Preparation of Lactobacillus Competent Cells
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.
