Bio-Rad Gene Pulser Xcell™ Electroporation Systems - Manual (Page 75)

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.
7.5.2 Electroporation
1. Pipette the DNA samples (up to 50 µg) to be electroporated into sterile 1.5 ml microfuge tubes.
Place tubes on ice.
2. Add 800 µl of the competent cells to each DNA sample and pipette up and down to mix; incubate
on ice ~1 min.
3. From the Home Screen on Gene Pulser Xcell open the Fungal Protocol Screen (press 4, Enter, 2,
Enter). To select D. discoideum, press 6 or the Down Arrow to highlight D. discoideum, then press
Enter to open the D. discoideum Protocol Detail screen. See Section 3.4 for operating instructions.
4. Transfer the DNA - cell samples to 0.4 cm electroporation cuvettes that have been chilled in ice
and tap the suspension to the bottom of the tube. Place the cuvette in the ShockPod. Push the
chamber lid down to close. Pulse once (the program delivers two pulses, each with a ~1 msec
time constant approximately 5 sec apart).
5. Remove the cuvette from the chamber and immediately dilute the cells to 10 ml with the appropri-
ate media.
6. Check and record the pulse parameters. The time constant should be 1 millisecond. The voltage
should be approximately 1.0 kV.
7. When selecting for complementation of an auxotrophic mutant, the cells may be plated
immediately into selective media lacking the appropriate nutrient. When selecting for antibiotic
resistance, incubate the cells overnight at 21°C prior to adding the selective agent.
7.5.3 Solutions and Reagents
1. HL5 media: 17.8 g bacteriological peptone (Oxoid, Ogdensburg, NY), 7.2 g yeast extract, 0.54 g
Na
2
HPO
4
, 0.4 g KH
2
PO
4
, 130 µl B12/Folic acid mix; bring to 1L with water and adjust to pH
6.3–6.5. Autoclave for 25 min on two successive days. Prior to use, add 20 ml of 50% glucose
and 10 ml of 100 X Antibiotic-Antimycotic (Life Technologies, Gaithersburg, MD).
2. B12/Folic acid mix: 5 mg B12, 200 mg folic acid; add 95 ml water, then pH to 6.5–6.8 with 5N
NaOH; bring to 100 ml with water. Filter sterilize and store at -20°C protected from light.
3. E buffer: 10 ml 100 mM NaH
2
PO
4
, adjusted to pH 6.1 with KOH, 10 ml 0.5 M sucrose, 80 ml
water; autoclave.
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