Preparation of Lactobacillus Competent Cells
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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
-
- 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.
