Bio-Rad MicroPulser™ Electroporator - Manual (Page 22)

9.2 Electroporation
1. Pipette the DNA samples (up to 1 µg) to be electroporated into sterile 1.5 ml microfuge
tubes. Place tubes on ice.
2. Add 200 µl of the competent cells to each DNA sample and mix gently.
3. Set the MicroPulser to "ScS". 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 0.8 ml of ice cold 1.2 M
sorbitol to the cuvette; 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. Incubate the tubes at room temperature for 40–60 min. Plate aliquots of the electroporated
cells on minimal agar plates containing 1.2 M sorbitol. Incubate plates for 60–96 hrs at
30 °C.
9.3 Solutions and Reagents for Electroporation
1. YCD media: 10 g yeast extract, 2 g casamino acids, dissolve in 900 ml water. Autoclave.
Add 100 ml 20% glucose
2. 1.2 M sorbitol: 218.6 g sorbitol, dissolve in 700 ml water. Add water to 1.0 L.
Section 10
Electroporation of
D. discoideum
10.1 Preparation of Electrocompetent Cells
See Howard et al. (1988) and Knecht & Pang (1995) for additional information.
1. Inoculate D. discoideum cells at a concentration of 5–7 x 10 5 cells/ml into 40 ml of HL5
media in a 500 ml flask. The cells may either be scraped from a plate or transferred from
liquid media. The doubling time of D. discoideum is approximately 12 hrs at 21 °C.
2. Incubate the culture at 21 °C for about 24 hrs, shaking at 125 rpm. About 16–20 hrs prior
to preparing the competent cells, dilute the cells to 7 x 10 5 cells/ml with HL5 media.
Incubate at 21 °C overnight, shaking at 125 rpm.
3. Transfer 100 ml of the cells into two sterile, disposable, 50 ml centrifuge tubes and incu-
bate on ice for 15 min to stop growth.
4. Pellet the cells by centrifugation at 400 x g for 5–7 min at room temperature.
5. Carefully pour off and discard the supernatant; place the centrifuge bottles with the cell
pellets on ice.
6. Pool the cell pellets and resuspend in 50 ml of sterile, ice-cold E buffer. Pellet the cells
by centrifugation at 400 x g for 5–7 min at room temperature.
7. Carefully pour off and discard the supernatant; place the centrifuge bottles with the cell
pellets on ice and resuspend the cells at a concentration of 1 x 10 7 cells/ml. Keep the cells
on ice and use as soon as possible for electroporation.
18