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Competent Cell Preparation

by Tannon Yu

Contents

Background

Competency is the state at which cells can intake free-floating environmental DNA and use it as its own in a process called transformation. While natural competency occurs under various environmental stresses, artificial competency can be induced in a laboratory environment.

Several different protocols for induced competent cells are widely available. The protocol below utilizes rubidium chloride preparation (RbCl) to allow for higher transformation efficiency and requires less time than other protocols. While SOC medium is recommended, LB medium can also be used.

Competent cell preparation is typically a two-day process. The first day can be used to prepare by making the necessary buffers, media, and autoclaving supplies such as tips and microcentrifuge tubes. The rest of the protocol should be completed on the second day, without interruption. Ideally, the protocol should completed in a cold room.

Required Materials

  • Bacterial strain of choice (DH5α, BL21 DE3, etc.)
  • SOC medium
  • Liquid nitrogen
  • TFB1 buffer, pH 5.8
    • Rubidium chloride (RbCl), 100 mM
    • Manganese chloride (MnCl2), 50 mM
    • Potassium acetate (CH3CO2K), 30 mM
    • Calcium chloride (CaCl2), 10 mM
    • Glycerol, 15%
  • TFB2 buffer, pH 6.5
    • Rubidium chloride, 10 mM
    • MOPS buffer, 10 mM
    • Calcium chloride, 75 mM
    • Glycerol, 15%

Protocol

Preparations

  1. Make TFB1 and TFB2 buffers (see Required Materials above for recipe). Filter sterilize both and store at 4 °C for use the next day.
  2. Autoclave 1.7 mL microcentrifuge tubes and store covered at −20 °C.
  3. Autoclave 0.5 L of SOC media. Store covered at room temperature.
  4. Inoculate 5 mL of SOC media with desired bacterial strain. Grow overnight at 37 °C.

Growing large culture

  1. Inoculate 0.5 L culture with 500 µL of starter culture.
  2. Grow the large culture at 37 °C until the optical density (OD) is between 0.4–0.6.
  3. Transfer into 10 Falcon tubes, 50 mL each.
  4. Incubate on ice for 15 minutes.
  5. Centrifuge the cells at 2500 rpm for 30 minutes at 4 °C. Completely decant the supernatant.

Washing and competency induction

  1. Gently resuspend pellets in 16.67 mL of chilled TFB1 buffer per Falcon tube.
  2. Incubate on ice for 15 minutes.
  3. Centrifuge the cells at 1950 rpm for 15 minutes at 4 °C. Completely decant the supernatant.
  4. Gently resuspend pellets in 1.67 mL of chilled TFB2 buffer per Falcon tube.
  5. Incubate on ice for 15 minutes.
  6. Aliquot 100 µL in chilled microcentrifuge tubes and immediately freeze in liquid nitrogen.
  7. Cells are immediately available for transformation, or can be stored at −80 °C.

Competency test

  1. Test the transformation efficiency of the competent cells using 1 ng of plasmid.
  2. Calculate the colony-forming unit (CFU) rate per µg of DNA. Ideally, competent cells should have a transformation efficiency of at least 108 CFU/µg.

Troubleshooting

If there are issues with the competency of the cells, try the following troubleshooting tips:

  1. Cells are more competent at lower ODs. Try growing the large culture until OD is between 0.4–0.5.
  2. Cells grown at lower temperatures are highly competent when compared to 37 °C. If time is not an issue, try growing the large culture at 18–26 °C.