Bio-Rad Gene Pulser Xcell™ Electroporation Systems User Manual
Page 70
8.
Plate aliquots of the electroporated cells on selective agar plates containing 1 M sorbitol. Incubate
plates for 48–72 hrs at 30°C.
7.1.3 Solutions and reagents
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.
7.2 Schizosaccharomyces pombe
7.2.1 Preparation of Electrocompetent Cells
See Prentice (1991) for additional information.
Using this method we have obtained transformation efficiencies up to 3 x 10
5
transformants / µg
electroporating S. pombe CHP408 with pART1 (Apolinario, et al, 1993).
Gene Pulser Xcell conditions: C = 25 µF; PC = 200 ohm; V = 2.3 kV.
This procedure requires a Gene Pulser Xcell main unit and PC Module.
1.
Inoculate 200 ml of YCD in a 2.8 L Fernbach flask with an aliquot from an overnight culture of S.
pombe. (An initial OD
600
of 0.1 is usually a good starting point.) The doubling time of S. pombe is
approximately 2 hrs at 30°C.
2.
Incubate at 30°C overnight, shaking at 250 rpm, to late log phase (~2 x 10
8
cells/ml; OD
600
(1:20 dil)
= 0.4–0.5).
3.
Chill the cells in an ice water bath for 15 min to stop growth.
4.
Decant the cells into a sterile 250 ml centrifuge bottle and pellet the cells by centrifugation at 4000
x g for 5 min at 4°C.
5.
Carefully pour off and discard the supernatant; keep the cells on ice.
6.
Add ~50 ml of sterile, ice-cold 1.2 M sorbitol and vortex to resuspend the cell pellet; bring the
volume in each of the centrifuge bottles to ~250 ml. Pellet the cells by centrifugation at 4000 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 ~200 ml sterile, ice-cold 1.2 M sorbitol.
8.
Resuspend the cell pellet with ~10 ml of sterile, ice-cold 1.2 M sorbitol and transfer to a chilled 30
ml Oakridge tube; rinse the centrifuge bottle with 15–20 ml of 1.2 M ice cold 1.2 M sorbitol and
transfer to the Oakridge tube. Pellet the cells by centrifugation at 4000 x g for 5 min at 4°C; pour
off and discard the supernatant.
9.
Resuspend the cell pellet in 3 ml of sterile, ice-cold 1.2 M sorbitol. Keep the cells on ice and use
as soon as possible for electroporation.
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