342
HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
14. Inspect with a fluorescence microscope equipped with appropriate filter sets for the visualization ofgreen, red and blue fluorescence. Images
may be recorded with conventional microphotography or digital image
acquisition systems. Double band pass filters for green and red excitation are recommended for visual analysis.
Troubleshooting
- Store cell culture media at 4°C for no more than 2 weeks.
- Since the success of the experiments, in particular meiotic time
courses, depends on the culture conditions and since the quality of
the various cell culture components varies depending on the supplier,
it is recommended to always test the performance of the components
used.
- Set up new experiments when wild-type budding rates do not exceed
40%.
- In meiotic time courses, sporulation rates of more than 80% should be
achieved to allow for acquisition and meaningful interpretation of
quantitative FISH data.
- Complete cell wall removal is imperative for a successful FISH experiment, since incomplete cell wall degradation results in high background fluorescence. This is particularly the case in experiments
with structurally preserved nuclei, which are fixed prior to spheroplasting. One should always ensure that the cell wall is maximally degraded
but at the same time, too much cell lysis has to be avoided.
- Another source for high background in yeast FISH is remnants of RNA
in the probe and/or in the cells.
- Cells which have been grown to stationary phase exhibit a thickened
cell wall. Therefore, prolonged spheroplasting times as compared to
the ones given in the protocol above, are required to obtain nuclei suitable for FISH.
- Successful FISH detection of chromosome regions depends on the access of the DNA probe to its target sequence. In preparations fixed with
cross-linking agents like formaldehyde, access is generally achieved
by limited proteolytic treatment and/or treatment with chaotropic
reagents, strong detergents or pre-incubation with 4xSSC/Tween
HARRY SCHERTHAN and EDGAR TRELLES-STICKEN
14. Inspect with a fluorescence microscope equipped with appropriate filter sets for the visualization ofgreen, red and blue fluorescence. Images
may be recorded with conventional microphotography or digital image
acquisition systems. Double band pass filters for green and red excitation are recommended for visual analysis.
Troubleshooting
- Store cell culture media at 4°C for no more than 2 weeks.
- Since the success of the experiments, in particular meiotic time
courses, depends on the culture conditions and since the quality of
the various cell culture components varies depending on the supplier,
it is recommended to always test the performance of the components
used.
- Set up new experiments when wild-type budding rates do not exceed
40%.
- In meiotic time courses, sporulation rates of more than 80% should be
achieved to allow for acquisition and meaningful interpretation of
quantitative FISH data.
- Complete cell wall removal is imperative for a successful FISH experiment, since incomplete cell wall degradation results in high background fluorescence. This is particularly the case in experiments
with structurally preserved nuclei, which are fixed prior to spheroplasting. One should always ensure that the cell wall is maximally degraded
but at the same time, too much cell lysis has to be avoided.
- Another source for high background in yeast FISH is remnants of RNA
in the probe and/or in the cells.
- Cells which have been grown to stationary phase exhibit a thickened
cell wall. Therefore, prolonged spheroplasting times as compared to
the ones given in the protocol above, are required to obtain nuclei suitable for FISH.
- Successful FISH detection of chromosome regions depends on the access of the DNA probe to its target sequence. In preparations fixed with
cross-linking agents like formaldehyde, access is generally achieved
by limited proteolytic treatment and/or treatment with chaotropic
reagents, strong detergents or pre-incubation with 4xSSC/Tween