Some notes on electroporation

20 Sep

Molecular cloning: a laboratory manual, Volume 3

 By Joseph Sambrook, David William Russell,+David+W.+Russell&printsec=frontcover&source=bl&ots=CttWVkjWNt&sig=VEkJ7tFxipKJUUkQqIi0KZUguIk&hl=en&ei=xYK1SuefDIrkNeCS2NoO&sa=X&oi=book_result&ct=result&resnum=1#v=onepage&q=electroporation&f=false

Page 1.162

Length of pulse determined by capacitor and conductivity of medium

Field strength proportional to applied voltage, and inversely proportional to electrode spacing

Shape determined by design of electroporator

For most common strains of e-coli, a single pulse of 12.5-15KV/cm and pulse length of 4.5-5.5msec result in cell survival rate of 50%.


Highly efficient transfection of mammalian cells by electric field pulses

Marie-Pierre ROLS 1 Denis COULET 1 Justin TEISSIÉ 1

1 Centre de Recherches de Biochimie et de Génétique Cellulaires du CNRS, Toulouse, France
Correspondence to J. Teissié, Centre de Recherches de Biochimie et de Génétique Cellulaires du CNRS, 118 route de Narbonne, F-31062 Toulouse cédex, France
Fax: + 33 61 33 5886.

We have transfected mammalian cells with plasmid DNA by application of electric pulses. Chinese hamster ovary cells were chosen as a model in order to study and to optimize the transfection protocol. A plasmid carrying the gene coding for β-galactosidase activity was used to determine transient expression of the electrotransferred acitivity at the cell level. Optimum transient expression for cells in suspension was obtained by application of 10 square wave pulses of 5-ms duration and 0.6-kV/cm intensity. Under the best conditions, transfection frequencies as high as 50–60% could be obtained and appeared to be highly dependent on the age of the cell culture. The method was applicable to plated cells growing in a petri dish or on microcarriers. The possibility of extension of the technique to large volumes of cells is presented. A flow system, composed of a peristaltic pump connected to the electropulser chamber, allowed large volumes of cells to be treated with flow rates in the order of several milliliters/minute. Transfection frequencies for the large volumes were 25% for cells in suspension and 35% for cells on microcarriers. These results open new perspectives in large-scale transfection technology of cells however they are grown.






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