HLE cells were cultured in 6-well plates to approximately 80% confluency, 1ug of either PGL4-CRYAA Promoter or PGL4 plasmid and 30ng pGL4.75 [hRluc/CMV] were transfected into cells using a LipoJet Transfection Kit (SignaGen). Seventy two hours after transfection luciferase activities were checked using a dual-luciferase reporter assay system (Promega) according to the supplier’s protocol.
Lipojet transfection kit
Manufactured by SignaGen
Sourced in United States
The LipoJet Transfection Kit is a laboratory product designed to facilitate the delivery of genetic material, such as DNA or RNA, into cells. The kit provides the necessary reagents and protocols to perform lipid-mediated transfection, a widely used technique in molecular biology and cell biology research.
Automatically generated - may contain errors
Lab products found in correlation
Dual luciferase reporter assay system, by Promega (2 mentions)
Pcr2.1 topo, by Thermo Fisher Scientific (1 mentions)
Hindiii, by New England Biolabs (1 mentions)
Pgl4.17 vector, by Promega (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Rq1 dnase, by Promega (1 mentions)
4d nucleofector system, by Lonza (1 mentions)
Rnase a, by Thermo Fisher Scientific (1 mentions)
5 protocols using lipojet transfection kit
1
Luciferase Assay of CRYAA Promoter
2
Cloning and Characterizing EPHA2 Promoter
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The human EPHA2 proximal promoter region was amplified by the primers EPHA2-Promoter-F and EPHA2-Promoter-R (above). The PCR product was then cloned into the PCR 2.1-TOPO (Invitrogen, Grand Island, NY) vector. After sequencing for verification, the EPHA2 promoter region was then cloned into the PGL4.17 vector (Promega, Madison, WI) with the restriction enzymes of Hind III and XhoI (NEB, Ipswich, MA). HLE cells were grown to 80% confluence in 6-well plates. One ug of either PGL4-EPHA2 Promoter or PGL4 plasmid were transfected into HLE cells along with 30 ng pGL4.75 [hRluc/CMV] using a LipoJet Transfection Kit (SignaGen, Gaithersburg, MD). Forty-eight or Seventy-two hours after transfection luciferase activities were tested using dual-luciferase reporter assay system (Promega, Madison, WI) per the manufacturer’s suggested protocol.
3
Plasmid and siRNA Transfection Protocols
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Plasmid transfections (2 µg for each plasmid) were performed with FuGENE HD transfection reagent (Roche Diagnostics) for CaSki and C33A cells in a 6-cm dish and with lipod293 transfection reagent (version II) (SignaGen Laboratories, Gaithersburg, MD) for HEK293 and HeLa cells. siRNA (40 nM) was transfected with a LipoJet transfection kit (SignaGen Laboratories), with a 48-h interval if the second transfections were needed for an efficient knockdown. For human primary keratinocytes, 2 µg of each plasmid was transfected for 1 × 106 cells with a 4D-Nucleofector system (Lonza Cologne GmbH, Cologne, Germany) according to the nucleofection protocol designed for human keratinocytes. For the immunoprecipitation, cells were collected in RIPA buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 0.5% NP-40). Following sonication, the cell lysate was treated with RQ1 DNase (Promega, Fitchburg, WI) and RNase A (Life Technologies) and incubated with antibody-conjugated Sepharose 4B (Sigma-Aldrich) for 1 h at 4°C. Precipitated products were then washed with RIPA buffer and eluted with a 2.5× SDS protein gel loading solution containing 10% β-mercaptoethanol for Western blotting.
4
Genotyping and siRNA Transfection in Mice
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All mice were genotyped by PCR analysis of genomic DNA extracted from tails or embryonic tissues using the primers listed in Table S1 . PCR reactions were carried out by standard protocols.
Specific siRNA sequences and controls were designed and synthesized by GenePharma (Shanghai, China), as indicated inTable S2 . Transfection was carried out at 60-70% confluency. Transfection of melb-a cells was carried out using a LipoJet Transfection Kit (SignaGen Laboratories, SL100468), according to the manufacturer's instructions.
Specific siRNA sequences and controls were designed and synthesized by GenePharma (Shanghai, China), as indicated in
5
Modulating SIRT1 Expression in Human Lymphatic Endothelial Cells
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HLECs were seeded in 6-well plates and transfected with small interfering RNAs (siRNAs) or lentiviral vectors (LVs). The siRNAs (40 nM) were transfected into HLECs using a lipojet transfection kit (SignaGen, USA) according to the manufacturer's protocol. The siRNA sequences were designed and synthesized by Gene Pharma Co., Ltd (Shanghai, China). The pCMV-EGFP-puro vector and pCMV-SIRT1-EGFP-puro vector, which encoded full-length human SIRT1, were obtained from Genechem Co., Ltd (Shanghai, China). HLECs were infected with the lentivirus at a MOI = 100 in the DMEM containing Polybrene (5 g/mL). The HCEC cell line stably overexpressing SIRT1 was obtained by puromycin selection (2 µg/mL). Sequences of the siRNAs are given in the Supplementary Table S1 .
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