Decoy oligonucleotides containing the palindromic p53 cis-element 5′-RRRCWWGYYY-3′ (R = A or G, Y = C or T, W = A or T), which allows self-hybridization and formation of a duplex hairpin that competes with p53 enhancers for binding of transcription factors, were used to inhibit p53-directed transcription in vivo, as previously described by us57 (link). The sequences of the p53 decoy and control phosphorothioate oligonucleotides (underscored) (Sigma-Aldrich) were as follows: p53 decoy, 5′-GGACA TGCC CGGGC ATGTCC-3′; control nonsense sequence, 5′-CTA GCTA GCTA GCTA GCTA GCTAG-3′. Cells were seeded at a density of 106 cells onto in 100 mm Petri dishes (Sigma-Aldrich), differentiated as above. After that cells were collected and 800 μl of 2.5 × 107 cell/ml, electroporated in presence of 500 μg sheared salmon sperm DNA (Sigma-Aldrich) and 1 μM of each phosphorothioate oligonucleotide at 330 V and 1000 uF using an Electroporator II (Invitrogen). Next, cells were plated onto 6-well plates (Sigma-Aldrich), grown in a complete medium overnight followed by its replacement with 1% FCIII containing 100 μM OT for 72 h and analysis using RT-PCR as said above.
Electroporator 2
Manufactured by Thermo Fisher Scientific
The Electroporator II is a laboratory instrument used for the introduction of nucleic acids, proteins, or other molecules into cells through a process called electroporation. It generates controlled electrical pulses to temporarily permeabilize the cell membrane, allowing the transfer of the desired substance into the cell.
Automatically generated - may contain errors
2 protocols using electroporator 2
1
Inhibiting p53-directed Transcription In Vivo
2
Transfection of PC12 Cells by Electroporation
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PC12 cells were cultured in DMEM (Sigma-Aldrich) supplemented with 5% horse serum and 5% calf serum at 37°C in a 10% CO2 atmosphere at constant humidity. PC12 cells (grown to ∼80% confluency in a 10-cm dish) were suspended in 0.5 ml of cytomix buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], 120 mM KCl, 10 mM KH2PO4, 0.15 mM CaCl2, 5 mM MgCl2, 2 mM ethylene glycol tetraacetic acid, pH 7.6) and were transfected with 10–50 μg of plasmid DNA. Transfections for plasmid DNA or siRNA were performed by electroporation using an ECM 830 (BTX, Holliston, MA) set at 8 ms, one pulse at 230 or 90 V, respectively. Alternatively, transfections were conducted with 10 μg of CAPS shRNA plasmid or empty vector using an Electroporator II (Invitrogen, Carlsbad, CA) set at 71 μF and 330 V. Cells were plated on poly-l -lysine and collagen-coated 35-mm glass-bottom dishes (MatTek, Ashland, MA) or six-well dishes for 72–96 h with subsequent TIRF analysis or Western blotting.
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