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Chemical transformation

Overview

Sterile technique must be strictly observed – work next to an open flame. All centrifuge bottles, tubes, solutions, pipets, etc. must be sterile.

Materials

  • E. coli strain
  • 100 mM CaCl2 (ice cold, filter-sterilized)
  • 100 mM CaCl2 with 15% glycerol (filter-sterilized):
    • 1 mL of 1 M CaCl2 (1.1g)
    • 1.89 g 100% glycerol
    • 7.11 g Milli-Q
  • LB medium
  • (Optional) Antibiotics

Procedure

Chemically competent cell preparation

Yields about 4 tubes with 50 uL of chemically competent cells.

Carry out all steps aseptically.

  1. Prepare 4 mL of overnight cell culture.
  2. Transfer 40 uL (diluting ratio 1:100) to a fresh tubes with LB medium and antibiotic (if needed) and incubate shaking at 250 rpm/37ºC until OD600 reaches .45-.6.
    • Monitor growth starting at 2 h. For a strain with no plasmids, you should reach the required OD600 in 2-2.5 h. However, it may take 3-4 h if your strain already contains high-copy number plasmids.
    • OD600 on the lower side is preferable, but up to .8 is still useable (with decreasing efficiency).
  3. Chill on ice for about 10 min.
  4. Chill the centrifuge to 4C.
  5. Pelleting: Repeat twice:
    1. Add 1 mL culture into two 1.5 mL microtubes (either fresh or on top of the previous pellet).
    2. Centrifuge at 6000 g / 4C for 5 min.
    3. Discard supernatant.
  6. Washing:
    1. Resuspend each pellet in .5 ml ice cold 100 mM CaCl2.
    2. Centrifuge at 6000 g / 4C for 5 min and discard supernatant.
    3. Resuspend each pellet in .5 ml ice cold 100 mM CaCl2 and incubate on ice for at least 30 min.
    4. Centrifuge at 6000 g / 4C for 5 min and discard supernatant.
    5. Can repeat the previous incubation/pelleting step once more to get even more competent cells.
  7. Resuspend the first microtube in 200 uL ice cold CaCl2:glycerol solution, then transfer to the second microtube and resuspend the other pellet.
  8. Prepare 50 uL aliquots in sterile microfuge tubes and snap freeze in liquid nitrogen.
  9. Store in -80ºC freezer and use within 3 months.

Transformation

  1. Prewarm water bath to 42C.
  2. Prewarm 1 mL SOC to 37C.
  3. Thaw a tube containing 50 uL chemically competent cells on ice for 2-5 min and gently flick to resuspend them.
    • Competent cells are very fragile!
  4. Add 10-100 ng of plasmid DNA to cells and mix without vortexing.
    • Total plasmid volume should not exceed 1/10 of the cell volume.
    • Choose to add closer to 100 ng of plasmid when competent cells are not fresh.
  5. Incubate on ice for 10-30 min.
    • Longer incubation generally leads to more colonies.
  6. Heat shock tubes in water bath (42C) for 45 sec.
  7. Place on ice for 3 min.
  8. Add 900 uL prewarmed SOC and recover in a shaker for 1 h (250 rpm).
  9. Proceed to plating.

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