Invitrogen™ pSCREEN-iT™/lacZ-DEST Gateway™ Vector Kit

Description

For RNAi target screening

Use the pSCREEN-iT™/lacZ-DEST Gateway™ Vector Kit to assess the potency of any RNAi knockdown reagent using a fast, sensitive, and cost-effective β-gal reporter assay or identify the best knockdown reagent for RNAi experiments without knowing protein function, western blotting, or qRT-PCR.
How it Works: The BLOCK-iT RNAi Target Screening system method uses a β-galactosidase activity assay to measure the knockdown ability of an RNAi reagent (Stealth RNAi™ siRNA, siRNA, dicer pools, or shRNA-containing plasmid). To identify the most effective knockdown reagent for your target, simply clone your target gene into the pSCREEN-iT /lacZ-DEST Gateway vector. Co-transfect the resulting construct with the knockdown reagent being tested. Expressed genes will be fused to β-galactosidase, enabling you to use β-galactosidase levels to measure the amount of RNAi-induced degradation of the target gene. The more effective any specific knockdown reagent is at mediating RNAi, the more degradation of the fusion mRNA will occur, lowering β-gal activity.
The BLOCK-iT RNAi Target Screening System includes the pSCREEN-iT /lacZ-DEST Gateway vector for generating a construct containing a lacZ/gene of interest fusion. This vector features:

• attR sites immediately following the lacZ ORF for fast and efficient recombination with any attL-flanked Gateway entry vector to generate the lacZ/target gene fusions
• The CMV promoter for strong expression of the lacZ/target gene fusion, leading to increased assay sensitivity and better discrimination between knockdown reagents of varied effectiveness
• Positional cloning to fuse a gene or fragment to the 3' end of the lacZ gene so that β-galactosidase will be expressed even if the target fragment being tested contains stop codons

In addition, the pSCREEN-iT /lacZ-DEST vector is fully compatible with the Ultimate™ ORF Clone Collection. This makes a wide selection of potential target genes readily available for generating lacZ fusions.

RNAi, RNAi, Epigenetics and Non-Coding RNA Research, Vector-Based RNAi

Specifications

• Format: Kit
• Promoter: CMV
• Product Type: β-Galactosidase Reporter Gene Assay System
• Reporter Gene: Beta-Gal (lacZ)
• Content And Storage: The pSCREEN-iT™ /lacZ-DEST Gateway™ Vector Kit contains 6 μg of vector and 10 μg of pSCREEN-iT™/lacZ-GW/CDK2 control vector, the positive control Stealth™RNAi lacZ, a negative control Scrambled Stealth RNAi™ siRNA, and 1X annealing/dilution buffer. Store at -20°C. The BLOCK-iT™ RNAi Target Screening Kit contains the pSCREEN-iT™/lacZ-DEST Gateway™ Vector Kit, 250 μl of Lipofectamine™ 2000 Reagent, and the FluoReporter™ lacZ/Galactosidase Quantification Kit. Store the Lipofectamine™ 2000 Reagent at +4°C. Store the FluoReporter™ Kit at -20°C protected from light. The BLOCK-iT™ RNAi Target Screening System contains the BLOCK-iT™ RNAi Target Screening Kit, LR Clonase™ enzyme mix, and pCR ¤ 8/GW/TOPO ¤ TA Cloning Kit. Store the LR Clonase™ enzyme mix at -80°C. The pCR™ 8/GW/TOPO™ TA Cloning Kit includes the ready-to-use pCR™ 8/GW/TOPO™ vector, dNTPs, salt solution, water, forward and reverse sequencing primers, control template and PCR primers, One Shot™ TOP10 Chemically Competent E. coli, S.O.C. medium, and a pUC19 control plasmid. Store the Competent Cells at -80°C. Store all other components at -20°C. All components are guaranteed stable for 6 months when properly stored.
• Sample Type: Synthetic siRNA, RNAi Plasmids (shRNA, miR)
• Quantity: 20 Reactions
• Product Line: BLOCK-iT™, Gateway™
• For Use With (Application): RNAi Target Screening

For Research Use Only. Not for use in diagnostic procedures.