitro phosphorylation experiments.  (3136)

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J. Biol. Chem. 275(33), 25061-25064. Association of calcium/calmodulin-dependent kinase II with developmentally regulated splice variants of the postsynaptic density protein densin-180 2000

Strack, S., Robison, A.J., Bass, M.A., and Colbran, R.J.

Notes: To demonstrate an interaction of activated CaM KII with the postsynaptic density protein densin-180, a gst-fusion protein of densin was produced and reacted with autophosphorylated CaM KII. The glutathione matrix-bound material was eluted and immunoblotted for phospho-Thr286-CaM KII. The autophosphorylated CaM KII was pulled down on the Glutathione only when the densin-GST fusion was present. The phosphoCaM KII was detected with the Anti-ACTIVE® CaM KII pAb. The clone of densin was generated by RT-PCR of rat forebrain total RNA with the Access RT-PCR System. (0076)

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Genetics 154, 1115-1123. Three subfamilies of pheromone and receptor genes generate multiple B mating specificities in the mushroom Coprinus cinereus. 2000

Halsalla, J., Milnera, M., Casseltona, L.

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)

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Hum. Gene Ther. 11, 247-261. The CXC chemokine, monokine-induced by interferon-gamma, inhibits non-small cell lung carcinoma tumor growth and metastasis 2000

Addison, C.L., Arenberg, D.A., Morris, S.B., Xue, Y.-Y., Burdick, M.D., Mulligan, M.S., Iannettoni, M.D. and Strieter, R.M.

Notes: The pTARGET™ Mammalian Expression Vector was used to clone and express MIG (monokine-induced by Interferon-gamma) and IP-10 (interferon-inducible protein 10) in A549 human adenocarcinoma cells. Stable transfectants were obtained through G418 selection. (2058)

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J. Biol. Chem. 275, 38589–38596. Identification of a functional nuclear export sequence in BRCA1. 2000

Rodríguez, J.A. and Henderson, B.R. 

Notes: Several cancer-associated proteins, such as the tumor suppressor p53 and the oncoproteins c-ABL and hdm2, have been shown to shuttle between the nucleus and the cytoplasm. Due to this information and the controversy regarding BRCA1 localization, the authors wanted to determine whether BRCA1 is also capable of nuclear-cytoplasmic shuttling.

To this end, a plasmid encoding untagged BRCA1 was created by subcloning the full-length BRCA1 cDNA into a vector as a NotI/ClaI fragment. To generate pYFP-BRCA1, a DNA fragment encoding the yellow fluorescent protein (YFP) was amplified by polymerase chain reaction (PCR) and inserted in frame at the end of the BRCA1 cDNA in the plasmid, using the unique NotI restriction site.

Human breast cancer cell lines T47D and MCF-7, the human breast epithelial cell line HBL-100, and mouse NIH3T3 fibroblasts were seeded onto glass coverslips and transfected at 50–70% confluency with 0.5–2µg of plasmid DNA using the FuGENE® 6 Transfection Reagent. After 48 hours, cells were fixed and processed for fluorescence microscopy. (4283)

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J. Immunol. 162, 871-877. Alternative splicing and hypermutation of a nonproductively rearranged TCR alpha-chain in a T cell hybridoma. 1999

Marshall, B., Schulz, R., Zhou, M., Mellor, A.

Notes: The Access RT-PCR System was used to amplify Vα1024 mRNA of the TCR alpha chain and the resulting product was cloned with the pGEM®-T Vector System. (0734)

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J. Immunol. 162, 6562-6571. Molecular cloning and characterization of a novel CD1 gene from the pig. 1999

Chun, T., Wang, K., Zuckermann, F.A., Gaskins, H.R.

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)

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J. Physiol. 519, 323–333. Cloning and functional expression of a novel degenerin-like Na+ channel gene in mammals. 1999

Sakai, H., Lingueglia, E., Champigny, G., Mattei, M.-G. and Lazdunski, M.

Notes: Taq DNA Polymerase was used extensively to clone the BLINaC cDNA. The resulting clone was put into the pCI Mammalian Expression Vector. Total RNA was isolated from rat liver and primary hepatocytes with the SV Total RNA Isolation System. The RNA was used for RT-PCR. Northern blotting was performed with poly-A(+) RNA isolated with the PolyATtract® mRNA Isolation System. (0472)

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J. Biol. Chem. 274, 20034-20039. Photocross-linking of an oriented DNA repair complex: Ku bound at a single DNA end. 1999

Yoo, S., Kimzey, A., Dynan, W.S.

Notes: The pGEM®-3Zf(+) Vector was used for routine subcloning and the resulting plasmid was used as a template for PCR. (0114)

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Genetics 153, 1257-1269. Structural requirements for the tissue-specific and tissue-general functions of the Caenorhabditis elegans epidermal growth factor LIN-3. 1999

Liu, J., Tzou, P., Hill, R.J., Sternberg, P.W.

Notes: Before RT-PCR reactions, the RNA template was treated with RQ1 RNase-free DNase. (0776)

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J. Biol. Chem. 274, 8391-8404. Cyclic AMP- and Cyclic GMP-dependent protein kinases differ in their regulation of cyclic AMP response element-dependent gene transcription. 1999

Collins, S.P., Uhler, M.D.

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

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J. Biol. Chem. 274, 2271-2278. Antioxidant function of the mitochondrial protein SP-22 in the cardiovascular system. 1999

Araki, M., Nanri, H., Ejima, K., Murasato, Y., Fujiwara, T., Nakashima, Y. and Ikeda, M.

Notes: The authors used the Tfx™-50 Reagent to transfect bovine aortic endothelial cells. They also used the pGEM®-T Easy Vector System to clone PCR products. (1482)

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J. Biol. Chem. 274, 21830–21839. Purification, cDNA cloning, and expression of GDP-L-Fuc:Asn-linked GlcNAc alpha1,3-fucosyltransferase from mung beans. 1999

Leiter, H., Mucha, J., Staudacher, E., Grimm, R., Glössl, J. and Altmann, F.

Notes: Total RNA was isolated from 3-day-old mung bean hypocotyls using the SV Total RNA Isolation System. RT for RT-PCR was performed with the Reverse Transcription System. (0825)

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J. Biol. Chem. 274, 29720-29725. Expression and functional interaction of the catalytic and regulatory subunits of human methionine adenosyltransferase in mammalian cells. 1999

Halim, A.-B., LeGros, L., Geller, A., Kotb, M.

Notes: Subunits of the cDNA for methionine adenosyltransferase were amplified from a plasmid using Taq DNA Polymerase and directly cloned into the pTARGET™ Mammalian Expression T-Vector. The sequence of the cloned inserts were confirmed with the fmol® DNA Cycle Sequencing System. The constructs were transiently transfected into COS-1 cells with the TransFast™ Reagent at a 1:1 ratio. Excellent detail is provided for the transfection. (1094)

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J. Biol. Chem. 273, 11556-11562. cDNA cloning and expression of bovine UDP-N-acetylglucosamine: alpha1,3-D-mannoside beta1,4-N-acetyl glucosaminyltransferase IV. 1998

Minowa, M.T., Oguri, S., Yoshida, A., Hara, T., Iwamatsu, A., Ikenaga, H. , Takeuchi, M.

Notes: The Access RT-PCR System was used to compare the abundance of GnT-IV mRNA in various bovine tissues. (0694)

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Genetics 149, 1081-1088. Gene silencing by DNA methylation and dual inheritance in Chinese hamster ovary cells. 1998

Paulin, R. P., Ho, T., Balzer, H. J., Holliday, R.

Notes: Bisulphite-treated DNA was purified using the Wizard® DNA Clean-Up System prior to PCR. The amplified DNA was then cleaned using a Wizard® PCR Preps DNA Purification System prior to cloning and the cloned DNAs were sequenced using the fmol® DNA Cycle Sequencing System. (0001)

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J. Neurosci. 18, 16-25. Regulation of Ca2+-dependent K+ channel expression in rat cerebellum during postnatal development. 1998

Muller, Y.L., Reitstetter, R., Yool, A.J.

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)

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J. Biol. Chem. 273, 18826-18834. Binding of Ca2+ and Zn2+ to human nuclear S100A2 and mutant proteins. 1998

Franz, C., Durussel, I., Cox, J.A., Schäfer, B.W., Heizmann, C.W.

Notes: The S100A2 protein cDNA was subcloned into the pGEMEX®-2 Vector and expressed in BL21(DE3)pLysS E.coli cells. The protein was purified by a referenced method. (1173)

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Proc. Natl. Acad. Sci. USA 95, 9654-9659. Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzymes. 1998

Ohno, K., Brengman, J., Tsujino, A., Engel, A.G.

Notes: The entire coding region of the T isoform of the catalytic subunit of the acetycholinesterase (ACHET)was amplified and cloned into the pTargeT™ Vector and sequenced. The ColQ cDNA was cloned into the pTargeT™ Vector as well and mutated with a Pfu DNA polymerase-based mutagenesis system. The ACHET and ColQ mutants were coexpressed in COS cells and interactions examined with sedimentation analysis through sucrose gradients. (0595)

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Biochemistry 37, 4420-4428.. Silent nucleotide substitution in the sterol 27-hydrosylase gene (CYP 27) leads to alternative pre-mRNA splicing by activating a crytic 5' splice site at the mutant codon in cerebrotendinous xanthomatosis patients. 1998

Chen, W., Kubota, S., Teramoto, T., Nishimura, Y., Yonemoto, K., Seyama, Y.

Notes: Genomic analysis noted a single base change between wild type and mutant. To see if this base change was enough for alternative mRNA splicing, minigenes were constructed and subcloned into the pTargeT™ Vector. The mutant and wild-type minigenes were transfected into COS-1 cells. Forty-eight hours post-transfection total RNA was isolated and analyzed by RT-PCR. Transfectants with the mutant sequence did produce a different product due to alternative splicing. (1331)

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