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SIG10 5-alpha Chemically Competent Cells

Preparation for Transformation

SIG10 5-alpha Chemically Competent Cells are transformed by heat shock at 42oC, followed by incubation on ice.

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.
  • Prepare nutrient agar plus antibiotic.
  • 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 plates with appropriate antibiotic.
  2. Chill sterile culture tubes on ice (17 mm x 100 mm tubes, one tube for each transformation reaction).
  3. Remove SIG10 5-alpha cells from the -80°C freezer and thaw completely on wet ice (10-20 minutes).
  4. Add 40 µl of the cells to the chilled culture tube.
  5. Add 1-4 µl of ligation reaction or DNA sample to the 40 µl of cells on ice. (Failure to purify or heat-inactivate, 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 30 seconds.
  8. Return the cells to ice for 2 minutes.
  9. Add 950 µl of room temperature Recovery Medium to the cells in the culture tube.
  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.
  12. Incubate the plates overnight at 37°C.
  13. Transformed clones can be further grown in any rich culture medium.

Materials

     
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