Bio-Rad MicroPulser™ Electroporator - Manual (Page 17)

4. Transfer the mixture of cells and DNA to a cold electroporation cuvette and tap the
suspension to the bottom. Place the cuvette in the chamber slide. Push the slide into the
chamber until the cuvette is seated between the contacts in the base of the chamber. Pulse
once.
5. Remove the cuvette from the chamber and immediately add 1 ml of SOC medium to the
cuvette. Quickly but gently resuspend the cells with a Pasteur pipette. (The period between
applying the pulse and transferring the cells to outgrowth medium is crucial for recovering
E. coli transformants (Dower et al., 1988). Delaying this transfer by even 1 minute causes
a 3-fold drop in transformation. This decline continues to a 20-fold drop by 10 minutes.
6. Transfer the cell suspension to a 17 x 100 mm polypropylene tube and incubate at 37 °C
for 1 hour, shaking at 225 rpm.
7. Check and record the pulse parameters. The time constant should be close to 5 millisec-
onds. The field strength can be calculated as actual volts (kV) / cuvette gap (cm).
8. Plate on selective medium.
5.3 Solutions and Reagents For Electroporation
1. L-Broth: 10 g Bacto tryptone, 5 g Bacto yeast extract, 5 g NaCl; dissolve in 1.0 L water.
Autoclave.
2. 10% (v/v) Glycerol: 12.6 g glycerol (density = 1.26 g/cc) in 90 ml of water. Autoclave
or filter sterilize.
3. TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTA.
4. SOC: 2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM
MgCl2, 10 mM MgSO4, 20 mM glucose.
Section 6
Electroporation of
Staphylococcus aureus
6.1 Preparation of Electrocompetent Cells
See Lee (1995) for additional information.
1. Inoculate 3 ml of B2 broth in a 17 x 100 mm tube with a colony from a fresh
S. aureus plate.
2. Incubate at 37 °C overnight, shaking at 250 rpm.
3. Inoculate 1.5 ml of the overnight culture into 150 ml of fresh B2 broth in a 1 liter flask.
Incubate at 37 °C, shaking at 250 rpm, to ~2 x 10 8 cells/ml. The doubling time of
S. aureus is about 30 min at 37 °C.
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.
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