Electro-transformation of Lactobacillus spp.: Difference between revisions
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#Prepare the following: | #Prepare the following: | ||
:*25mL MRS media | :*25mL MRS media | ||
:*25mL MRS media with 4g glycine(4%) and 15g (1.8M) sucrose added. | :*25mL Treatment Media (MRS media with 4g glycine(4%) and 15g (1.8M) sucrose added). | ||
:*50mL Water | :*50mL Water | ||
:*50ml 50mM EDTA | :*50ml 50mM EDTA | ||
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#Cap the flasks with foil and autoclave. | #Cap the flasks with foil and autoclave. | ||
#Once the MRS has cooled, inoculate the flask without glycine with ''L.plantarum'' culture and grow overnight at 30°C. | #Once the MRS has cooled, inoculate the flask without glycine with ''L.plantarum'' culture and grow overnight at 30°C. | ||
===Day 2=== | ===Day 2=== | ||
#Add the 25mL MRS plus Glycine. | #Add the 25mL MRS plus Glycine. |
Revision as of 18:12, 23 September 2010
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:
- 25mL MRS media
- 25mL Treatment Media (MRS media with 4g glycine(4%) and 15g (1.8M) sucrose added).
- 50mL Water
- 50ml 50mM EDTA
- 50mL Electroporation Buffer (0.9M Sucrose, 10%(v/v) glycerol)
- Cap the flasks with foil and autoclave.
- Once the MRS has cooled, inoculate the flask without glycine with L.plantarum culture and grow overnight at 30°C.
Day 2
- Add the 25mL MRS plus Glycine.
- 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.
Notes
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.
References
Relevant Papers and Books
Alegre et al (FEMS Microbiology Letters 241 (2004) 73-77)
Contact
- morto077@uottawa.ca
or instead, discuss this protocol. -->