pEco-Lenti-U6-shRNA-(Blasticidin) Cloning Kit is designed to cloning of double-strand DNA encoding a short hairpin RNA. Once transcribed, the shRNA was processed into short RNA in vivo for RNAi analysis. To make shRNA expressing vector, two synthetic oligonucleotides were first annealed to form the double stand DNA duplex which was then cloned into the Ready-to-use, linear pEco-Lenti-U6-(Bsd) vector via T4 enzyme ligation.
The transcription of shRNA was driven by a consitutive human U6 promoter. This Kit provides materials for generating 10 of your own shRNA expression lentivectors with the following advanced features. Please see product manual for details.
shRNA vector cloning scheme:
pEco-Lenti-U6-(Blastidicin) linear vector: 10ul (10rxn, 1ul/rxn)
10x shRNA oligo annealing solution: 50 ul
5X ligation buffer: 50 ul
T4 ligase enzyme: 10 ul (10rxn, 1ul/rxn)
Cloning control insert: annealed Luc-shRNA duplex: 5 ul (5rxn, 1ul/rxn)
Sh-Sequencing primer: 10 ul (10rxn, 1ul/rxn)
Material required but not included:
DH5a or other chemical competent cells: 10 vials (10xn, 1 vial/rxn)
Linearized vector and ready for use, no need for the tedious bench works for preparation of vector backbone;
Precisely directional cloning of your DNA duplex encoded shRNA structure;
Rapid, high efficient cloning with low background (RT 30min, >90% positive rate);
- Long-term stable silencing effect: the vector encoded Blasticidin (Bsd) resistance marker which allow to generate stable cell lines for long-term knockdown of your target;
Lentiviral shRNA vector allows generate shRNA lentiviral particles for delevering shRNA into any kinds of mammalina cell types for both in vivo or in vitor analysis of gene silencing effects.
Please see product manual for details.
- Lee, R. C., et al, The C-elegans Heterochronic Gene lin-4 Encodes small RNAs with antisense complementarily to lin-14. Cell, 75(843-854), 1993.
- Hannon, G.J., RNA interference. Nature, 418(6894): p. 244-51, 2002.
- Bosher, M., et al, RNA interference, Nature Cell Biol. 2 E31-E36, 2000.
- Meister, G. and T. Tuschl, Mechanisms of gene silencing by double-stranded RNA. Nature, 2004. 431(7006): p. 343-9.
- Paddison, P.J., A.A. Caudy, and G.J. Hannon, Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci U S A, 2002. 99(3): p. 1443-8.
- SHEILA A. STEWART et al, RNA (2003), 9:493-501. 2003.