Psuperior puro vector

Manufactured by Oligoengine

The PSUPERIOR.puro vector is a laboratory equipment product designed for molecular biology applications. It serves as a vehicle for the introduction and expression of genetic material in cells. The core function of this product is to facilitate the delivery and integration of target DNA sequences into host cells.

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Lab products found in correlation

Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions) Lipofectamine ltx with plus reagent, by Thermo Fisher Scientific (1 mentions) Sirna oligonucleotides, by Eurofins (1 mentions) Puromycin, by Thermo Fisher Scientific (1 mentions) Sigenome non targeting sirna, by Thermo Fisher Scientific (1 mentions) Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions) Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions) Plus reagent, by Thermo Fisher Scientific (1 mentions)

Close spelling variants of this product

PSUPERIOR.retro.puro vector PSUPERIOR-puro

4 protocols using psuperior puro vector

BRUCE Knockdown in U2OS Cells

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siRNA targeting BRUCE (GGCACAGCAGCTCTTATCA) was used to generate the shRNA: 5’-atcccGGCACAGCAGCTCTTATCAttcaagagaTGATAAGAGCTGCTGTGCCttttta-3’ and 5’-tcgaatttttCCGTGTCGTCGAGAATAGTaagttctctACTATTCTCGACGACACGGcc-3’. Eight tandem repeats of each shRNA, with an H1 promoter, were cloned into pSUPERIOR.puro vector following the manufacturer’s instruction (Oligoengine). The construct was then transfected with Lipofectamine 2000 into a U2OS cell line in which a Tet repressor (TetR) was stably expressed. The transfected cells were selected for clones that integrated with both the shRNA construct and TetR by puromycin (1 μg/ml) and blasticidin (5 μg/ml), respectively. More than six positive stable clones were identified by immunoblotting in which DOX (1 μg/ml) treatment for 4–6 days ablated BRUCE expression, and clone #16 was used in this study and results confirmed in other clones.

Ge C., Che L, & Du C. (2015). The UBC Domain Is Required for BRUCE to Promote BRIT1/MCPH1 Function in DSB Signaling and Repair Post Formation of BRUCE-USP8-BRIT1 Complex. PLoS ONE, 10(12), e0144957.

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siRNA Knockdown and Plasmid Rescue Assay

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The siRNA transfections were performed with 50 nM siRNA duplexes using Lipofectamine RNAiMAX (Invitrogen). Samples were collected 60 h after transfection unless otherwise stated. The siRNA oligonucleotides were obtained from Eurofins Genomics (MWG). For annotations and sequences see Supplementary Table S1. The TCOF1 STTT, S1216A+S1199A and S1216A expression plasmids were previously described (28 (link),29 (link)) and primer sequences used for the generation of the ATM-null construct can be found in Supplementary Table S1. The shRNA against TCOF1 was generated by insertion of an oligonucleotide into the pSUPERIOR.puro vector (Oligoengine) (28 (link)). Cells were transfected in a 4:1 ratio of shTCOF1 and the rescue plasmid (either TCOF1 WT, TCOF1 STTT, TCOF1 ATM-null, TCOF1 S1216A+S1199A or TCOF1 S1216A) using Lipofectamine LTX with plus reagent (Invitrogen) according to the manufacturer's specifications. After 72 h cells were transfected with gRNAs and collected at indicated time points.

Korsholm L.M., Gál Z., Lin L., Quevedo O., Ahmad D.A., Dulina E., Luo Y., Bartek J, & Larsen D.H. (2019). Double-strand breaks in ribosomal RNA genes activate a distinct signaling and chromatin response to facilitate nucleolar restructuring and repair. Nucleic Acids Research, 47(15), 8019-8035.

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Silencing PARP-1 and Snail in MDA-MB-231 Cells

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MDA-MB-231 were transfected with Lipofectamine 2000 reagent (Life Technologies, Invitrogen) adopting the manufacturer's protocol. For silencing experiments, cells were transfected with siGENOME SMARTpool PARP-1 and siGENOME Non-Targeting siRNA (Thermo Scientific, Dharmacon) at a final concentration of 50 nM. To generate Snail-deficient cells, MDA-MB-231 were transfected with shRNA containing specific oligonucleotide sequences against EGFP (22 nt) or human Snail (19 nt: 5′-GATGCACATCCGAAGCCAC-3′) cloned into the pSuperior-Puro vector (Oligoengine). The selection was obtained with puromycin 1 μg/ml (Invitrogen) for 2–4 weeks. Appropriate expression levels of Snail were confirmed by immunoblotting.

Mariano G., Ricciardi M.R., Trisciuoglio D., Zampieri M., Ciccarone F., Guastafierro T., Calabrese R., Valentini E., Tafuri A., Del Bufalo D., Caiafa P, & Reale A. (2015). PARP inhibitor ABT-888 affects response of MDA-MB-231 cells to doxorubicin treatment, targeting Snail expression. Oncotarget, 6(17), 15008-15021.

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siRNA Transfection and Mutagenesis Protocol

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The siRNA oligonucleotides were obtained from Microsynth AG. For annotations and sequences see Supplementary Table 1. siRNA oligonucleotides targeting MRE11 were from Ambion (MRE11 siRNA-59: AMBION 4427038, siRNA ID#:8959; MRE11 siRNA-60, AMBION 4427038, siRNA ID#:8960). The siRNA transfections were performed with 50 or 100 nM siRNA duplexes using Lipofectamine RNAiMAX (Invitrogen). Samples were harvested 72 h after initiation of transfection unless stated otherwise. For mutagenesis primer sequences see Supplementary Table 1. shRNA was generated by insertion of an oligo (see Supplementary Table 1) into the pSUPERIOR.puro vector (Oligoengine). Cells were transfected with Lipofectamine LTX with plus reagent (Invitrogen) according to manufacturer’s specifications and harvested 4-5 days post transfection.

Larsen D.H., Hari F., Clapperton J.A., Gwerder M., Gutsche K., Altmeyer M., Jungmichel S., Toledo L.I., Fink D., Rask M.B., Grøfte M., Lukas C., Nielsen M.L., Smerdon S.J., Lukas J, & Stucki M. (2014). The NBS1-Treacle complex controls ribosomal RNA transcription in response to DNA damage. Nature cell biology, 16(8), 792-803.

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