Last data update:
22 October 2019 04:14 CEST
|Name:||pLKO.1-puro-shRNA Add to cart|
|Accession number:||LMBP 7210|
|Sequence file:||Depositor's sequence: p7210.gb View with Genome Compiler|
|Status:||GeneCorner non-core plasmid|
|Cloned DNA:||Target (human or mouse) short hairpin RNA (shRNA) sequence
Human immunodeficiency virus pol gene; central polypurine tract (HIV-1 cPPT)
|Promoter:||Human U6 small nuclear RNA gene promoter
Human phosphoglycerate kinase 1 promoter (PGK1)
Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 5' LTR)
Phage T7 gene 10 promoter (T7g10)
Phage T3 promoter
Escherichia coli lac operon promoter
|Terminator:||Rous sarcoma virus/human immunodeficiency virus hybrid long terminal repeat (RSV/HIV-1 3' LTR); modified HIV-1 3' LTR with deleted U3 region (ΔU3/3' LTR)
Simian virus 40 polyadenylation signal (SV40 polyA)
|Selection marker:||Ampicillin (amp)
|Replicon:||Escherichia coli plasmid pMB1 origin
Simian virus 40 bidirectional origin (SV40)
Phage f1 origin
|Host range:||Escherichia coli
Mammalian cells; SV40 permissive cells
|Parental clone:||SHC001 (empty vector)|
|Further information:||This datasheet is representative for all the MISSION TRC1/1.5 shRNA clones developed at the Broad Institute of MIT and Harvard (USA), purchased from Sigma-Aldrich (Germany) and available at BCCM/GeneCorner. Consequently, the accession number '7210' of this template clone is fictitious.
pLKO.1-puro-shRNA is a lentiviral plasmid derived from pLKO.1-puro (= SHC001 (empty vector)) by cloning a short hairpin RNA (shRNA) sequence into the AgeI and EcoRI sites.
For all TRC1.5 clones and for most but not all TRC1 clones, the EcoRI site was restored during ligation.
The shRNA insert is expressed under control of the human U6 promoter and targets a human or a mouse gene.
The data related to a specific shRNA insert is online accessible via www.sigma.com/yfg.
Search Your Favorite Gene to locate shRNAs that target your gene. Look for the validated symbol on the shRNA clones detail pages to identify functionally validated shRNAs.
For each target gene, a set of minimum 3 to 5 shRNA clones is available, providing varying levels of knockdown and targeting different regions of mRNA transcript. Sigma-Aldrich guarantees that at least one of the clones of such a set should yield more than 70% knockdown.
For a given RefSeq, there is often a shRNA clone targeting the 3' UTR, which is useful in phenotypic rescue studies using cDNA expression constructs.
Grow for 20-22 hours in TB-8gly medium (modified Terrific Broth medium, containing 8 ml/l glycerol instead of 4 ml/l) with 100 μg/ml carbenicillin, resulting in an increased plasmid stability as compared to ampicillin.
BCCM/GeneCorner uses the 'Terrific Broth, modified' from Sigma (cat. nr. T0918, Fluka). You can also prepare the medium yourself:
1. add the following to 800 ml distilled H2O:
- 12,0 g tryptone (enzymatic casein digest)
- 24,0 g yeast extract
- 8 ml glycerol
2. adjust to 900 ml with distilled H2O
3. sterilize by autoclaving (20 minutes at 121°C)
4. allow to cool to room temperature
5. adjust volume to 1000 ml with 100 ml of a filter-sterilized solution of 0,17M KH2PO4 and 0,72M K2HPO4
HOST STRAIN INFORMATION
DH5αT1R is a T1 bacteriophage resistant DH5α derivative. Sigma sells the identical host strain (i.e. identical genotype) as GC5 (catalogue number: G3169).
Genotype of DH5αT1R: Φ80lacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 relA1 gyrA96 hsdR17(rk-, mk+) phoA supE44 thi-1 tonA (Ref.: http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/shclngbul.Par.0001.File.tmp/shclngbul.pdf)
The presence of a G nucleotide at the end of the nucleotide sequence of the shRNA insert on the Your Favorite Gene (http://www.sigmaaldrich.com/life-science/your-favorite-gene-search.html) website indicates the EcoRI site has been restored.
In the compiled nucleotide sequence of this template shRNA plasmid, the shRNA sequence is replaced by a 57 bp stretch 'CCGGNNNNNNNNNNNNNNNNNNNNNCTCGAGNNNNNNNNNNNNNNNNNNNNNTTTTT', consisting of a 21 base stem and a 6 base loop.
The shRNA insert of each clone was confirmed by sequence analysis at the Broad Institute of MIT and Harvard.
|EMBL Accession number:||-|
|Latest sequence update:||28/02/2011|
|History of deposit:||This plasmid was purchased from Sigma-Aldrich(1) and deposited in the frame of the Hercules project 'RNAi collection and associated facilities' by Prof. Dr R. Beyaert(2)(3).
(1) Schnelldorf, Germany, www.sigmaaldrich.com
(2) Inflammation Research Center, VIB, Ghent, Belgium
(3) Department of Biomedical Molecular Biology, Ghent University, Ghent, Belgium
|Related plasmid reference:||Moffat et al., Cell 124 (2006), 1283-1298 [PMID: 16564017]
Stewart et al., RNA 9 (2003), 493-501 [PMID: 12649500]
|Restricted distribution:||- Restricted to project-related users
- The buyer cannot sell or transfer this product, its components nor materials made using this product or its components to another research group.
- Visit www.sigma.com/shRNA for more, up-to-date info on the limited use of this product.
- Acknowledge BCCM/GeneCorner (supplier) and the Hercules Foundation (funder) in any publication resulting from the use of a shRNA clone
|Distributed as:||H/P active culture or plasmid DNA|
|Host for distribution:||Escherichia coli K12 DH5αT1R|
|Cultivation medium:||TB-8gly + ampicillin (100 μg/ml)|
|Biosafety level:||L1 in E. coli; L2 in mammalian cells|
|Other culture collection numbers:||-|
Note: Up-to-date, validated data are enclosed with the biological material. Nevertheless, these data are a snapshot at a given moment; further updates are always possible.