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JAIME GOSALVEZ et al.
Procedure
Restriction endonucleases
REs recognise specific DNA targets. REs such as AluI, MboI, Sau3A, MspI,
HpaII, HaeIII, DdeI have been extensively used for "in situ" DNA digestion (Gosalvez et al. 1997a); all of them have been able to induce longitudinal differentiation of the chromosomes of different species.
In situ digestion with REs
1. Chromosome preparations are made following standard procedures of
spreading or squashing. The slides are left at room temperature for at
least 2 h in order to ensure that they are completely dried before digestion.
2. Digestion of chromosomes with REs is always performed on freshly
dried preparations (no more than 12-24 h after having been prepared).
The standard procedure was described by Mezzanotte et al. (1985).
However, slight modifications of the standard procedure may be required for each type of chromosome.
10-50 units of the RE diluted to 20-50 III in the buffer recommended by
the supplier are usually used per slide. The enzyme solution is placed
on the slide and covered with a coverslip to spread the enzyme evenly.
3. The slides are incubated in a humid box for a selected time and at the
temperature specified by the manufacturer. Usually, overnight incubation at 37°C is a good standard for most REs. In order to prevent evaporation during long-term incubations, the coverslip can be sealed
with rubber solution.
4. Control experiments are performed under identical conditions by
treating the preparations with the incubation buffer without the enzyme.
5. The reaction is stopped by washing the slides at room temperature for
5 min in 2xSSC (pH 7).
6. Those preparations to be used for FISH are dehydrated in an ethanol
series (70%, 85%, 100%) and air dried.
JAIME GOSALVEZ et al.
Procedure
Restriction endonucleases
REs recognise specific DNA targets. REs such as AluI, MboI, Sau3A, MspI,
HpaII, HaeIII, DdeI have been extensively used for "in situ" DNA digestion (Gosalvez et al. 1997a); all of them have been able to induce longitudinal differentiation of the chromosomes of different species.
In situ digestion with REs
1. Chromosome preparations are made following standard procedures of
spreading or squashing. The slides are left at room temperature for at
least 2 h in order to ensure that they are completely dried before digestion.
2. Digestion of chromosomes with REs is always performed on freshly
dried preparations (no more than 12-24 h after having been prepared).
The standard procedure was described by Mezzanotte et al. (1985).
However, slight modifications of the standard procedure may be required for each type of chromosome.
10-50 units of the RE diluted to 20-50 III in the buffer recommended by
the supplier are usually used per slide. The enzyme solution is placed
on the slide and covered with a coverslip to spread the enzyme evenly.
3. The slides are incubated in a humid box for a selected time and at the
temperature specified by the manufacturer. Usually, overnight incubation at 37°C is a good standard for most REs. In order to prevent evaporation during long-term incubations, the coverslip can be sealed
with rubber solution.
4. Control experiments are performed under identical conditions by
treating the preparations with the incubation buffer without the enzyme.
5. The reaction is stopped by washing the slides at room temperature for
5 min in 2xSSC (pH 7).
6. Those preparations to be used for FISH are dehydrated in an ethanol
series (70%, 85%, 100%) and air dried.