Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 268

4. Add 360 l of the NaOH/SDS solution, mix by inverting sev-

eral times, and incubate at room temperature for 5 min.
5. Add 270

μ

l of the KAc/HAc solution, mix by inverting several
times, and centrifuge at full speed for 10 min.
6. Transfer supernatant carefully to a fresh tube, add 560

μ

l of
isopropanol, and mix. Centrifuge at full speed for 5 min and
remove supernatant.
7. Wash the pellet with 75 % ethanol and centrifuge as above.
Remove supernatant carefully.
8. Dissolve the pellet in 20

μ

l of sterile water and use an aliquot
for transformation of competent E. coli cells ( see Note 9 ).

μ

4 Notes

1. A dedicated incubator is better than one shared with other cul-
tures, although 37 °C cultures can be grown together with E.
coli and low temperature ones with Agrobacterium . However,
beware that yeast cultures produce CO 2 that might affect other
contents of the incubator and that they also attract free-living
fruit fl ies that may become a nuisance. Depending on incuba-
tor type, you will probably have to seal the plates with Parafi lm
to prevent drying out during prolonged cultivation.

2. Commonly used selection markers are auxotrophies (e.g., the
trp1 , leu2 , his3, ade2, ura3 mutations conferring dependence on
exogenous tryptophan, leucine, histidine, adenine, or uracil,
respectively).The standard yeast nomenclature labels recessive
mutations in lowercase italics and dominant alleles in uppercase
italics; distinct genes of the same or related function are num-
bered (i.e., ADE2 and ADE3 are two different genes), and dis-
tinct alleles of the same locus are denoted by numbers after a
hyphen (i.e., leu2-3,112 is a recessive allele of the LEU2 gene
carrying two different point mutations). Beware of multiple
mutations in the same pathway (i.e., a double ade2 ade3 mutant
would have to be complemented by two genes to regain adenine
independence) and of undocumented mutations introduced by
crossing in the strains' history. If using a galactose-inducible
expression system, make sure your strain does not carry gal
mutations preventing utilization of galactose.

3. DNA constructs may be maintained in yeasts either integrated
into the yeast chromosome via homologous recombination
(integrative vectors) or as episomes of a varying copy number
(replicative plasmid vectors). Avoid integrative vectors if you
plan to rescue the construct back from yeasts. Replicative vectors
usually contain the replication origin of the yeast endogenous
2

μ m plasmid, or 2 μ ORI (leading to high-copy amplifi cation,