Electroporation-mediated transformation

Scott, P.T. and Rood, J.I. (1989) Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens. Gene, 82, 327-333. 

Composition of electroporation buffer is an important factor affecting electroporation yields. Ionic strength of cell suspension medium needs control, which determines resistance of the cell suspension and resultant RC time constant of the field pulse. Medium supplemented with Ca and Mg in mM concentration range is found to promote efficiency of transformation and cell viability. Erythrocytes electroporated in isotonic buffer in the presence of EDTA or membrane specific drugs showed significant modification in hemolysis response to electroporation [33,34]. Use of square wave pulse removes the medium conductivity mediated effects on cell/tissue electroporation outcome. Generally, cells are pulsed in suspensions of sucrose, mannitol, or sorbitol. Electroporation as well as incubation of pulsed cells can be carried out in medium containing usual cell culture recipes. 

Restriction enzyme-mediated integration (REMI) is a commonly used method for random mutagenesis that has been adapted for the use in D. discoideum by Kuspa and Loomis [40]. A plasmid containing an appropriate selection marker is Hn-earized with a restriction enzyme, and the linear plasmid is transformed by electroporation along with the restriction enzyme (Fig. 5.2). It is assumed that the restriction enzyme will cut certain chromosomal loci at specific recognition sites. If a transformed plasmid would Hgate with its sticky ends to the double-strand break introduced by the restriction enzyme, mutants can be isolated based on the selection marker present on the integrated REMI plasmid. 

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