Electrotransformation of Lactobacillus Spp.
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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
Procedure
- Ensure that the in vitro modification of DNA protocol for Lactobacillus plantarum has been followed.
- Thaw 50 ul of competent cells on ice from aliquots in -80°C freezer.
- 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).
- 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 MgCl2 and 55mM Glucose
- Incubate for 2 hours at 30°C.
- Plate cells on MRS plates + antibiotic resistance.
