1. Establishment and application of a standard method of electroporation for introduction of plasmid and cosmid DNAs to mammalian cells
- Author
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T. Nishimoto, M. Ohtsubo, Koki Sato, H. Hama-Inaba, and M. Kasai
- Subjects
Chemistry ,Electroporation ,Clone (cell biology) ,Biophysics ,Pulse duration ,Molecular biology ,Transformation (genetics) ,chemistry.chemical_compound ,Plasmid ,Cosmid ,Electrochemistry ,Physical and Theoretical Chemistry ,DNA ,Transformation efficiency - Abstract
In order to establish a simple and efficient method of electroporation applicable to gene transfer in mammalian cells, the effects of various pulsing conditions on viability and transformation efficiency were studied. By investigating the effects of temperature during and after the pulse, field strength, pulse length, and pulsing media, we have established a standard method: a single decaying pulse with a duration of around 1 ms at room temperature in a medium containing physiological concentrations of salts, including Ca2+ and Mg2+. Application of the standard method to various types of cells, both floating and adherent, resulted in an efficient transformation by pSV2neo DNA. Furthermore, the electroporation-mediated transformation of tsBN2 cells by a cosmid clone (∼ 45 kb) carrying the human gene RCC1 was successful.
- Published
- 1989
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