Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 263

2. To revive a strain from storage, scratch the surface of the still
frozen stock (kept in a freezer block or on ice) with the blunt
end of a sterile toothpick and streak on a fresh YEPD plate to
obtain single colonies (use a repeatedly fi re-sterilized wire
loop or additional two new toothpicks to ensure colony sepa-
ration). Alternatively, keep the stock in liquid nitrogen and
scratch its surface with a yellow pipette tip; while this is more
hassle, it is less destructive for the stock.
3. Use a refrigerator for short-term storage of yeast cultures on
plates (up to 2 weeks).

3.2 High-Effi ciency
Yeast Transformation
for Library Screening

This protocol is based on the standard library transformation pro-
tocol from Dualsystems Biotech ( http://www.dualsystems.com );
other transformation protocols for two hybrid library screening
may work as well.

1. Inoculate yeast from the master plate into 10 ml YEPD and
grow for 8 h at 28 °C with shaking (25 °C should be used for
temperature-sensitive strains in this and all following steps).
2. Inoculate 100 ml YEPD with the entire 10 ml culture from
step 1 and grow overnight at 28 °C with shaking.
3. Take a 1 ml aliquot of the culture, centrifuge at 2,500 × g for
5 min, and resuspend the pellet in 1 ml of water. Measure
OD 550 against a water blank.
4. Calculate the amount of culture needed for 30 OD units.
Aliquot this amount of the overnight culture into 50 ml Falcon
tubes and spin down at 700 × g for 5 min.
5. Resuspend the pellet in 200 ml 2×YEPD (pre-warmed to
28 °C) in a 1 l Erlenmeyer fl ask and remove a 1 ml aliquot.
Centrifuge this 1 ml aliquot at 2,500 × g for 5 min, discard the
supernatant, and resuspend the pellet in water. Measure OD 550
against a water blank; the value should be around 0.15 (30
OD units in 200 ml correspond to OD = 0.15).
6. Grow the cells at 28 °C with vigorous shaking to OD 550 = 0.6
(two cell divisions; typically this takes 3-5 h but may vary
depending on the yeast strain used). Boil the single-stranded
carrier DNA in the meantime.
7. After reaching the required OD, divide the 200 ml culture
into four 50 ml Falcon tubes and centrifuge at 700 × g for
5 min.
8. Resuspend each pellet in 30 ml of sterile water by vortexing.
Centrifuge at 700 × g for 5 min.
9. Remove the supernatant, resuspend each pellet in 1 ml LiAc/
TE mix, and transfer to an Eppendorf tube. Centrifuge at
700 × g for 5 min.