Preparation of Competent Bacterial Cells

Based on a protocol from Neil Olszewski and Scott Medberry.

They based their protocol on the one by Dower, et al. in Nucleic Acids Research 16:6127-6145 (1988).

Protocol

  1. Grow E. coli in X ml of your favorite bacterial media at 37°. Shake the cells vigorously.

  2. Harvest the cells when they are in mid log growth (A600 is between 0.5 and 1.0)

  3. Chill the cells on ice for a few minutes.

  4. Pellet the cells and resuspend in X ml of cold, sterile H2O.

  5. Pellet the cells again and resuspend in 0.5X ml of cold, sterile H2O.

  6. Pellet the cells again and resuspend in 0.02X ml of cold, sterile 10% glycerol.

  7. Pellet the cells again and resuspend in 0.002X ml of cold, sterile 10% glycerol.

  8. Pipet 60 to 100 µl aliquots into 0.5 ml Epindorf tubes. Quick freeze in liquid nitrogen and store at -70°.

This page is, as always, Copyright (c) 1998 by Craig D. Amundsen. All rights reserved.