294
SUSANN NEUBAUER
3. Put the slides in a Coplin jar, and wash three times with 2 x SSC for
5 min at room temperature (RT).
4. Immerse the slides in 1x PBS for 5 min.
5. Incubate the slides for 5-10 min at 37°C with 0.005% pepsin solution
(prewarm 99 ml of distilled water and 1 ml of 1 M HCI at 37°C in a
water bath and dilute 50 /ll of a 10% pepsin stock solution just before
performing the pepsin step).
6. Add 5 ml 1 M MgCh to 95 ml 1x PBS and wash the slides for 5 min
at RT.
7. For post-fIxation, discard MgCh/PBS and replace it with 1% formaldehyde MgCh/PBS solution for 10 min at RT.
8. Wash the slides with lxPBS for 5 min, dehydrate the slides through an
ethanol series (70 %, 90% and 100% ethanol for 5 min each) and air dry.
Three-color fluorescence in situ hybridization (FISH)
For the three-color FISH approach described here, different wcp probes
for chromosomes 1,2 and 4 can be used. When unlabeled probes are available (e.g. derived from American Type Culture Collection) the probes have
to be biotinylated and/or digoxigenated according to the points 1 to 5
mentioned below. On the other hand, labeled probes are available
from different commercial resources (e.g. ONCOR or Vysis) - when using
such probes start with point 6.
labeling reaction 1. Plasmid or phage DNA from the DNA libraries of these chromosomes
are isolated according to manufacturers instructions (e.g. Qiagen). Depending on the quality ofpurifIed DNA, in summary about 7.5 /lg DNA
(2.5/lg DNA from chromosome 1, 3/lg DNA from chromosome 2 and
2 times l/lg DNA from chromosome 4) allows "painting" of metaphase
spreads on a whole slide (up to 1000 metaphases).
2. SpecifIc labeling of the chromosomes 1,2 and 4 is performed using a
modifIed approach of Lichter et al. (1988). The nick translation procedure for each chromosome is performed separately and on ice. In a
reaction volume of 24 III the DNA of the chromosome library of chromosome 1was digoxigenated, of chromosome 2 biotinylated and chromosome 4 was biotinylated and digoxigenated (1:1) by nick translation. In detail:
SUSANN NEUBAUER
3. Put the slides in a Coplin jar, and wash three times with 2 x SSC for
5 min at room temperature (RT).
4. Immerse the slides in 1x PBS for 5 min.
5. Incubate the slides for 5-10 min at 37°C with 0.005% pepsin solution
(prewarm 99 ml of distilled water and 1 ml of 1 M HCI at 37°C in a
water bath and dilute 50 /ll of a 10% pepsin stock solution just before
performing the pepsin step).
6. Add 5 ml 1 M MgCh to 95 ml 1x PBS and wash the slides for 5 min
at RT.
7. For post-fIxation, discard MgCh/PBS and replace it with 1% formaldehyde MgCh/PBS solution for 10 min at RT.
8. Wash the slides with lxPBS for 5 min, dehydrate the slides through an
ethanol series (70 %, 90% and 100% ethanol for 5 min each) and air dry.
Three-color fluorescence in situ hybridization (FISH)
For the three-color FISH approach described here, different wcp probes
for chromosomes 1,2 and 4 can be used. When unlabeled probes are available (e.g. derived from American Type Culture Collection) the probes have
to be biotinylated and/or digoxigenated according to the points 1 to 5
mentioned below. On the other hand, labeled probes are available
from different commercial resources (e.g. ONCOR or Vysis) - when using
such probes start with point 6.
labeling reaction 1. Plasmid or phage DNA from the DNA libraries of these chromosomes
are isolated according to manufacturers instructions (e.g. Qiagen). Depending on the quality ofpurifIed DNA, in summary about 7.5 /lg DNA
(2.5/lg DNA from chromosome 1, 3/lg DNA from chromosome 2 and
2 times l/lg DNA from chromosome 4) allows "painting" of metaphase
spreads on a whole slide (up to 1000 metaphases).
2. SpecifIc labeling of the chromosomes 1,2 and 4 is performed using a
modifIed approach of Lichter et al. (1988). The nick translation procedure for each chromosome is performed separately and on ice. In a
reaction volume of 24 III the DNA of the chromosome library of chromosome 1was digoxigenated, of chromosome 2 biotinylated and chromosome 4 was biotinylated and digoxigenated (1:1) by nick translation. In detail: