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 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)

Cap the flasks with foil and autoclave. Once the MRS has cooled, inoculate one of the flasks with L.plantarum culture and grow overnight at 30°C. Day 2 Inoculate the remaining 4 flasks of MRS each with 5mL of the overnight culture. Incubate cells for 3-4 hrs at 30°C until OD600 is approximately 0.85. Put the buffers on ice and pre-chill the centrifuge. Pour Cultures into 50mL centrifuge tubes. Centrifuge for 2 minutes at 5000g or until supernatant is clear. Pour off supernatant and resuspend pellet in 10mL ice-cold Wash Buffer. Centrifuge for 2 minutes at 5000g or until supernatant is clear. Pour off supernatant and resuspend pellet in 10mL ice-cold Wash Buffer. Centrifuge for 2 minutes at 5000g or until supernatant is clear. Pour off supernatant and resuspend pellet in 10mL ice-cold Electroporation Buffer. Centrifuge for 2 minutes at 5000g or until supernatant is clear. Pour off supernatant and resuspend cells in 1-2mL ice-cold Electroporation Buffer. Can then be stored in an ice bath for no more than 4hrs before use. Store in aliquots and store in a -80°C freezer.