Lactobacillus transformation (Berthier 1996): Difference between revisions

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==Procedure==
==Procedure==
#Ensure that the [[In vitro modification of DNA for L. plantarum]] protocol has been followed.
#Grow cells overnight in MRS Broth at 30°C
#Thaw 50μL of competent cells on ice from aliquots in -80°C freezer.
#Use overnight culture to inoculate 100ml MRS Broth
#Add between 1 ng and 3 ug of plasmid DNA in 5 ul of TE buffer to 50 ul of freshly prepared competent cells and transfer to prechilled cuvette for electroporation (interelectrode distance 1 mm).
#Incubate at 30°C until OD<sub>600</sub> = 0.4-0.6
#Electroporation done at 13 Kv cm-1 (time constant: 2-4 ms), Set electroporation machine to 25μF and 200 Ω.
#Add 500 ul of MRS media containing 80mM MgCl<sub>2</sub> and 55mM Glucose
#Incubate for 2 hours at 30°C.
#Plate cells on MRS plates + antibiotic resistance.


==Notes==
==Notes==

Revision as of 18:00, 16 September 2011

Back to Protocols
Back to Electro-transformation of Lactobacillus spp.

Overview

General guidelines for the electro-transformation of Lactobacillus sake as described by Berthier et al. and as used by Alegre et al. with Lactobacillus plantarum.

Materials

  • 50 ul of L.plantarum competent cells
  • In vitro modified plasmid DNA
  • MRS media + antibiotic
  • Stock solution of MgCl2
  • Stock Solution of Glucose
  • Electroporation Buffer (0.5M Sucrose, 10% Glycerol)

Procedure

  1. Grow cells overnight in MRS Broth at 30°C
  2. Use overnight culture to inoculate 100ml MRS Broth
  3. Incubate at 30°C until OD600 = 0.4-0.6

Notes

All questions, input and feedback are welcome!

References

Berthier, F., Zagorec, M., Champomier-Verge`s, M., Ehrlich, S.D. and Morel-Deville, F. (1996) Efficient transformation of Lactobacillus sake by electroporation. Microbiology 142, 1273–1279.

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

Contact

  • morto077@uottawa.ca

or instead, discuss this protocol. -->

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