Heterologous Expression in Budding Yeast as a Tool for Studying the Plant Cell Morphogenesis Machinery - Plant Cell Morphogenesis: Methods and Protocols - page 270

and use only fresh plate cultures (preferentially less than a
week old) to start experiments. Avoid using cultures exhibit-
ing obvious heterogeneity in colony appearance. In liquid cul-
tures, beware of unusual smell or too fast growth, as both may
be signs of bacterial contamination. Normal doubling time of
a haploid, polyauxotrophic laboratory yeast strain in liquid
YEPD at 28 °C is around 2 h.
11. It is possible to use 1.5 ml Eppendorf tubes at this and follow-
ing steps, adding an extra round of centrifugation: spin down
half of the sample, remove supernatant, add a second half of
the sample to the pellet, and spin again.
12. Empty vector transformation introduces the vector's selective
marker, eliminating thus effects of differing auxotrophic
requirements of the original (non-transformed) strain and the
transformant carrying the gene to be expressed.
13. Consider using larger, square Petri dishes when evaluating
many strains. In this case, you also can use mutichannel
pipettes and sterile microtiter plate for serial dilutions and
plating. Make sure the layout of drops is identical for all dishes.
14. Use a fresh toothpick for each colony. To maintain the same
layout on both plates, draw two copies of a template on two
disposable Petri dish lids using a permanent marker and lay
these templates underneath the dishes when transferring the
colonies.

Acknowledgments

This work has been supported by the MSM0021620858 project.
We thank Marta adyová for expert technical assistance and mem-

bers of the A. Ragnini laboratory (University of Vienna, Austria)
for the rapid yeast transformation protocol.

Č

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