pEco-Lenti-H1-shRNA-(RFP-Puro) 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-H1-(RFP-Puro) vector via T4 enzyme ligation.
The expression of shRNA was driven by an optional inducible human H1 promoter, a RNA polymerase III 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-H1-(RFP-Puro) linear vector: 10 ul (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 (10rxn, 1ul/rxn)
Sh-Sequencing primer: 10 ul (10xn, 1ul/rxn)
Material required but not included:
DH5a 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);
Internal transfection efficiency reference: the vector encode a RFP fluorescent protein, allowing real-time monitoring and normalizing the transfection differences;
Long-term stable silencing effect: the vector encoded Puromycin (Puro) resistance marker which is fusioned with RFP, allowing to generate stable cell lines for long-term knockdown of your target;
- Optional inducible knockdown: the vector’s human H1 promoter was integrated with two Tet-repressor binding-sites (TRBS), allowing inducible expression of shRNA by tetracycline. Note: this vector can be used for constitutive high level expression of shRNA. If used as inducible expression, you need another repressor (TetR) expression vector or virus. The shRNA expression was repressed in the presence of TetR and induced by tetracycline. The expression of TetR can be achieved by using the Tet-repressor stable cell line (Cat# SC005 ) or using Gentarget’s pre-made lentiviral particles, Tet-repressor, or co-transfection with the TetR expression vectors.
- 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.
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