We have developed a convenient Escherichia coli transformation method, termed colony-based transformation, in which a single fresh colony of plasmid-containing donor strain is used instead of extracted plasmid to transform E. coli recipient cell. Thus the need for plasmid extraction and competent cell preparation is avoided. Additionally, a high-throughput transformation process based on this method was designed in which samples are prepared in a 96-well PCR plate and hundreds of transformations can be performed simultaneously in a thermocycler. We therefore suggest that this method may serve as a substitute of current transformation methods based on plasmid extraction.