Electro-transformation of Lactobacillus spp.

Overview

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

Materials

• MRS media
• Overnight culture of L. plantarum cells
• 20 ml solution of MgCL2 (10mM)
• 20 ml solution of sucrose (0.5 M) and glycerol (10%w/v)

Procedure

1. Inoculate L. plantarum overnight using 10 ml of MRS media.
2. The next morning inoculate 100 ml of MRS with the 10 ml overnight culture.
3. Incubate cells for 3-4 hrs at 30°C unitl absorbance reaches 600nm=0.85.
4. Pellet cells by centrifugation at max speed (keep cells cold on ice in between centrifugations) for approx. 2 minutes or until supernatant is clear.
5. Wash cells twice with 10 ml of chilled MgCL2 solution.
6. Resuspend cells in 3-4ml of sucrose/glycerol solution used to wash cells.
7. Can then be stored in an ice bath for no more than 4hrs before use.
8. Store in aliquots and store in a -80°C freezer.

Notes

All questions, input and feedback are welcome!

1. Centrifugation at 4000 rpm for 2 minutes was sufficient to pellet the competent cells to give a clear supernatant.

References

Relevant Papers and Books

Alegre et al (FEMS Microbiology Letters 241 (2004) 73-77)

Contact

• morto077@uottawa.ca

or instead, discuss this protocol. -->