pEco-Lenti-U6-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-U6-(RFP-Puro) vector via T4 enzyme ligation.
The expression of shRNA was driven by a constitutive human U6 promoter, a RNA polymerase III promoter. This Kit provides materials for generating 10 of your own shRNA expression lentivectors with the following advanced features.
shRNA vector cloning scheme:
pEco-Lenti-U6-(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;
- Lentiviral shRNA vector allows generate shRNA lentiviral particles for delivering shRNA into any kinds of mammalian cell types for both in vivo or in vitro analysis of gene silencing effects.
Please see Product Manual for details.
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