Cell Signaling RNA Interference GeneClip™ U1 Hairpin Cloning Systems Product GeneClip™ U1 Hairpin Cloning System— Puromycin GeneClip™ U1 Hairpin Cloning System— Hygromycin GeneClip™ U1 Hairpin Cloning System— Neomycin Size Cat.# Price (Fr) GeneClip™ U1 Hairpin Cloning System—Basic 1 system C8750 557.00 1 system C8760 557.00 1 system C8770 557.00 1 system C8780 557.00 GeneClip™ U1 Hairpin Cloning System—hMGFP 1 system C8790 649.00 For Research Use Only. Not for Use in Diagnostic Procedures. Description: The GeneClip™ U1 Hairpin Cloning Systems consist of linearized plasmids designed for fast and easy cloning of human target sequences to express short hairpin RNAs (shRNAs) in human cells. After transfection into human cells, in vivo expression of short interfering RNAs (siRNAs) can be effectively achieved from DNA constructs that contain a U1 RNA polymerase promoter and a siRNA template. The U1 promoter has been used successfully to generate hairpin siRNAs in vivo. To insert hairpin siRNAs into the pGeneClip™ Vectors, two short DNA oligonucleotides are annealed to form a DNA insert that contains the hairpin siRNA target sequence. After annealing, the oligonucleotides form overhangs that are compatible with the pGeneClip™ Vector ends and facilitate sticky-end ligation. Once transfected, RNA polymerase II transcribes the hairpin insert sequences to generate hairpin siRNAs in vivo. Features: • More Vector Choices: These systems provide vectors containing a variety of eukaryotic antibiotic-selectable markers for stable transfection or hMGFP for determination of transfection efficiency. • Time Savings: Vectors are supplied predigested to eliminate timeconsuming vector preparation. • Convenience: Each system includes T4 DNA Ligase, 2X Rapid Ligation Buffer, Oligo Annealing Buffer and the pGeneClip™ Vector. • Easier Identification of Desired Clones: A PstI digestion quickly identifies positive recombinants. Storage Conditions: Store at –20°C. Protocol GeneClip™ U1 Hairpin Cloning Systems Technical Manual T7 RiboMAX™ Express RNAi System Product Size Cat.# Price (Fr) T7 RiboMAX™ Express RNAi System 50 × 20µl reactions P1700 832.00 For Research Use Only. Not for Use in Diagnostic Procedures. Description: The T7 RiboMAX™ Express RNAi System is an in vitro transcription system designed for quickly producing milligram amounts of double-stranded RNA (dsRNA). The dsRNA is free of protein and other contaminants and is suitable for use in RNA interference (RNAi) in both mammalian and nonmammalian systems. The T7 RiboMAX™ Express RNAi System can be used to synthesize short interfering RNAs (siRNAs) of 21bp for use in mammalian systems. siRNAs synthesized in vitro have been demonstrated to be as effective as chemically synthesized siRNAs for inducing RNAi in mammalian cells. In addition, the T7 RiboMAX™ Express RNAi System can be used for the synthesis of dsRNA molecules of 200bp or greater, for use in nonmammalian systems. Two complementary RNA strands are synthesized from DNA template and annealed after the transcription reaction to form dsRNA. Any remaining single-stranded RNA and DNA template are removed with a nuclease digestion step. The dsRNA is then purified by isopropanol precipitation and can be introduced into the organism of choice for RNAi applications. For complete and up-to-date product information visit: www.promega.com/catalog 291 Part# TM256 100 20 40 60 80 0 Syn #1 IVT #1 Syn #2 IVT #2 siRNAs Comparison of RNA interference induced by siRNAs synthesized chemically and by in vitro transcription. Two different target luciferase sequences were synthesized by in vitro transcription using the T7 RiboMAX™ Express RNAi System (IVT #1 and #2) and synthesized chemically (Syn #1 and #2). After transfection using CodeBreaker™ Transfection Reagent, these siRNAs were evaluated for RNA interference in CHO cells stably expressing luciferase. Site 1 Site 2 Features: • Save Time: The T7 RiboMAX™ Express RNAi System produces milligram amounts of RNA in as little as 30 minutes. • Minimize Pipetting Errors: The four rNTPs and 2X transcription buffer have been combined, thus minimizing pipetting errors and setup time. Storage Conditions: Store all components at –20°C, except RNase A, which should be stored at 22–25°C after the initial thaw. Protocol T7 RiboMAX™ Express RNAi System Technical Bulletin Part# TB316 16 RNA Analysis % Decrease in Renilla Activity 4218MA06_3A Pagina 294
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