Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 269

which makes plasmid rescue easier), or a chromosome-derived
autonomously replicating sequence ( ARS ) that is copied exactly
once per cell cycle, resulting in a single copy plasmid easily lost
unless it also contains a centromere ( CEN ).

4. Strains carrying the ade1 or ade2 mutations, and no additional
mutations in the adenine biosynthesis pathway, accumulate a
red metabolic intermediate on media with a limited amount of
adenine (provided by the yeast extract without an extra sup-
plement). While this may serve as a visible marker for strain
verifi cation, red biomass color is a sign of futile attempts to
synthesize adenine and a cause of concern for the culture's
well-being. Adding extra adenine to the media is important
especially when transforming adenine auxotrophs and aiming
for high effi ciency (i.e., in library screens).
5. Mix all components of the medium except the sugar in half of
the volume of water in a suffi ciently large autoclavable bottle
with an autoclavable stirring bar until any clumps disappear
(agar will not dissolve at room temperature). Dissolve the
sugar in another bottle in the remaining half of the water.
Autoclave both components (leaving the stirring bar in) and
mix them together after the content has cooled below 60 °C.
For agar-containing media, pour plates (approx. 40 from 1 l if
using 9 cm Petri dishes) immediately after mixing. Keep liquid
media in reasonably sized aliquots to avoid prolonged storage
of opened bottles.
6. A 40 % solution of suffi ciently pure (p.a.) glucose may be auto-
claved without adverse effects. Check with a small amount of
your sugar stock—if the solution does not turn yellow during
autoclaving, you can use this method of sterilization.
7. If your strain has an additional auxotrophic requirement, add
the appropriate amino acid at 100 mg/100 ml of solution B.
8. Flat toothpicks are better than round ones. Sterilize them ver-
tically in a small glass beaker covered by an aluminum foil.
Random orientation ensures that you can choose between the
fl at end for transferring larger amount of yeasts or spreading
them over a large surface and the pointed end for picking small
colonies.
9. Use the bacterial strain and transformation protocol you
would routinely use for cloning. In our hands, 0.5-1

μ l work

l aliquots of E. coli cells.
10. Yeast cultures can undergo epigenetic or genetic changes that
may lead to phenotypic alterations upon prolonged cultivation
(this is sometimes referred to as “picking up modifi ers”).
They also tend to lose plasmids on nonselective media and lose
viability on selective ones upon storage. Retest the strains'
phenotype (including microscopic appearance) periodically

well for electroporation in 50 μ