Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 260

may be obtained on request by laboratories that produced them.
The “Lacroute library” of young A. thaliana seedling-derived
cDNAs under the control of the constitutive PGK1 promoter in
the pFL61 vector including the URA3 selection marker [ 12 ] can
be acquired from the ATCC (No. 77500).

2.3 Yeast Cultivation,
Storage, and
Manipulation

1. YEPD medium: 10 g/l yeast extract, 20 g/l peptone, 20 g/l
glucose. For strains with an adenine requirement, add 40 mg/l
adenine to improve growth ( see Note 4 ). Autoclave the glu-
cose solution separately ( see Note 5 ).
2. YEPD plates: 10 g/l yeast extract, 20 g/l peptone, 20 g/l
agar, 20 g/l glucose. For strains with an adenine requirement,
add 40 mg/l adenine to improve growth ( see Note 4 ).
Autoclave the glucose solution separately and pour plates
( see Note 5 ).
3. YEPGal plates: 10 g/l yeast extract, 20 g/l peptone, 20 g/l
agar, 20 g/l galactose. Autoclave the galactose solution sepa-
rately and pour plates ( see Note 5 ).
4. −URA plates: 7.3 g/l yeast nitrogen base without amino acids,
20 g/l agar, 10 g/l casamino acids, 50 mg/l tyrosine, 50 mg/l
adenine, 20 g/l glucose (replace with galactose if required for
galactose-inducible expression under selective conditions).
Autoclave the sugar solution separately and pour plates
( see Note 5 ).
5. +URA plates: prepare like −URA plates but add uracil to fi nal
concentration 50 mg/l from an autoclaved or fi lter-sterilized
100× concentrated stock prior to pouring plates.
6. 10× Dropout solutions: 20 mg/100 ml L -adenine hemisulfate
salt, 20 mg/100 ml L -arginine HCl, 20 mg/100 ml L -histi-
dine HCl monohydrate, 30 mg/100 ml L -isoleucine,
100 mg/100 ml L -leucine, 30 mg/100 ml L -lysine HCl,
20 mg/100 ml L -methionine, 50 mg/100 ml L -phenylalanine,
200 mg/100 ml L -threonine, 20 mg/100 ml L -tryptophan,
30 mg/100 ml L -tyrosine, 20 mg/100 ml L -uracil,
150 mg/100 ml L -valine. Leave out the appropriate amino
acid(s) or nucleotides to obtain selective media (e.g., leaving
out tryptophan produces a −TRP dropout for use in media
selecting for clones carrying the TRP1 marker in a trp1 back-
ground); a complete solution without leaving out any compo-
nents should be used for controls.
7. SD dropout medium: 6.7 g yeast nitrogen base without amino
acids, 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 a fi nal concentration of
2 % ( see Note 6 ).