Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 60)

5. SOB: 2.0 g Tryptone peptone, 0.5 g Yeast extract, 0.2 ml 5 M NaCl, 0.25 ml 1 M KCl; dissolve
in 90 ml water. Adjust pH to 7.0. Bring volume to 100 ml. Autoclave. Add 1.0 ml sterile
1 M MgCl
2
and 1.0 ml sterile 1 M MgSO
4
.
6. SOC: to 100 ml SOB, add 2.0 ml sterile 1 M glucose (sterilize by filtration).
6.2 Staphylococcus aureus
6.2.1 Preparation of Electrocompetent Cells
See Lee (1995) for additional information.
Using this method, we have obtained transformation efficiencies of >1 x 10 6 transformants/µg
electroporating S. aureus RN4220 with pLI50.
Gene Pulser Xcell conditions: C = 25 µF; PC = 100 ohm; V = 2.9 kV.
This procedure requires a Gene Pulser Xcell main unit and PC Module.
1. Inoculate a colony of S. aureus from a TSA plate into 3 ml of B2 broth in a 17 x 100 mm tube.
2. Incubate at 37°C overnight, shaking at 250 rpm.
3. Inoculate 1.5 ml of the overnight culture into 150 ml of B2 broth in a 1 liter flask. Incubate at 37°C,
shaking at 250 rpm, to 3–5 x 10
8 cells/ml (OD
600
~ 0.8–0.85). The doubling time of S. aureus is
about 30 min at 37°C; growth will take about 4 hrs.
4. Chill the cells in an ice water bath for 15 min to stop growth. Decant the cells into a sterile 500 ml
centrifuge bottle. Harvest the cells by centrifugation at 12,000 x g for 15 min at 4°C.
5. Carefully pipette off the supernatant, keeping the cell pellet on ice.
6. Resuspend the cell pellet in 500 ml of sterile, ice-cold water. Pellet the cells by centrifugation at
12,000 x g for 15 min at 4°C; carefully remove the supernatant. Wash the cells 2 more times in
500 ml of sterile, ice-cold water.
7. Resuspend the cell pellet in 25 ml of sterile, ice-cold 10% glycerol. Transfer to a 30 ml sterile
Oakridge tube. Pellet the cells by centrifugation at 12,000 x g for 15 min at 4°C; carefully remove
the supernatant.
8. Resuspend the cells in 20 ml of 10% glycerol. Incubate at 20°C for 15 min. Pellet the cells by
centrifugation at 12,000 x g for 15 min at 4°C; carefully remove the supernatant.
9. Resuspend the cell pellet in 2 ml of 10% glycerol; the final cell concentration should be
~1 x 10
10 cells/ml.
10. Dispense 250 µl aliquots of the electrocompetent cells into sterile 1.5 ml microfuge tubes; freeze
the cells in an isopropanol-dry ice bath, then store at -70°C. The cells are stable for several months
under these conditions.
6.2.2 Electroporation
1. Pipette the DNA samples (5 ng - 2 µg in no more than 3 µl) to be electroporated into sterile 1.5 ml
microfuge tubes.
2. Thaw the competent cells at room temperature for several minutes. Add 50 µl of cells to each DNA
sample; gently pipette up and down to mix. Incubate the samples at room temperature for 30 min.
3. For each sample to be electroporated, add 1 ml of SMMP medium containing a subinhibitory con-
centration of antibiotic to a 17 x 100 mm tube at room temperature and place a 0.2 cm cuvette at
room temperature.
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