Steady Chemically Competent Cells

Preparation for Transformation

To ensure successful transformation results, the following precautions must be taken:

  • For best results, ligation reactions must be heat killed at 70ºC for 15 minutes before transformation. Alternately, the reactions may be purified, if desired. For other ligation reactions, follow the manufacturer’s recommendations.
  • Prepare nutrient agar plus antibiotic for selection.
  • All microcentrifuge tubes must be thoroughly pre-chilled on ice before use.
  • The cells must be completely thawed on ice before use.
  • For highest transformation efficiency, use the provided Recovery Medium to resuspend the cells after transformation.

Transformation Protocol

  1. Prepare nutrient agar (e.g., LB) plates with antibiotic for selection.
  2. Chill sterile culture tubes on ice (17 mm x 100 mm tubes, one tube for each transformation reaction).
  3. Remove cells from the -80°C freezer and thaw completely on wet ice (10-20 minutes).
  4. Add 40 μL of cells to the chilled culture tube.
  5. Add 1-4 μl of heat-inactivated ligation reaction or DNA sample to the 40 μL of cells on ice. (Failure to heat-inactivate—70°C for 15 minutes—or otherwise purify, the ligation reaction may prevent transformation.) Stir briefly with pipet tip; do not pipet up and down to mix, which can introduce air bubbles and warm the cells.
  6. Incubate on ice for 30 minutes.
  7. Heat shock cells by placing them in a 42oC water bath for 45 seconds.
  8. Return the cells to ice for 2 minutes.
  9. Add 960 μL of room temperature Recovery Medium to the cells in the culture tube. When using these cells with a cloning kit, follow the Recovery Medium volume given in that kit manual.
  10. Place the tubes in a shaking incubator at 250 rpm for 1 hour at 37 oC.
  11. Plate up to 100 μL of transformed cells on nutrient agar plates containing the appropriate antibiotic. Note: the quality of LB plates varies widely. Transformants plated on LB may grow slowly.
  12. Incubate the plates overnight at 37°C.
  13. Transformed clones can be further grown in any rich culture medium (e.g., LB, or TB).

Materials

     
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