We are trying to do a simple plasmid electroporation on L. rhamnosus GG but we hae had some problems with. We do not know if all the problems come from the machine configuration, the cells, the buffer....
Tania Cristina Gaviria Cantin
here you can found protocol: Lactobacillus species & further more details can be found in below shared PDF: Gene Pulser Xcell™
Electroporation System instruction manual- https://www.bio-rad.com/webroot/web/pdf/lsr/literature/4006217A.pdf
6.7.1 Preparation of Electrocompetent Cells
From Bringel & Hubert (1990). Using this method, we have obtained transformation efficiencies of 4 x 104
transformants/μg electroporating L.plantarum NCIB7220 with pGK12.
Gene Pulser Xcell conditions: C = 25 μF; PC = 400 ohm; V = 2.0 kV.
This procedure requires a Gene Pulser Xcell main unit and PC Module.
1. Inoculate L. plantarum into 50 ml of MRS media in a 500 ml flask; incubate overnight at 30°C shaking
at 300 RPM. The cells should be in exponential growth (OD600 < 1.5).
2. Inoculate a sample of the overnight culture into 500 ml of MRS/SG in a 2.8 L Fernback flask to an
OD600 = 0.05. Incubate at 30°C shaking at 80 RPM.
3. Monitor the culture until OD 600 = 0.3 (usually ~ 4 hrs).
4. Transfer the cells into a sterile 500 ml centrifuge bottle at room temperature.
5. Pellet the cells by centrifugation at 18°C for 10 min at 3000 x g.
6. Discard the supernatant. Resuspend the cell pellet in 250 ml of sterile water at room temperature.
Centrifuge the cells as above but increase the speed of to 4000 x g.
7. Discard the supernatant. Repeat the water wash as above. Pellet the cells as above.
8. Discard the supernatant. Resuspend the cell pellet in a final volume of 2 ml of 30% PEG. Keep the
cells on ice and use as soon as possible for electroporation.
6.7.2 Electroporation
1. For each sample to be electroporated, pipette 0.1–1.0 μg of plasmid into a sterile 1.5 ml microfuge
tube (
