Electro-transformation of Lactobacillus spp.: Difference between revisions

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===Day 2===
===Day 2===
#Add the 25mL MRS plus Glycine.
#Incubate cells for 3-4 hrs at 30°C until OD<sub>600</sub> is approximately 0.85.
#Put the buffers on ice and pre-chill the centrifuge.
#Put the buffers on ice and pre-chill the centrifuge.
#Pour Cultures into 50mL centrifuge tubes.  
#Add the 25mL Treatment Media to the overnight culture.
#Centrifuge for 2 minutes at 5000g or until supernatant is clear.
#Incubate cells for 1-1.5 hr at 30°C.
#Pour off supernatant and resuspend pellet in 10mL ice-cold Wash Buffer.
#Divide culture into two 50mL centrifuge tubes.  
#Centrifuge for 2 minutes at 5000g or until supernatant is clear.
#Centrifuge for 2 minutes at 5000g.
#Pour off supernatant and resuspend pellet in 10mL ice-cold Wash Buffer.
#Pour off supernatant and resuspend pellet in 10mL ice-cold water.
#Centrifuge for 2 minutes at 5000g or until supernatant is clear.
#Centrifuge for 2 minutes at 5000g.
#Pour off supernatant and resuspend pellet in 10mL ice-cold water.
#Centrifuge for 2 minutes at 5000g.
#Pour off supernatant and resuspend pellet in 25mL EDTA solution.
#Let cell suspension sit on ice for 30 mins.
#Centrifuge for 2 minutes at 5000g.
#Pour off supernatant and resuspend pellet in 10mL ice-cold Electroporation Buffer.
#Pour off supernatant and resuspend pellet in 10mL ice-cold Electroporation Buffer.
#Centrifuge for 2 minutes at 5000g or until supernatant is clear.
#Centrifuge for 2 minutes at 5000g or until supernatant is clear.
#Pour off supernatant and resuspend cells in 1-2mL ice-cold Electroporation Buffer.
#Pour off supernatant and resuspend cells in 500μL ice-cold Electroporation Buffer.
#Can then be stored in an ice bath for no more than 4hrs before use.
#Dispense into 100μL aliquots.
#Store in aliquots and store in a -80°C freezer.
#Store at -20°C for use that day.


==Notes==
==Notes==

Revision as of 18:21, 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

  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)
  1. Cap the flasks with foil and autoclave.
  2. Once the MRS has cooled, inoculate the flask without glycine with L.plantarum culture and grow overnight at 30°C.

Day 2

  1. Put the buffers on ice and pre-chill the centrifuge.
  2. Add the 25mL Treatment Media to the overnight culture.
  3. Incubate cells for 1-1.5 hr at 30°C.
  4. Divide culture into two 50mL centrifuge tubes.
  5. Centrifuge for 2 minutes at 5000g.
  6. Pour off supernatant and resuspend pellet in 10mL ice-cold water.
  7. Centrifuge for 2 minutes at 5000g.
  8. Pour off supernatant and resuspend pellet in 10mL ice-cold water.
  9. Centrifuge for 2 minutes at 5000g.
  10. Pour off supernatant and resuspend pellet in 25mL EDTA solution.
  11. Let cell suspension sit on ice for 30 mins.
  12. Centrifuge for 2 minutes at 5000g.
  13. Pour off supernatant and resuspend pellet in 10mL ice-cold Electroporation Buffer.
  14. Centrifuge for 2 minutes at 5000g or until supernatant is clear.
  15. Pour off supernatant and resuspend cells in 500μL ice-cold Electroporation Buffer.
  16. Dispense into 100μL aliquots.
  17. Store at -20°C for use that day.

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. -->

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