Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 264

10. Remove the supernatant and resuspend each pellet in 600 μ l

of LiAc/TE mix.
11. Set up four 50 ml Falcon tubes, each of them containing, in
this order: cDNA library 7 μ g, single-stranded carrier DNA

solution 200 μ g (in a volume to up to 100 μ l), cell suspension

l, PEG/LiAc mix 2.5 ml.
12. Vortex for 1 min to thoroughly mix all components and incu-
bate at 28 °C for 45 min, mixing briefl y every 15 min.
13. In the meantime, rinse one of the Falcon tubes containing the
rest of cells from step 10 with 100

from the previous step 600 μ

l of sterile water and plate
this suspension on a clearly labeled selection plate (same as in
step 18 ) as DNA free control.
14. Add 160

μ

l DMSO to each tube and mix immediately by shak-
ing. Incubate at 42 °C for 20 min.
15. Pellet cells at 700 × g for 5 min. Resuspend each pellet in 3 ml
of 2×YEPD and pool all cell suspensions. Let the cells recover
at 28 °C for 90 min with shaking.
16. Pellet the cells at 700 × g for 5 min and resuspend the pellet in
4.5 ml of 0.9 % NaCl.
17. Plate onto the appropriate selection plates (depends on the
plasmid used in the library; if using an inducible expression
system, make sure that the plates contain the right inducer,
e.g., galactose). Use 300

μ

μ l of resuspended cells per 15 cm

plate or 200 μ l per 9 cm plate. Take aside a 45 μ l aliquot

l of 0.9 % NaCl
(resulting in 10 −2 dilution), repeat 4-5 times to obtain serial
dilutions with the factor of 10, and plate three to four highest
dilutions on a separate clearly labeled control plate for estimat-
ing transformation effi ciency. This protocol can give about
10 6 -10 7 transformants in total, depending on the strain
and library.
18. If complementing a temperature-sensitive defect, keep all
plates for a couple of hours (at most overnight) at the permis-
sive temperature and transfer the library screen to the restric-
tive temperature next morning. Colonies may appear gradually
in the course of a week. Leave both controls at the permissive
temperature; the DNA-free control should produce no colo-
nies, while the effi ciency control is expected to show abundant
growth. Count colonies on the later and calculate transforma-
tion effi ciency.
19. If the growth defect of the mutant to be complemented is not
temperature sensitive but, e.g., elicited by a pharmacological
treatment, include the appropriate chemicals into the selective
media.

(1/100 of total volume), dilute it into 450 μ