Electro-transformation of Lactobacillus spp.
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(New page: ==Overview== Instructions on how to prepare ''Lactobacillus plantarum'' competent cells before electrotransformation. =='''Materials'''== *MRS media *Overnight culture of L. plantarum c...)
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Revision as of 13:20, 18 October 2009
Instructions on how to prepare Lactobacillus plantarum competent cells before electrotransformation.
- 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)
- Inoculate L. plantarum overnight using 10 ml of MRS media.
- The next morning inoculate 100 ml of MRS with the 10 ml overnight culture.
- Incubate cells for 3-4 hrs at 30°C unitl absorbance reaches 600nm=0.85.
- Pellet cells by centrifugation at max speed (keep cells cold on ice in between centrifugations) for approx. 2 minutes or until supernatant is clear.
- Wash cells twice with 10 ml of chilled MgCL2 solution.
- Resuspend cells in 3-4ml of sucrose/glycerol solution used to wash cells.
- 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.
All questions, input and feedback are welcome!
- Centrifugation at 4000 rpm for 2 minutes was sufficient to pellet the competent cells to give a clear supernatant.
Relevant Papers and Books
Alegre et al (FEMS Microbilogy Letters 241 (2004) 73-77)
or instead, discuss this protocol. -->