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Transformation of Escherichia coli JM109 using pUC19 by the Yoshida effect.

Journal of microbiological methods (2015-05-15)
G P Mendes, P S Vieira, S Lanceros-Méndez, L D Kluskens, M Mota
ABSTRACT

Transformation of non-competent Escherichia coli JM109 was accomplished using pUC19 as donor plasmid and sepiolite as the acicular material to promote cell piercing via application of friction with a polystyrene stick or a magnetic bar on the surface of a hydrogel containing agar. An automatic spreading setup was built with a conventional stirring plate and compared to manual spreading. Several parameters were optimized, namely, the agar content of the hydrogel (2%), concentration of cells (OD=1.3 corresponding to 1.4×10(9) bacterial cells/mL), concentration of sepiolite (0.01%), manual versus mechanical spreading (automatic spreading more consistent) and spreading time (30s). Efficiency values up to 4.1×10(4) CFU/μg pUC19 were obtained. The method proved to be suitable for a rapid and low cost transformation of non-competent E. coli JM109, where higher values of efficiency do not need to be attained.

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