Hancock Lab

CaCl2 Transformation of E. coli

Categorized: Bacterial Genetics

  1. Grow cells overnight in 5ml LB broth (10g tryptone, 5g yeast extract, 5g NaCl per liter).
  2. Inoculate into fresh LB broth (50ml will yield 1ml of competent cells; 0.1 ml/transformation) at a 1:200 dilution (OD550 should be 0.05 or less). Grow at 37oC with vigorous shaking in a large flask to facilitate aeration.
  3. When cells reach an OD550 of 0.2 to 0.4 put them on ice for 10 minutes.
  4. Centrifuge the cells 10,000 rpm and 4oC for 5 minutes in a cold, sterile tube in the SS34 rotor. Discard the supernatant and resuspend the cells in a half volume of sterile, ice-cold (4oC) 0.1 M CaCl2. Leave the cells on ice for 20 to 40 minutes.
  5. Centrifuge the cells as above. Gently resuspend the cells in 1/50 of the original culture volume in cold, sterile 0.1 M CaCl2. Leave the cells on ice for 1 to 40 hours (24 hours is optimal for many common strains but spells death to really sick ones). The cells are now competent for transformation.
  6. Aliquot 0.1ml of cells to a prechilled eppendorf tube and add the DNA in 10µl or less volume. Make sure to mix the tube gently before aliquoting the cells as they tend to settle at the bottom of the tube. Leave the cells on ice for 20-40 minutes to allow the DNA to adhere to the cells.
  7. Heat shock the cells in a 42oC water bath for 45 to 60 seconds and return to ice for 1 minute.
  8. Add 1.2ml of LB broth to the tube, mix and place the eppendorf tube in a small glass test tube and secure with parafilm. Put the tube in the tube roller in the 37oC room for 1 hour.
  9. Plate 0.1ml of the transformed cells on selective plates, spin the remaining cells in the microfuge for 20 seconds and pour off all the media except the last drop (i.e. do not shake out the tube). Resuspend the pellet in the last drop and plate this “neat” on a selective plate. If you anticipate lots of transformants you could plate a 1/10 dilution of the original tube. Incubate the cells overnight at 37oC. Screen, etc.

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