超净台

Preparation of Lactobacillus Competent Cells

Instructions on how to prepare Lactobacillus plantarum competent cells before electrotransformation.

  • MRS media
  • Culture of L. plantarum cells
  • MgCL2 (10mM)
  • sucrose
  • glycerol
  • Centrifuge capable of holding four 50mL centrifuge tubes.

Day 1

  1. Prepare the following:
  • 250mL MRS media divided equally into 5 flasks (50mL each)
  • 100mL Wash buffer (10mM MgCL2)
  • 50mL Electroporation Buffer (0.5M Sucrose, 10%(v/v) glycerol)
  1. Cap the flasks with foil and autoclave.
  2. Once the MRS has cooled, inoculate one of the flasks with L.plantarum culture and grow overnight at 30°C.

Day 2

  1. Inoculate the remaining 4 flasks of MRS each with 5mL of the overnight culture.
  2. Incubate cells for 3-4 hrs at 30°C until OD600 is approximately 0.85.
  3. Put the buffers on ice and pre-chill the centrifuge.
  4. Pour Cultures into 50mL centrifuge tubes.
  5. Centrifuge for 2 minutes at 5000g or until supernatant is clear.
  6. Pour off supernatant and resuspend pellet in 10mL ice-cold Wash Buffer.
  7. Centrifuge for 2 minutes at 5000g or until supernatant is clear.
  8. Pour off supernatant and resuspend pellet in 10mL ice-cold Wash Buffer.
  9. Centrifuge for 2 minutes at 5000g or until supernatant is clear.
  10. Pour off supernatant and resuspend pellet in 10mL ice-cold Electroporation Buffer.
  11. Centrifuge for 2 minutes at 5000g or until supernatant is clear.
  12. Pour off supernatant and resuspend cells in 1-2mL ice-cold Electroporation Buffer.
  13. Can then be stored in an ice bath for no more than 4hrs before use.
  14. Store in aliquots and store in a -80°C freezer.

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