CHANGER Electrocompetent Cells

Preparation for Transformation

Transformation is carried out in a 1.0 mm gap cuvette using 25 μL of CHANGER Electrocompetent Cells. Optimal settings for electroporation are listed in the table below. Typical time constants are 3.5 to 4.5 msec.

Optimal Setting Alternate Settings
(~ 20-50% lower efficiencies)
For 2.0 mm cuvettes
1.0 mm cuvette 1.0 mm cuvette 2.0 mm cuvette
10 μF 25 μF 25 μF
600 Ohms 200 Ohms 750 Ohms
1800 Volts 1400 – 1600 Volts 2400 Volts

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

  • Ligation reactions must be heat killed at 70ºC for 15 minutes before transformation.
  • DNA samples in other buffers must be purified and dissolved in water or a buffer with low ionic strength, such as TE. The presence of salt in the electroporation sample leads to arcing at high voltage, resulting in loss of the cells and DNA.
  • Microcentrifuge tubes and electroporation cuvettes 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 electroporation. Use of SOC or other media will result in lower transformation efficiencies.
  • Prepare nutrient agar plus appropriate antibiotic.

Transformation Protocol

Use the following protocol for cells provided in microfuge tubes.

  1. Have Recovery Medium and 17 mm x 100 mm sterile culture tubes readily available at room temperature (one tube for each transformation reaction). Transformation efficiency may decrease with the use of SOC or other media.
  2. Place electroporation cuvettes (0.1 cm gap) and microcentrifuge tubes on ice (one cuvette and one microfuge tube for each transformation reaction).
  3. Remove cells from the -80°C freezer and place on wet ice until they thaw completely (10-20 minutes).
  4. When cells are thawed, mix them by tapping gently. Aliquot 25 μL of cells to the chilled microcentrifuge tubes on ice.
  5. Add 1 μL of the heat-denatured ligation reaction to the 25 μL of cells on ice. Failure to heat-inactivate the ligation reaction will prevent transformation. Stir briefly with pipet tip; do not pipet up and down to mix, which can introduce air bubbles and warm the cells. Use of more than 2 μL of ligation mix may cause electrical arcing during electroporation.
    For ligation reactions using other commercial kits, please refer to the manufacturer’s instructions.
  6. Carefully pipet 25 μL of the cell/DNA mixture into a chilled electroporation cuvette without introducing bubbles. Quickly flick the cuvette downward with your wrist to deposit the cells across the bottom of the well. Electroporate according to the conditions recommended above.
  7. Within 10 seconds of the pulse, add 975 μL of Recovery Medium to the cuvette and pipet up and down three times to resuspend the cells. Transfer the cells and Recovery Medium to a culture tube.
  8. Place the tube in a shaking incubator at 250 rpm for 1 hour at 37°C.
  9. Spread up to 100 μl of transformed cells on nutrient agar plates containing the appropriate antibiotic.
  10. Incubate the plates overnight at 37°C.
  11. Transformed clones can be further grown in any rich culture medium.

Materials

     
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