PreparationofLactobacillusCompetentCells

Overview

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

Materials

• MRS media

• Culture of L. plantarum cells

• MgCL₂ (10mM)

• sucrose

• glycerol

• Centrifuge capable of holding four 50mL centrifuge tubes.

Procedure

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.