Bio-Rad MicroPulser™ Electroporator - Manual (Page 24)

5. Add 50 ml of sterile YPD/HEPES to each of the bottles and vortex to resuspend the cell
pellets; add 1.25 ml of 1M DTT to each bottle; mix gently. Incubate the cells for 15 min
at 30 °C.
6. Add 200 ml of sterile, ice-cold 1 M sorbitol to each centrifuge bottle. Pellet the cells by
centrifugation at 3000 x g for 5 min at 4 °C; pour off and discard the supernatant.
7. Add ~50 ml of sterile, ice-cold 1 M sorbitol to each of the bottles and vortex to resuspend
the cell pellets; bring the volume in each of the centrifuge bottles to 250 ml with sterile,
ice-cold 1 M sorbitol. Pellet the cells by centrifugation at 3000 x g for 5 min at 4 °C; pour
off and discard the supernatant.
8. Resuspend each cell pellet in 10 ml of sterile, ice-cold 1 M sorbitol and pool in 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 9 cells/ml. Keep the cells
on ice and use as soon as possible for electroporation.
11.2 Electroporation
1. Pipette the DNA samples (up to 10 µg) to be electroporated into sterile 1.5 ml microfuge
tubes. Place tubes on ice.
2. Add 40 µl of the competent cells to each DNA sample and mix gently.
3. Set the MicroPulser to "Pic". See Section 4 for operating instructions.
4. Transfer the DNA—cell samples to 0.2 cm electroporation cuvettes that have been chilled
in ice and tap the suspension to the bottom of the tube. 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 1.0 ml of ice cold 1.0 M
sorbitol to the cuvette when selecting for complementation of an auxotrophic mutant, or
1.0 ml of ice cold YPD/sorbitol when selecting for antibiotic resistance. Gently transfer
the diluted cells to a sterile tube.
6. Check and record the pulse parameters. The time constant should be close to 5 milliseconds.
The field strength can be calculated as actual volts (kV) / cuvette gap (cm).
7. When selecting for complementation of an auxotrophic mutant, the cells may be plated
immediately onto minimal agar plates containing 1 M sorbitol but lacking the appropriate
nutrient. When selecting for antibiotic resistance, incubate the cells at 30 °C for 1–2 hrs
without shaking; plate aliquots of the electroporated cells on YPD agar plates containing
1 M sorbitol with the appropriate antibiotic. Incubate the plates for 72–96 hrs at 30 °C.
11.3 Solutions and Reagents for Electroporation
1. YPD/HEPES: 100 ml YPD media, 20 ml 1 M HEPES, pH 8.0
2. 1M DTT: 1.55 g dithiothreitol, dissolve in 8 ml water. Bring the volume to 10 ml with
water. Filter sterilize.
3. YPD/sorbitol: 10 g yeast extract, 20 g peptone, 182.2 g sorbitol, dissolve in 700 ml water;
bring volume to 900 ml with water. Autoclave. Add 100 ml sterile 20% glucose.
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