Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 63)

8. Incubate the cells 3 hr at 30°C, shaking at 250 rpm. Plate aliquots of the electroporated cells on
YM agar plates containing the appropriate selective media. Incubate plates for 48 hrs at 30°C.
6.3.3 Solutions and reagents
1. YM broth: 0.4 g yeast extract, 10 g mannitol, 0.1 g NaCl, 0.1 g MgSO
4
, 0.5 g K
2
HPO
4
.3H
2
0,
dissolve in 1.0 L of water and adjust to pH 7.0. Autoclave. For YM plates, add 15 g agar/1 L of YM
broth.
2. 10% (v/v) Glycerol: 252 g glycerol (density = 1.26 g/cc) in 1800 ml water. Autoclave or filter sterilize.
6.4 Bacillus cereus
6.4.1 Preparation of Electrocompetent Cells
Slight modification of the method of Silo-Suh, et al. (1994).
Using this method, we have obtained transformation efficiencies of 2 x 10 6 transformants/µg
electroporating B. cereus UW85 with pAD123.
Gene Pulser Xcell conditions: C = 50 µF; PC = 200 ohm; V = 1.0 kV.
This procedure requires a Gene Pulser Xcell main unit and PC Module.
1. Inoculate a colony of B. cereus into 10 ml of LB in a 125 ml flask; incubate overnight at 28°C
shaking at 300 RPM.
2. Inoculate 2 ml of the overnight culture into 500 ml of BHI in a 2.8 L Fernbach flask; incubate at
28°C shaking at 300 RPM.
3. Monitor the culture until OD
600
= 0.3 (cell density is ~1 x 10 7 cells/ml).
4. Chill the culture on ice for 10 min.
5. Transfer the cells into 2 chilled, sterile 250 ml centrifuge bottles.
6. Pellet the cells by centrifugation at 4°C for 10 min at 10,000 x g in a chilled rotor.
7. Discard the supernatant. Resuspend each pellet in 50 ml of sterile, ice cold EP buffer. Pellet the
cells as above.
8. Discard the supernatant. Resuspend each pellet in 15 ml of sterile, ice cold EP buffer. Transfer to a
chilled 30 ml Oakridge tube.
9. Pellet the cells by centrifugation at 4°C for 10 min at 10,000 x g in a chilled rotor.
10. Discard the supernatant. Resuspend the cell pellet in 0.5 ml of EP buffer. Keep the cells on ice and
use as soon as possible for electroporation. Cells cannot be frozen and used for electroporation.
6.4.2 Electroporation
1. For each sample to be electroporated, pipette the DNA (0.05 - 1 ug in up to 10 µl of either water
or TE) into a sterile 1.5 ml microfuge tube. Place tubes on ice. Add 100 µl electrocompetent cells
to each plasmid sample; mix, and incubate on ice for 5–10 min.
2. Transfer 100 µl of plasmid/cell suspension to chilled 0.2 cm cuvettes on ice; tap the solution to the
bottom of the cuvette.
3. For each DNA sample to be electroporated, prepare a 17 x 100 tube containing 2.0 ml of LB at
room temperature.
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