336
HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
2. Stop cell wall digest by adding ice cold Sol II.
3. Sediment cells and resuspend them in a volume of Sol II corresponding
to a 1/20 of the starting volume.
4. Place an aliquot of spheroplasts (e.g. 20 fll) on a clean glass slide.
5. Add 1% Lipsol and ftxative in the ratio: 1 vol spheroplasts: 2 vol Lipsol:
4 vol ftxative.
6. Briefly tilt the slide after each step to mix the solutions evenly within
the resulting drop.
7. After addition of the ftxative, distribute the suspension evenly by
streaking with the side of a pipette tip over the slide without touching
the surface.
8. The degree of nuclear spreading may be altered in two ways: Changing
the given ratios in favor of Lipsol results in nucleoids with a bigger
diameter, while the resulting diameter will be smaller when the relative
volume of detergent is reduced in the mixture.
Preparation of
extensively spread
nuclei
Extensively spread nuclei are obtained when the aliquots removed from
the culture are directly transferred to ice and the pre-ftxation step is
omitted. Spheroplasting and detergent spreading of unftxed cells is
then performed as described above, with the exception that ftxation is performed according to the following scheme:
- Successively mix: 1 vol spheroplasts/2 vol ftxative/4 vol Lipso1l4 vol
ftxative.
- Tilt and air dry.
I Subprotocol 2
Probe labeling and FISH
Nick translation, random priming and long range PCR protocols have proven effective methods to label probe DNAs in our hands. For ease of handling, we recommend the use of commercially available labeling kits. For a
detailed description of the set up of a nick translation reaction, the reader
may refer to detailed protocols published elsewhere (Lichter and Cremer
1992). A critical aspect for successful yeast FISH is that all probes used are
completely free of RNA. With the availability of the complete DNA se-
HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
2. Stop cell wall digest by adding ice cold Sol II.
3. Sediment cells and resuspend them in a volume of Sol II corresponding
to a 1/20 of the starting volume.
4. Place an aliquot of spheroplasts (e.g. 20 fll) on a clean glass slide.
5. Add 1% Lipsol and ftxative in the ratio: 1 vol spheroplasts: 2 vol Lipsol:
4 vol ftxative.
6. Briefly tilt the slide after each step to mix the solutions evenly within
the resulting drop.
7. After addition of the ftxative, distribute the suspension evenly by
streaking with the side of a pipette tip over the slide without touching
the surface.
8. The degree of nuclear spreading may be altered in two ways: Changing
the given ratios in favor of Lipsol results in nucleoids with a bigger
diameter, while the resulting diameter will be smaller when the relative
volume of detergent is reduced in the mixture.
Preparation of
extensively spread
nuclei
Extensively spread nuclei are obtained when the aliquots removed from
the culture are directly transferred to ice and the pre-ftxation step is
omitted. Spheroplasting and detergent spreading of unftxed cells is
then performed as described above, with the exception that ftxation is performed according to the following scheme:
- Successively mix: 1 vol spheroplasts/2 vol ftxative/4 vol Lipso1l4 vol
ftxative.
- Tilt and air dry.
I Subprotocol 2
Probe labeling and FISH
Nick translation, random priming and long range PCR protocols have proven effective methods to label probe DNAs in our hands. For ease of handling, we recommend the use of commercially available labeling kits. For a
detailed description of the set up of a nick translation reaction, the reader
may refer to detailed protocols published elsewhere (Lichter and Cremer
1992). A critical aspect for successful yeast FISH is that all probes used are
completely free of RNA. With the availability of the complete DNA se-