Bio-Rad MicroPulser™ Electroporator - Manual (Page 20)

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.
7.3 Solutions and Reagents For Electroporation
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.
Section 8
Electroporation of
Saccharomyces cerevisciae
8.1 Preparation of Electrocompetent Cells
See Becker & Guarantee (1991) and Ausubel et al. (1987) for additional information.
1. Inoculate 500 ml of YPD in a 2.8 L Fernbach flask with an aliquot from an overnight
culture of S. cerevisiae. The doubling time of S. cerevisiae is approximately 2 hrs at 30 °C.
2. Incubate at 30 °C overnight, shaking at 250 rpm, to a density of ~1 x 10 8 cells/ml.
3. Chill the cells in an ice water bath for 15 min to stop growth.
4. Decant the cells into two sterile 250 ml centrifuge bottles and pellet the cells by
centrifugation at 3000 x g for 5 min at 4 °C.
5. Carefully pour off and discard the supernatant; place the centrifuge bottles with the cell
pellets on ice.
6. Add ~50 ml of sterile, ice-cold water to each of the bottles and vortex to resuspend the cell
pellets; bring the volume in each of the centrifuge bottles to 250 ml. Pellet the cells by cen-
trifugation at 3000 x g for 5 min at 4 °C; pour off and discard the supernatant.
7. Wash the cells again as in step 6 with a total of 250 ml sterile, ice-cold water.
8. Resuspend the cell pellet in 20 ml of sterile, ice-cold 1 M sorbitol and transfer to a chilled
30 ml Oakridge tube. Pellet the cells by centrifugation at 3000 x g for 5 min at 4 °C; pour
off and discard the supernatant.
9. Resuspend the cell pellet in 0.5 ml of sterile, ice-cold 1 M sorbitol; the final cell volume
should be ~1.3 ml and the cell concentration should be ~1 x 10 10 cells/ml. Keep the cells
on ice and use as soon as possible for electroporation.
8.2 Electroporation
1. Pipette the DNA samples (5–100 ng in a volume of 5 µl) to be electroporated into sterile
1.5 ml microfuge tubes. Place tubes on ice.
2. If 0.2 cm cuvettes are used, add 40 µl of the competent cells to each DNA sample; if
0.4 cm cuvettes are used, add 80 µl of the competent cells to each DNA sample. Mix
gently and incubate on ice for ~5 min.
3. Set the MicroPulser to “Sc2” when using 0.2 cm cuvettes or to “Sc4” when using 0.4 cm
cuvettes. See Section 4 for operating instructions.
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