Cell Signaling pRL Renilla Luciferase Control Reporter Vectors Product pRL-SV40 Vector pRL-TK Vector pRL-CMV Vector pRL-null Vector For Research Use Only. Not for Use in Diagnostic Procedures. Description: The pRL Vectors are wildtype Renilla luciferase (Rluc) control reporter vectors. The pRL Vectors, which provide constitutive expression of Renilla luciferase, can be used in combination with a firefly luciferase vector to cotransfect mammalian cells. Expression of Renilla luciferase provides an internal control value to which expression of the experimental firefly luciferase reporter gene may be normalized. The pRL Vectors contain the cDNA encoding Renilla luciferase (Rluc) cloned from the anthozoan coelenterate Renilla reniformis (sea pansy). Four different promoter configurations are available. The HSV-thymidine kinase promoter (pRL-TK) is relatively weak and may be particularly useful in providing neutral constitutive expression of the Renilla luciferase control reporter. The early SV40 enhancer/promoter region (pRLSV40) and the CMV immediate early enhancer/promoter region (pRL-CMV) typically provide high-level transcription and, therefore, may be less suitable for co-reporter applications involving experimental vectors with robust regulatory elements. In general, we recommend validating the performance of specific co-reporter combinations in the desired target cells. In addition to the modified Rluc reporter gene, all pRL Vectors are isolated from a dam–/dcm– E. coli K host strain, allowing digestion with restriction enzymes that are sensitive to dam and dcm methylation. Features: • A T7 promoter is located immediately upstream of Rluc, allowing in vitro synthesis of Renilla luciferase. • The SV40 late poly(A) signal sequence is positioned downstream of Rluc to provide efficient transcription termination and mRNA polyadenylation. • A prokaryotic origin of replication and β-lactamase gene allow selected propagation of the pRL vectors in E. coli host strains. • To avoid DNA methylation, all pRL Vectors are isolated from a dam–/dcm– E. coli K host strain. Storage Conditions: Store vectors at –20°C. Protocol pRL Renilla Luciferase Reporter Vectors Technical Bulletin Part# TB550 2448 2442 BglII XhoI SacI Ampr ori BamHI 1499 SV40 late poly(A) 1247 XbaI Rluc (3320bp) pRL-null Vector T7 Promoter 1 5 EcoICR I HindIII NdeI NsiI SphI SpeI NarI SalI MluI EcoRI XmaI SmaI PstI NheI 299 Csp45I 315 12 10 15 24 30 32 34 41 47 51 58 65 67 77 SalI BamHI SV40 Enhancer (for luc+ reporter) HpaI 2094 poly(A) signal SV40 late XbaI 1934 ori pGL3-Control Vector (5256bp) luc+ Ampr f1 ori Size Cat.# Price (Fr) 20 µg E2231 244.00 20 µg E2241 244.00 20 µg E2261 244.00 20 µg E2271 244.00 pGL3 Luciferase Reporter Vectors Product pGL3-Basic Vector pGL3-Control Vector pGL3-Enhancer Vector pGL3-Promoter Vector For Research Use Only. Not for Use in Diagnostic Procedures. Description: The pGL3 Luciferase Reporter Vectors provide a basis for the quantitative analysis of factors that potentially regulate mammalian gene expression. These may be cis- or trans-acting factors. The backbone of the pGL2 Luciferase Reporter Vectors was redesigned for the pGL3 Vectors for increased expression, with a modified coding region for firefly (Photinus pyralis) luciferase that has been optimized for monitoring transcriptional activity in transfected eukaryotic cells. The assay of this genetic reporter is rapid, sensitive and quantitative. In addition, the Luciferase Reporter Vectors contain numerous features aiding in the structural characterization of the putative regulatory sequences under investigation. For the most advanced reporter vectors and widest selection of features, please see the pGL4 Luciferase Reporter Vectors. Features: • Easy to Use: NcoI site located at 5´ end of luc+ gene allows creation of fusions with reporter gene using a unique NcoI site. • Flexible: Placement of SmaI site in the MCS allows blunt-ended inserts to be ligated into the MCS and restricted on either side by other restriction endonucleases. • Versatile: XbaI site just downstream of luc+ gene facilitates insertions into the 3´ untranslated region of mRNA or subcloning of the luciferase gene. Storage Conditions: Store vectors at –20°C. Protocol pGL3 Luciferase Reporter Vectors Technical Manual Part# TM033 Synthetic poly(A) signal / transcriptional pause site (for background reduction) KpnI SacI MluI NheI SmaI XhoI BgIII 5 11 15 21 28 32 36 SV40 Promoter HindIII 245 NcoI 278 NarI 313 Size Cat.# Price (Fr) 20 µg E1751 592.00 20 µg E1741 592.00 20 µg E1771 592.00 20 µg E1761 592.00 15 For complete and up-to-date product information visit: www.promega.com/catalog 275 Reporter Assays and Transfection 1352VA01_6A 0747VA08_4A Pagina 278
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