For example, in this protocol for E. coli competent cell preparation, it says:

Plate 10 uL E. coli BL21(DE3) cells on a LB-agar plate; incubate overnight (12 hours).

Prepare 500 mL SOB medium so it can be pre-warmed in the 37 degrees Celsius incubator the day before.

Pick a single selected colony and inoculate in 5 mL of SOB medium and grow for 16 hours at 37 degrees Celsius and 250 rpm in a shaker.

Inoculate 500 mL of prewarmed SOB medium with 5 mL of the fresh overnight culture.

and then in the protocol for overexpressing proteins after transformation of these cells, I basically follow similar steps as above but this time, without prewarming the large-scale inoculation lqiuid medium. Why is that?

  • 1
    $\begingroup$ Dramatically changing the temperature quickly can shock cells, both prokaryotic and eukaryotic. This could, for example, cause quickly-growing log phase cells to arrest for a period of time, depending on the temperature of the large volume of media (i.e., RT or 4C). $\endgroup$
    – MattDMo
    Jun 11, 2015 at 23:20
  • $\begingroup$ Growth speed also changes by incubation temp. If you put large, medium, and small sizes of culture media kept at room temp into 37 degrees incubator, times when you get the desired density of E coli may vary among the sizes $\endgroup$
    – 243
    Jun 12, 2015 at 1:05

1 Answer 1


It's because E.coli BL21(DE3) are competent cells. The competent is the key here as the cells were chemically treated so the transfection can be performed by heat-shock with high efficiency.

This means these bacteria are quite fragile, due to the chemical treatment, and therefore are very sensitive to both mechanical and thermal shocks.

Pre-heating the media simply avoids damaging the cells and maximizes the growth rate.


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