Science Catalog 2012 Life Cell Signaling pSP73 Vector Product pSP73 Vector For Research Use Only. Not for Use in Diagnostic Procedures. Description: The pSP73 Vector offers a wide range of restriction sites, providing greater versatility in cloning and transcription of RNA in vitro. The pSP73 Vector contains the SP6 and T7 RNA polymerase promoters and a unique multiple cloning region, which includes restriction sites for BglII, EcoRV, ClaI, EcoRI, SacI, KpnI, SmaI, BamHI, XbaI, SalI, AccI, PstI, SphI, HindIII, PvuII and XhoI. The pSP72 and pSP73 Vectors are essentially identical except for the orientation of the multiple cloning region. Features: • Versatile: This vector can be used for standard cloning and in vitro transcription from SP6 and T7 RNA polymerase promoters flanking the multiple cloning region. • Convenient: Multiple cloning site provides a selection of restriction sites for cloning. Storage Conditions: Store vector at –20°C. Protocol pSP73 Vector Technical Bulletin XmnI 1809 ScaI 1690 PvuI 1580 FspI 1432 BglI 1330 Ampr (2,464bp) pSP73 Vector HpaI 138 ori SspI 2014 AatII 2132 NdeI 2381 SP6 BglII EcoRV ClaI EcoRI SacI KpnI SmaI BamHI XbaI SalI AccI PstI SphI HindIII PvuII XhoI T7 103 14 19 24 34 40 42 45 51 57 58 67 73 75 83 87 1 start 6 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000 10,000 0 BL21(DE3) BL21(DE3) pLysS B. 10,000 1,700 1,000 100 14 10 1 8 Rosetta™ 2 pLysS KRX Part# TB041 Size Cat.# Price (Fr) 20 µg P2221 186.00 Bacterial Strains and Competent Cells Product Bacterial Strain ES1301 mutS, Glycerol Stock (noncompetent) Bacterial Strain BMH 71-18 mutS, Glycerol Stock (noncompetent) Bacterial Strain JM109, Glycerol Stock Bacterial Strain JM109(DE3), Glycerol Stock Bacterial Strain LE392, Glycerol Stock Bacterial Strain NM522, Glycerol Stock Single Step (KRX) Competent Cells L-Rhamnose Monohydrate JM109 Competent Cells, >107cfu/µg JM109 Competent Cells, >108cfu/µg HB101 Competent Cells, >108cfu/µg BL21(DE3)pLysS Competent Cells, >106cfu/µg A. 100 120 140 20 40 60 80 0 Pre-Induction Expression Size Cat.# Price (Fr) 200 µl Q6131 79.00 500 µl Q6321 79.00 500 µl P9751 75.00 500 µl P9801 75.00 500 µl K9981 79.00 500 µl P2301 75.00 20 × 50 µl L3002 406.00 10 g L5701 68.00 50 g L5702 289.00 1 ml L1001 198.00 1 ml L2001 264.00 1 ml L2011 239.00 1 ml L1191 340.00 43 BL21(DE3) BL21(DE3) pLysS Rosetta™ 2 pLysS KRX Pre-induction and post-induction expression levels of firefly luciferase. Cells were transformed with the pF1K T7 Flexi® Vector containing the firefly luciferase gene. Cultures were grown at 37°C to an optical density (O.D.600) of 0.8–1.0 and then moved to a 25°C incubator shaker. When cultures reached an O.D.600 of 1.0–1.5, protein expression was induced using either 0.1% rhamnose or 1mM IPTG and grown overnight at 25°C. Samples for luciferase assays were removed prior to and after induction. Panel A. Firefly luciferase expression level was determined using the Bright-Glo™ Luciferase Assay Reagent. Pre- and post-induction firefly luciferase expression levels were normalized to cell number (n = 3). Panel B. Induction ratios were calculated by dividing the post-induction luminescence values by the pre-induction values. 104 For complete and up-to-date product information visit: www.promega.com/catalog 0300VA05_2A Induction Ratio Normalized luminescence (RLU/OD) Normalized luminescence (RLU/OD) 6032MA KRX Roset t a™ 2 pLysS BL21(DE3) BL21(DE3) pLysS Pagina 107
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