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High Electrotransformation Efficiencies Obtained With DNA From Ligation Mixtures

Paul Zoller, Research and Development Department, Genetic Systems Division, Bio-Rad Laboratories, 2000 Alfred Nobel Drive, Hercules, CA 94547


Introduction
It has become clear that electroporation of E. coli results in significantly higher transformation efficiencies than chemical transformation procedures by a factor of 10 to 100, and is therefore the method of choice for applications requiring high efficiencies. For example, preparation of cDNA libraries from a small amount of plasmid DNA necessitates the high transformation efficiencies obtained using electroporation. Problems in obtaining high efficiencies occur when electroporating DNA directly from ligation mixtures, due to components in the mixtures which interfere with transformation.1 Several approaches have been suggested that may overcome this interference problem, such as dilution, precipitation, microdialysis, and heat treatment of the ligation reaction.2-5 In this study I explored combinations of these approaches to determine the method that results in the highest electro-transformation efficiencies for the introduction of DNA directly from ligation reactions.


Methods
Sample Preparation
Electro-Competent E. coli JS5 cells and supercoiled pUC18 plasmid DNA (10 pg/l) were obtained from Bio-Rad Laboratories. Linear pUC18 plasmid was prepared by digestion of the supercoiled form with EcoRI restriction enzyme (New England Biolabs), followed by extraction with phenol/chloroform, ethanol precipitation, and resuspension in TE buffer (10 mM Tris, pH 8.0, 1 mM EDTA) to a concentration of 10 ng/l. All ligation reactions consisted of DNA
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