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

Catalog number:  V47020

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Using the pSCREEN-iT™/lacZ-DEST Gateway® Vector Kit, you can:

•Assess the potency of any RNAi knockdown reagent using a fast, sensitive, and cost-effective β-gal reporter assay
•Identify the best knockdown reagent for your 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 (Figure 1). The more effective any specific knockdown reagent is at mediating RNAi, the more degradation of the fusion mRNA will occur, lowering β-gal activity (Figure 2).

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


Control Type: Gene Knockdown Positive Control, Negative Control
Product Line: BLOCK-iT™, Gateway®
Product Size: 20 reactions
Promoter: CMV
Reporter: Beta-Gal (lacZ)
Sample Type (Specific): RNAi Plasmids (shRNA, miR), Synthetic siRNA
Validated Application: RNAi Target Screening

Contents & 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.


Manuals & protocols