250
2. Yeast cells are harvested by centrifugation (3 min, 500 × g),
washed once with sterile water (0.1–0.5 volumes) followed by
a washing step with sterile SORB buffer (0.1–0.2 volumes).
3. Cells are suspended in 450 μl SORB buffer and 50 μl of carrier
DNA is added. Cells are divided into appropriate aliquots and
placed at −80 °C (no shock freezing).
4. Usually, 50 μl of competent cells are used for the transformation with a PCR product. Thawed competent cells are mixed
with PCR product (5–15 μl of DNA for 50 μl of cells) and six
volumes of PEG buffer are added.
5. Cells are incubated at room temperature for approximately
30 min.
6. Cells are incubated at 42 °C for 5–20 min (15 min works well
with most strains).
7. Cells are sedimented (3 min, 500 × g) and washed once with
YPD.
8. The pellet is resuspended in YPD and spread onto an appropriate selection medium. Plates with yeast cells harboring a lightsensitive construct should be protected from ambient light
during growth and storage.
A modified standard method is used to transfer plasmids from yeast
to E. coli for further usage [21].
1. An amount of yeast cells corresponding to the size of a match
head is scraped off a plate and dissolved in 500 μl of breaking
buffer.
2. 200 μl of phenol–chloroform–isoamyl alcohol mixture
(25/24/1, buffered with TE, pH 7.5–8) and 0.3 g of glass
beads (~200 μl) are added.
3. Cells are disrupted by vortexing (5 min, highest speed) and
phases are separated in a microcentrifuge (10 min, 16,000 × g).
4. 400 μl of the aqueous layer are transferred to a new tube and
1 ml of ethanol is added to this tube.
5. The tube is incubated at −20 °C for 10 min and then subjected
to centrifugation (10 min, 16,000 × g).
6. The supernatant is removed and the pellet is washed with 70%
ethanol.
7. The tube is centrifuged again (3 min, 16,000 × g), the supernatant removed and the pellet air-dried for 3 min.
8. The pellet is dissolved in 20 μl of desalted water.
9. 1 μl of the solution is used for transformation of E. coli cells by
electroporation.
3.6 Plasmid Rescue
from Yeast
Christof Taxis
2. Yeast cells are harvested by centrifugation (3 min, 500 × g),
washed once with sterile water (0.1–0.5 volumes) followed by
a washing step with sterile SORB buffer (0.1–0.2 volumes).
3. Cells are suspended in 450 μl SORB buffer and 50 μl of carrier
DNA is added. Cells are divided into appropriate aliquots and
placed at −80 °C (no shock freezing).
4. Usually, 50 μl of competent cells are used for the transformation with a PCR product. Thawed competent cells are mixed
with PCR product (5–15 μl of DNA for 50 μl of cells) and six
volumes of PEG buffer are added.
5. Cells are incubated at room temperature for approximately
30 min.
6. Cells are incubated at 42 °C for 5–20 min (15 min works well
with most strains).
7. Cells are sedimented (3 min, 500 × g) and washed once with
YPD.
8. The pellet is resuspended in YPD and spread onto an appropriate selection medium. Plates with yeast cells harboring a lightsensitive construct should be protected from ambient light
during growth and storage.
A modified standard method is used to transfer plasmids from yeast
to E. coli for further usage [21].
1. An amount of yeast cells corresponding to the size of a match
head is scraped off a plate and dissolved in 500 μl of breaking
buffer.
2. 200 μl of phenol–chloroform–isoamyl alcohol mixture
(25/24/1, buffered with TE, pH 7.5–8) and 0.3 g of glass
beads (~200 μl) are added.
3. Cells are disrupted by vortexing (5 min, highest speed) and
phases are separated in a microcentrifuge (10 min, 16,000 × g).
4. 400 μl of the aqueous layer are transferred to a new tube and
1 ml of ethanol is added to this tube.
5. The tube is incubated at −20 °C for 10 min and then subjected
to centrifugation (10 min, 16,000 × g).
6. The supernatant is removed and the pellet is washed with 70%
ethanol.
7. The tube is centrifuged again (3 min, 16,000 × g), the supernatant removed and the pellet air-dried for 3 min.
8. The pellet is dissolved in 20 μl of desalted water.
9. 1 μl of the solution is used for transformation of E. coli cells by
electroporation.
3.6 Plasmid Rescue
from Yeast
Christof Taxis