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.