Generating stocks of competent cells is one of the most important tasks in the lab. Making certain stocks of bacteria are refreshed continuously utilizing proper techniques ensures no time is wasted for anybody wishing to amplify a plasmid, express a protein, or prepare bacmid. The following protocol is called “The Inoue Method” and was generated from the CSH Molecular Cloning Manual (Protocol 24, page 1.112). Expect the protocol to take at least two days, with the first day simply inoculating culture and the second day a combination of monitoring growth and snap freezing. This protocol can be used for all types of E. coli, with special considerations for certain strains at the end.

General Protocol

This protocol should be used for Bl21DE3, XL10Gold, and DH5alpha. This protocol will generally take two days.

Materials Required

  1. SOB Medium
    1. 3x 250mL and 1x 50mL
  2. DMSO
  3. Inoue Transformation Buffer
  4. Liquid nitrogen
  5. Sterilized microcentrifuge tubes

Protocol

Day 1

This is to prepare the starter culture and inoculate flasks.

  1. Dip a sterilized pipette tip into a stock of competent cells which have been stored at -80 °C.
    1. Can alternatively pick a single colony from a plate of competent cells
  2. Drop pipette tip into 50mL of SOB media with appropriate antibiotics (or no antibiotics). Agitate at 225 rpm at 37 °C for 6-8 hours.
  3. Transfer dilutions of starter culture to three, 250mL flasks containing SOB media. Dilutions can be somewhat arbitrary and are best determined empirically. More specialized strains such as RosettaGami2DE3 and DH10Bac which require multiple antibiotics seem to require higher dilutions. Simpler and more common strains such as BL21DE3 and DH5α seem to be fine with lower dilutions. The starter culture can be clear or turbid by end of day, but should generally be turbid. Do not forget to take an aliquot of blank culture for a blank OD reading.
    1. First flask will receive 2-5mL of starter culture
    2. Second flask will receive 6-8mL of starter culture
    3. Third flask will receive 10-12mL of starter culture
  4. Transfer flasks to shaking and refrigerated incubator. Shake overnight at 225 rpm and set temperature to 19 °C.

Day 2