Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 67)

8. Plate onto THY agar plates with selective antibiotic. Incubate 1–2 D at 37°C.
6.6.3 Solutions and reagents
1. THY media (Todd-Hewitt broth with 0.2% yeast extract): 30 g Todd Hewitt broth, 2 g Yeast
extract; dissolve in 1 L water. Autoclave.
2. THY + Glycine: 500 ml THY media, 5 ml 2 M Glycine (15 g/100 ml).
3. THY agar with antibiotic: 30 g Todd-Hewitt broth, 2 g Yeast extract, 15 g Bacto agar; dissolve in
1 L water. Autoclave; cool to 55–60°C and add antibiotic. Pour plates.
4. Elpo medium: 122 ml 20% Glucose, 0.5 ml 1 M MgCl
2
; bring to 500 ml with water and pH to 6.5.
Filter sterilize.
6.7 Lactobacillus plantarum
6.7.1 Preparation of Electrocompetent Cells
From Bringel & Hubert (1990). Using this method, we have obtained transformation efficiencies of 4 x 10 4
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 (OD
600
< 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
OD
600
= 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 (<2 µl). Place tubes on ice. Add 40 µl electrocompetent cells to each plasmid sample; mix.
2. Transfer 40 µl of plasmid/cell suspension to chilled 0.2 cm cuvettes on ice; tap the solution to the
bottom of the cuvette.
3. From the Home screen on Gene Pulser Xcell open the Pre-set Protocols screen, then the Bacterial
Protocol screen (press 4, then Enter twice). To select L. plantarum, press 9 or the Down Arrow to
highlight L. plantarum, then press Enter to open the L. plantarum Protocol Detail screen. See
Section 3.4 for operating instructions.
4. Place the cuvette in the ShockPod. Push the chamber lid down to close. Pulse once.
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