Lactobacillus transformation (Berthier 1996): Difference between revisions
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(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...) |
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==Materials== | ==Materials== | ||
*50 ul of L.plantarum competent cells | *50 ul of L.plantarum competent cells | ||
* | *In vitro modified plasmid DNA | ||
*MRS media + antibiotic | *MRS media + antibiotic | ||
*Stock solution of MgCl2 | *Stock solution of MgCl2 | ||
Revision as of 10:16, 19 October 2009
Overview
General guidelines for the electrotransformation of Lactobacillus plantarum as described by Alegre et al.
Materials
- 50 ul of L.plantarum competent cells
- In vitro modified plasmid DNA
- MRS media + antibiotic
- Stock solution of MgCl2
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 80 mM MgCl2
- Incubate for 2 hours at 30°C.
- 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. -->
