12. Homogenizer (use for example the Mixer Mill MM
400, Retsch).
13. Vacuum concentrator.
14. HPLC-PDA-HRMS apparatus.
3 Methods
3.1 Preparation
of Electrocompetent
Yeast Cells
1. Inoculate the yeast strain in 5–10 mL YPD medium. Grow
overnight (O/N) in a rotary shaker at 30
C, 150 rpm.
2. Inoculate 50 mL YPD with saturated culture (measure OD;
starting OD 600 should be about 0.2) (see Note 3).
3. Grow cells until OD 600 ¼ 0.6–0.8.
4. Collect the cells by centrifugation (1200 Â g, 5 min, 4
C).
5. Wash cells with 20 mL of sterile bd H 2 O and centrifuge
(1200 Â g, 5 min, 4
C).
6. Add 5 mL 1 M sorbitol and 0.1 mL 0.5 M DTT.
7. Incubate at 30
C, 10 min, 150 rpm.
8. Centrifuge (1200 Â g, 5 min, 4
C). From now on work on ice.
9. Wash cells with 5 mL of cold 1 M sorbitol (no DTT) and
centrifuge (1200 Â g, 5 min, 4
C). Repeat this step twice.
10. Resuspend cells in 0.4 mL cold 1 M sorbitol.
11. Aliquot 40 μL cells per reaction tube.
3.2 Yeast
Transformation by
Electroporation
1. Mix 40 μL cells with DNA (100 ng vector/300 ng insert) [the
volume of DNA should be 5 μL]. Use chilled/sterile
cuvettes.
2. Pulse at 1.5 kV. 25 μF, 200 Ω (5 ms).
3. Add immediately 0.2 mL 1 M sorbitol.
4. Plate on selective SD medium.
3.3 Isolation
of Microsomes from
Recombinant Yeast
Cells
1. Inoculate 5–10 mL liquid media (adapted to your yeast strain)
with one yeast colony.
2. Grow on selective SD medium O/N at 30
C, 150 rpm.
3. Inoculate 900 mL liquid culture (selective SD medium) with
an aliquot of the O/N culture (OD 600 ¼ at least 0.2) and let it
grow O/N.
4. Sediment the yeast cells by centrifugation 10 min at 1200 Â g.
Discard supernatant.
5. Resuspend the pellet in 900 mL YPD media and let them grow
for 1 h at 30
C, continuous shaking (This step helps to soften
the cell walls).
94
Olivia Costantina Demurtas et al.
400, Retsch).
13. Vacuum concentrator.
14. HPLC-PDA-HRMS apparatus.
3 Methods
3.1 Preparation
of Electrocompetent
Yeast Cells
1. Inoculate the yeast strain in 5–10 mL YPD medium. Grow
overnight (O/N) in a rotary shaker at 30
C, 150 rpm.
2. Inoculate 50 mL YPD with saturated culture (measure OD;
starting OD 600 should be about 0.2) (see Note 3).
3. Grow cells until OD 600 ¼ 0.6–0.8.
4. Collect the cells by centrifugation (1200 Â g, 5 min, 4
C).
5. Wash cells with 20 mL of sterile bd H 2 O and centrifuge
(1200 Â g, 5 min, 4
C).
6. Add 5 mL 1 M sorbitol and 0.1 mL 0.5 M DTT.
7. Incubate at 30
C, 10 min, 150 rpm.
8. Centrifuge (1200 Â g, 5 min, 4
C). From now on work on ice.
9. Wash cells with 5 mL of cold 1 M sorbitol (no DTT) and
centrifuge (1200 Â g, 5 min, 4
C). Repeat this step twice.
10. Resuspend cells in 0.4 mL cold 1 M sorbitol.
11. Aliquot 40 μL cells per reaction tube.
3.2 Yeast
Transformation by
Electroporation
1. Mix 40 μL cells with DNA (100 ng vector/300 ng insert) [the
volume of DNA should be 5 μL]. Use chilled/sterile
cuvettes.
2. Pulse at 1.5 kV. 25 μF, 200 Ω (5 ms).
3. Add immediately 0.2 mL 1 M sorbitol.
4. Plate on selective SD medium.
3.3 Isolation
of Microsomes from
Recombinant Yeast
Cells
1. Inoculate 5–10 mL liquid media (adapted to your yeast strain)
with one yeast colony.
2. Grow on selective SD medium O/N at 30
C, 150 rpm.
3. Inoculate 900 mL liquid culture (selective SD medium) with
an aliquot of the O/N culture (OD 600 ¼ at least 0.2) and let it
grow O/N.
4. Sediment the yeast cells by centrifugation 10 min at 1200 Â g.
Discard supernatant.
5. Resuspend the pellet in 900 mL YPD media and let them grow
for 1 h at 30
C, continuous shaking (This step helps to soften
the cell walls).
94
Olivia Costantina Demurtas et al.