The BLOCK-iT™ U6 RNAi Entry Vector provides a simple, streamlined approach to cloning short hairpin RNA (shRNA) sequences for testing in transient transfections for RNA interference (RNAi), a preferred technique for analyzing gene downregulation. An easy cloning process places an ~50-bp oligonucleotide DNA immediately following a U6 pol III type promoter (Figure 1). Expression of this RNAi cassette forms a shRNA molecule in the cell that will be processed and act as short interfering RNA (siRNA) that will generate the RNAi effect.
Delivery of the U6 RNAi Cassette Once cloning is complete, the U6 Entry Vector is ready to be used immediately in transient transfections using a reagent such as Lipofectamine™ 2000. This makes this system ideal for initial shRNA screenings in many mammalian cell types. As an alternative in hard-to-transfect or non-dividing cell types, or to deliver to animal model systems, the RNAi cassette can easily be recombined into a BLOCK-iT™ Viral RNAi vector. For stable shRNA lentiviral delivery and expression, recombine the BLOCK-iT™ U6 Entry Vector with the pLenti6/BLOCK-iT™ RNAi Vector. For transient delivery to challenging cell types using adenoviral transduction, recombine with the pAd/BLOCK-iT™ RNAi Vector.
For Research Use Only. Not for use in diagnostic procedures.
Constitutive or Inducible System:
BLOCK-iT RNAi Vectors
Contents & storage
The BLOCK-iT™ U6 Entry Vector Kit contains two boxes. The cloning box contains four tubes of linearized vector at 5 ng/tube, enough to complete 5 cloning reactions per tube. Also included are 250 µl 10X annealing buffer, 20 µl T4 DNA ligase, 80 µl 5X DNA ligase buffer, 1.4 ng lacZ control oligo, 10 µl lyophilized lacZ control plasmid, 1.5 ml DNase/RNase-free water, and 2 µg each of forward and reverse sequencing primer. Store the vectors, buffers, control oligo and plasmid, water, and sequencing primers at -20°C. The One Shot® box contains transformation reagents including twenty-one 50-µl aliquots of One Shot® TOP10 Chemically Competent E. coli, S.O.C. medium, and a pUC19 supercoiled control plasmid. Store at -80°C. All reagents are guaranteed stable for 6 months when properly stored.