Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 261

8. SD dropout plates: 6.7 g yeast nitrogen base without amino
acids, 20 g agar, 100 ml 10× dropout solution (see above),
850 ml water. Adjust the pH to 5.8 if necessary and autoclave.
Allow to cool to 55 °C and then add 50 ml of fi lter-sterilized
40 % glucose (or other appropriate carbon source to fi nal con-
centration 2 %, see Note 6 ) and pour plates.
9. 5-FOA plates: solution A—14 g/l yeast nitrogen base without
amino acids, 100 mg/l uracil, 110 mg/l adenine, 40 g/l glu-
cose, 20 ml/l solution B, sterilize by fi ltration; solution
B—400 mg/100 ml tryptophan, 600 mg/100 ml leucine,
100 mg/100 ml histidine, 100 mg/100 ml methionine, steril-
ize by fi ltration ( see Note 7 ); solution C—40 g/l agar, auto-
claved. For twenty 35 mm Petri dishes, which is a reasonable
amount, dissolve 70 mg of 5-FOA (e.g., Sigma F5013) in
35 ml of solution A. It is quite diffi cult to dissolve; heating the
solution to approx. 40 degrees is recommendable. Mix with
35 ml of melted agar (solution C) and pour approx. 3 ml per
35 mm dish (use larger dishes if treating many strains at the
same time).
10. 15 % glycerol, autoclaved.
11. Sterile 1.5 ml screw-top Eppendorf vials or cryogenic storage
tubes.
12. Sterile wooden toothpicks ( see Note 8 ), glass rods for plating,
and a bacteriological wire loop.
13. Sterile water.
14. Sterile plastic Petri dishes, pipette tips, Eppendorf vials, glass
bacteriological tubes with stoppers, and Erlenmeyer fl asks.

2.4 Yeast
Transformation

1. Sterile water.
2. 2×YEPD medium: 20 g/l yeast extract, 40 g/l peptone,
40 g/l glucose, 80 mg/l adenine. Autoclave the glucose solu-
tion separately ( see Note 5 ).
3. Single-stranded carrier DNA: can be obtained from different
sources as ready-to-use solution with concentration ranging
from 2 mg/ml to 10 mg/ml (e.g., Sigma D7656). Store ali-
quots in −20 °C. Immediately before transformation or while
cells are growing, thaw 1 ml carrier DNA, incubate for 5 min
at 99 °C (or boiling water bath), then place immediately on
ice. Repeat boiling and cooling twice for high-effi ciency trans-
formation (for rapid transformation, once is enough).
4. 10× TE: 100 mM Tris, 10 mM EDTA, pH 7.5. Use 0.5-1 M
Tris stock solution with pH adjusted to pH 7.5 by HCl to
prepare this buffer.
5. LiAc/TE mix: 1.1 ml/10 ml 1 M lithium acetate solution,
1.1 ml/10 ml 10× TE, 7.8 ml/10 ml water, sterilize by
fi ltration.