The Inoue Method for Preparation and Transformation of ...

This protocol differs from other procedures in that the bacterial culture is grown at 18 °C rather than the conventional 37 °C. Otherwise, the protocol is unremarkable and follows a fairly standard course. Why growing the cells at low temperature should affect the efficiency of transformation is unknown. Perhaps the composition or the physical characteristics of bacterial membranes synthesized at 18 °C are more favorable for uptake of DNA, or perhaps the phases of the growth cycle that favor efficient transformation are extended. Incubating bacterial cultures at 18 °C is a challenge. Most laboratories do not have a shaking incubator that can accurately maintain a temperature of 18 °C summer and winter. One solution is to place an incubator in a 4 °C cold room and use the temperature control to heat the incubator to 18 °C. Alternatively, there is almost no loss of efficiency if the cultures are grown at 20-23 °C, which is the ambient temperature in many laboratories. Cultures incubated at these temperatures grow slowly with a doubling time of 2.5 to 4 h. To avoid reaching desired OD late at night, set up cultures in the evening and harvest the bacteria early the following morning. The procedure works well with many strains of E. coli in common use in molecular cloning, including XL1-Blue, DH1, JM103, JM108/9, DH5a, and HB101.