Product List for "The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells Protocol

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Selected Protocol from "Molecular Cloning: A Laboratory Manual", Third Edition, Joseph Sambrook, David W. Russell. The Inoue Method for Preparation and Transformation of Competent E. coli: "Ultra Competent" Cells Joseph Sambrook Peter Maccallum Cancer Institute and The University of Melbourne, Australia David W. Russell University of Texas Southwestern Medical Center, Dallas

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ABSTRACT
     At its best, this method for preparing competent E. coli from Inoue et al. (1990) can challenge the efficiencies achieved by Hanahan (1983). However, under standard laboratory conditions, efficiencies of 1 x 10⁸ to 3 x 10⁸ transformed colonies/mg of plasmid DNA are more typical. The advantages of the procedure are that it is less finicky, more reproducible, and therefore more predictable than the original Hanahan method.
     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 anybody's guess. 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 hours. This can lead to frustration, especially late at night when it seems that the culture will never reach the desired OD₆₀₀ of 0.6. The answer to this problem is to 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.

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