312
HARRY SCHERTHAN
the development of reverse and forward comparative chromosome painting protocols by which a refined resolution of comparative chromosome
maps is achieved (Goureau et al. 1996, Wienberg et aI. 1997, Nash et al.
1998). Presently, it remains to be seen whether the Zoo-FISH approach
proves effective in other than eutherian species combinations. The
Zoo-FISH protocol outlined below displays the general procedure for
the preparation and hybridization of human plasmid WCPs (Collins et
al. 1991) to the chromosomes of a mammalian species of choice.
Materials
Equipment - Pasteur pipettes
- Plastic centrifuge tubes, 15 ml
- Glass slides. Remove impurities by submerging slides for several hours
in 80% ethanol in a Coplin jar. Dry with a Kim wipe prior to use
- Coverslips of 22x60 mm and round coverslips with 10 mm diameter
(Hecht Assistent)
- Coplin jars with covers
- Hot plate or heating block
- Moist chamber to prevent slides from drying out. This can be e.g. a
plastic box and lid with wet paper and glass rods to raise slides above
the moist surface.
- Water bath, preferably shaking. It is used to adjust the temperature of
the solutions in Coplin jars. Always measure temperatures inside Coplin jars.
- Fluorescence microscope with appropriate fllter sets and camera system
Reagents and
solutions
- Methanol and glacial acetic acid (Merck)
- Fixative; make fresh each time by mixing 3 parts of ice-cold methanol
with 1 part of glacial acetic acid, always keep on ice.
- 20xSSC (3 M NaCI, 0.3 M Na3citrate, pH 7.0). Store at RT. Make up
0.05 x SSC from this stock. Discard dilute solutions after use.
HARRY SCHERTHAN
the development of reverse and forward comparative chromosome painting protocols by which a refined resolution of comparative chromosome
maps is achieved (Goureau et al. 1996, Wienberg et aI. 1997, Nash et al.
1998). Presently, it remains to be seen whether the Zoo-FISH approach
proves effective in other than eutherian species combinations. The
Zoo-FISH protocol outlined below displays the general procedure for
the preparation and hybridization of human plasmid WCPs (Collins et
al. 1991) to the chromosomes of a mammalian species of choice.
Materials
Equipment - Pasteur pipettes
- Plastic centrifuge tubes, 15 ml
- Glass slides. Remove impurities by submerging slides for several hours
in 80% ethanol in a Coplin jar. Dry with a Kim wipe prior to use
- Coverslips of 22x60 mm and round coverslips with 10 mm diameter
(Hecht Assistent)
- Coplin jars with covers
- Hot plate or heating block
- Moist chamber to prevent slides from drying out. This can be e.g. a
plastic box and lid with wet paper and glass rods to raise slides above
the moist surface.
- Water bath, preferably shaking. It is used to adjust the temperature of
the solutions in Coplin jars. Always measure temperatures inside Coplin jars.
- Fluorescence microscope with appropriate fllter sets and camera system
Reagents and
solutions
- Methanol and glacial acetic acid (Merck)
- Fixative; make fresh each time by mixing 3 parts of ice-cold methanol
with 1 part of glacial acetic acid, always keep on ice.
- 20xSSC (3 M NaCI, 0.3 M Na3citrate, pH 7.0). Store at RT. Make up
0.05 x SSC from this stock. Discard dilute solutions after use.