27 Yeast FISH: Delineation of Chromosomal Targets in Vegetative and Meiotic Yeast Cells
333
2. Transfer a colony from the plate into an appropriate volume (e.g. 20 ml)
of liquid YPD medium and grow the culture at 30°C to an OD600=1.
3. Analyze budding rate (mitotic cells) by transferring an aliquot of cells
to a phase contrast microscope. Logarithmic growth is indicated by a
budding rate of 50-60%.
4. Transfer an aliquot ofyour culture (e.g. 9 ml) to a centrifuge tube on ice
containing 1/ 10 volume of acid-free formaldehyde (37%) (Pringle et al.
1991).
5. Incubate for 30 min on ice.
6. Sediment cells at 700 g for 3 min and wash them once in a large volume
of dH 2 0.
7. Sediment cells at 700 g for 3 min and resuspend them in 1/10 of the
original volume of Solution I. Cells are now ready for spheroplasting
(see procedure 1.3).
1.2 Preparation of meiotic yeast nuclei
Since the yeast cell cycle is regulated by the availability of nutrients, vegetative cells can be induced to switch from vegetative growth (budding)
to meiosis (sporulation) by the deprivation of nitrogen and fermentable
carbon sources. In fast sporulating strains like SKI (Kane and Roth 1974)
cells enter and progress through meiosis in a rather synchronous manner
(Padmore et aI. 1991) which ultimately yields haploid spores.
1. Streak out diploid ala strain of interest on a YPD agar plate (see procedure 1.1., step 1) and transfer a colony to pre-sporulation medium
(e.g. 20 mI). Grow cells at 30°C overnight to a density of OD 6oo =1-1.5.
Control the budding rate which should be 40% (see troubleshooting).
2. Sediment cells at 3000 rpm for 5 min at RT.
3. Wash the cells twice with sporulation medium at 30°C.
4. Resuspend the cell pellet in half the initial volume of sporulation medium (here 10 ml) and return the culture back to 30°C. This time point
represents t=O in a meiotic time course experiment.
5. Remove aliquots from the culture at t=O and subsequent time points.
333
2. Transfer a colony from the plate into an appropriate volume (e.g. 20 ml)
of liquid YPD medium and grow the culture at 30°C to an OD600=1.
3. Analyze budding rate (mitotic cells) by transferring an aliquot of cells
to a phase contrast microscope. Logarithmic growth is indicated by a
budding rate of 50-60%.
4. Transfer an aliquot ofyour culture (e.g. 9 ml) to a centrifuge tube on ice
containing 1/ 10 volume of acid-free formaldehyde (37%) (Pringle et al.
1991).
5. Incubate for 30 min on ice.
6. Sediment cells at 700 g for 3 min and wash them once in a large volume
of dH 2 0.
7. Sediment cells at 700 g for 3 min and resuspend them in 1/10 of the
original volume of Solution I. Cells are now ready for spheroplasting
(see procedure 1.3).
1.2 Preparation of meiotic yeast nuclei
Since the yeast cell cycle is regulated by the availability of nutrients, vegetative cells can be induced to switch from vegetative growth (budding)
to meiosis (sporulation) by the deprivation of nitrogen and fermentable
carbon sources. In fast sporulating strains like SKI (Kane and Roth 1974)
cells enter and progress through meiosis in a rather synchronous manner
(Padmore et aI. 1991) which ultimately yields haploid spores.
1. Streak out diploid ala strain of interest on a YPD agar plate (see procedure 1.1., step 1) and transfer a colony to pre-sporulation medium
(e.g. 20 mI). Grow cells at 30°C overnight to a density of OD 6oo =1-1.5.
Control the budding rate which should be 40% (see troubleshooting).
2. Sediment cells at 3000 rpm for 5 min at RT.
3. Wash the cells twice with sporulation medium at 30°C.
4. Resuspend the cell pellet in half the initial volume of sporulation medium (here 10 ml) and return the culture back to 30°C. This time point
represents t=O in a meiotic time course experiment.
5. Remove aliquots from the culture at t=O and subsequent time points.