Electrotransformation of Lactobacillus Spp.
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Last Update: 2021-02-15
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Source: Internet
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Author: User
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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
.Materials50 ul of L.plantarum competent cells
In vitro modified plasmid
DNAMRS media + antibiotic
Stock solution of MgCl
2Stock Solution of Glucose
ProcedureEnsure 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 MgCl
2
and 55mM Glucose
Incubate for 2 hours at 30°C.
Plate cells on MRS plates + antibiotic resistance.
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