p53 variants with residues replaced by alanine at positions S15, T18 and S20 were generated by QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). The PCR products were subcloned into the pEF/myc-His vector (Thermo Scientific). Sequencing (ABI 3730 XL Sequencer) was carried out to confirm all mutations. The plasmids were introduced into Saos-2 cells using CUY21 SC Square Wave Electroporator (Nepa Gene http://www.nepagene.jp/index2.html ).
Cuy21 sc square wave electroporator
Manufactured by Nepa Gene
Sourced in Japan
The CUY21 SC Square Wave Electroporator is a laboratory equipment designed for electroporation, a technique used to introduce foreign molecules into cells. The device generates square wave pulses to create temporary pores in cell membranes, facilitating the transfer of genetic material, proteins, or other substances into the cells.
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2 protocols using cuy21 sc square wave electroporator
1
Ala-Substitution of p53 Variants
2
In Utero Electroporation of Mouse Embryos
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In utero electroporation was performed as described before.14 (link) Time pregnant (E14) CD1 wild-type mice were deeply anesthetized with isoflurane. Ketoprofen (5 mg/kg - Mibe) and Buprenorphine (0.05 mg/kg - Bayer) were used as analgesia. Uterine horns were exposed to inject each embryo once into the lateral ventricle with 1–2 μL DNA with a concentration of 1.5 μg/μL and fast green (1 mg/mL, Sigma, USA) using a pulled and beveled glass capillary (Drummond Scientific, USA) and a microinjector (Picospritzer III, Parker Hannifin Corporation, USA). Five electric pulses with 50 ms duration were applied at 950 ms intervals with a triple-electrode using a CUY21SC Square Wave Electroporator charged to 30 V (Nepa Gene, Japan). Electrode forceps were positioned to IUE cortical progenitor cells in the somatosensory and motor cortex.58 (link)
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