Bio-Rad MicroPulser™ Electroporator - Manual (Page 21)

4. Transfer the DNA-cell samples to the appropriate 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 ml of ice cold 1 M
sorbitol to the cuvette; gently transfer the diluted cells into 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. Plate aliquots of the electroporated cells on selective agar plates containing 1 M sorbitol.
Incubate plates for 48–72 hrs at 30°C.
8.3 Solutions and Reagents For Electroporation
1. YPD: 10 g yeast extract, 20 g peptone, dissolve in 900 ml water. Autoclave. Add 100 ml
sterile 20% glucose.
2. 1M sorbitol: 182.2 g sorbitol, dissolve in 800 ml water. Bring volume to 1.0 L with water.
Autoclave.
3. 20% glucose: 20 g glucose, dissolve in 60 ml water. Adjust volume to 100 ml with water.
Sterilize through a 0.22 µ filter.
Section 9
Electroporation of
Schizosaccharomyces pombe
9.1 Preparation of Electrocompetent Cells
See Prentice (1991) for additional information.
1. Inoculate 500 ml of YCD in a 2.8 L Fernbach flask with an aliquot from an overnight
culture of S. pombe. The doubling time of S. pombe is approximately 2 hrs at 30 °C.
2. Incubate at 30 °C overnight, shaking at 250 rpm, to a density of 1 x 107 cells/ml (OD
600
~0.7).
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 1.2 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. Pellet the cells
by centrifugation 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 1.2 M sorbitol.
8. Resuspend the cell pellet in 20 ml of sterile, ice-cold 1.2 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 1.2 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.
17