pLenti-U6-shRNA-(GFP-Bsd) Cloning Kit

$1,250.00

Out of stock

SKU: SH-U6-GB Category:

Description

pLenti-U6-shRNA-(GFP-Bsd) Cloning Kit is designed to cloning 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-(GFP-Bsd) vector via T4 enzyme ligation.

The expression of shRNA was driven by a constitutive human U6 promoter. This Kit provides materials for generating 10 of your own shRNA expression lentivectors with the following advanced features.  

shRNA vector cloning scheme:

Kit contents:

pEco-Lenti-U6-(GFP-Bsd) 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 (5rxn, 1ul/rxn)
Sh-Sequencing primer:                                                  10ul (10rxn, 1ul/rxn)

DH5a chemical competent cells:                        10 vials (10xn, 1 vial/rxn)

Key Features:

    1. Linearized vector and ready for use, no need for the tedious bench works for preparation of vector backbone;
    2. Precisely directional cloning of your DNA duplex encoded shRNA structure;
    3. Rapid, high efficient cloning with low background (RT 30 min, >90% positive rate);
    4. Internal transfection efficiency reference: the vector encode a GFP fluorescent protein, allowing real-time monitoring and normalizing the transfection differences;
    5. Long-term stable silencing effect: the vector encoded Blasticidin (Bsd) resistance marker which is fusioned with GFP, allowing to generate stable cell lines for long-term knockdown of your target;
    6. Lentiviral shRNA vector allows generating 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. 

References:

  1. 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.
  2. Hannon, G.J., RNA interference. Nature, 418(6894): p. 244-51, 2002.
  3. Bosher, M., et al, RNA interference, Nature Cell Biol. 2 E31-E36, 2000.
  4. Meister, G. and T. Tuschl, Mechanisms of gene silencing by double-stranded RNA. Nature, 2004. 431(7006): p. 343-9.
  5. 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.
  6. SHEILA A. STEWART et al, RNA (2003), 9:493-501. 2003.

Cat#: SH-U6-GB

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