Lactobacillus transformation (Berthier 1996)

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search

Matthew Orton (Talk | contribs)
(New page: ==Overview== General guidelines for the electrotransformation of ''Lactobacillus plantarum'' as described by Alegre et al. ==Materials== *50 ul of L.plantarum competent cells *Plasmid DNA...)
Next diff →

Revision as of 13:15, 19 October 2009

Contents

Overview

General guidelines for the electrotransformation of Lactobacillus plantarum as described by Alegre et al.

Materials

  • 50 ul of L.plantarum competent cells
  • Plasmid DNA
  • MRS media + antibiotic
  • Stock solution of MgCl2

Procedure

  1. Ensure that the in vitro modification of DNA protocol for Lactobacillus plantarum has been followed.
  2. Thaw 50 ul of competent cells on ice from aliquots in -80°C freezer.
  3. 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).
  4. Electroporation done at 13 Kv cm-1 (time constant: 2-4 ms), Set electroporation machine to 25μF and 200 Ω.
  5. Add 500 ul of MRS media containing 80 mM MgCl2
  6. Incubate for 2 hours at 30°C.
  7. Plate cells on MRS plates + antibiotic resistance.

Notes

All questions, input and feedback are welcome!

References

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

Contact

  • morto077@uottawa.ca

or instead, discuss this protocol. -->

Personal tools