Bio-Rad MicroPulser™ Electroporator - Manual (Page 23)

10.2 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. Set the MicroPulser to "dic". See Section 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 chamber
slide. Push the slide into the chamber until the cuvette is seated between the contacts in
the base of the chamber. Pulse once (the program delivers two pulses approximately 5 sec
apart).
5. Remove the cuvette from the chamber and immediately dilute the cells to 10 ml with the
appropriate media.
6. Check and record the pulse parameters. The time constant should be 1 millisecond. 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 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.
10.3 Solutions and Reagents For Electroporation
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.
Section 11
Electroporation of
Pichia pastoris
11.1 Preparation of Eelectrocompetent Cells
See Cregg & Russell (1998) for additional information.
1. Inoculate 500 ml of YPD in a 2.8 L Fernbach flask with an aliquot from a fresh overnight
culture of P. pastoris. The doubling time of P. pastoris is approximately 2 hrs at 30 °C.
2. Incubate at 30 °C overnight, shaking at 300 rpm, to a density of 5–7 x 10 7 cells/ml.
3. Decant the cells into two sterile 250 ml centrifuge bottles and pellet the cells by cen-
trifugation at 3000 x g for 5 min at 4 °C.
4. Carefully pour off and discard the supernatant.
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