428
ANGELA NIETZEL et al.
solution 10% (w/v) (Serva 31855) and leave the Coplin jar at 37°C;
make fresh as required].
7. Replace fluid with 100 mIl x PBS/MgClz, incubate at RT for 5 min with
gentle agitation. MgCh will block the enzymatic activity of pepsin
(1 x PBS/MgCI 2 = 5% (v/v) 1 M MgCh in 1x PBS).
8. PostfIX nuclei on the slide surfaces by replacing 1x PBS/MgCh with 100
ml offormalin-buffer (3 ml acid free formaldehyde [37%; Roth 4979.1]
in 100 ml 1xPBS) for 10 min (RT, with gentle agitation).
9. Formalin-buffer is replaced by 100 mIl xPBS for 2 min (RT, with gentle agitation).
10. Finally, slides are dehydrated by an ethanol series (70%, 90%, 100%,
3 min each) and air dried (see comment 2).
Preparation of probes
1. Centromere-specific probes can be obtained (for research use only)
from Prof. Dr. Mariano Rocchi (internet: http://www.biologia.uniba.itlrmc).
2. Plasmid-DNA with centromere-specific DNA as inserts are labeled by
nick translation using commercially available kits according to the
manufacturers instructions (e.g. biotin labeling: Biotin-Nick Translation Kit, Boehringer, 1745824; labeling with DEAC-dUTP (NEN-Dupont; NEL455), Chromatide TexasRed-dUTP (MoL Probes; C-763l),
SpectrumOrange-dUTP (Vysis; 30-803000), SpectrumGreen-dUTP
(Vysis; 30-803400) can be done by Nick-Translation Kit, Boehringer,
976776). Labeling is done according to the labeling scheme in Fig. lB.
3. 1 Ilg of each probe can be labeled with one nick translation reaction;
each labeling should be tested separately for its efficiency by hybridizing lOng of the probe on normal metaphase spreads.
4. All labeled probes are mixed and 50 aliquots are placed in 1.5-ml microtubes. Each aliquot is precipitated together with 10 III of 1 Ilg/Il1
COTl-DNA (Gibco). The pellets are vacuum dried and stored until
use at -20°e.
5. Prior to use the pellet is dissolved in 20 III of the hybridization-buffer,
which is sufficient for 1 slide covered with a 24x50 mm coverslip. (Hybridization buffer: Dissolve 2 g dextran sulfate in 10 m150% deionized
ANGELA NIETZEL et al.
solution 10% (w/v) (Serva 31855) and leave the Coplin jar at 37°C;
make fresh as required].
7. Replace fluid with 100 mIl x PBS/MgClz, incubate at RT for 5 min with
gentle agitation. MgCh will block the enzymatic activity of pepsin
(1 x PBS/MgCI 2 = 5% (v/v) 1 M MgCh in 1x PBS).
8. PostfIX nuclei on the slide surfaces by replacing 1x PBS/MgCh with 100
ml offormalin-buffer (3 ml acid free formaldehyde [37%; Roth 4979.1]
in 100 ml 1xPBS) for 10 min (RT, with gentle agitation).
9. Formalin-buffer is replaced by 100 mIl xPBS for 2 min (RT, with gentle agitation).
10. Finally, slides are dehydrated by an ethanol series (70%, 90%, 100%,
3 min each) and air dried (see comment 2).
Preparation of probes
1. Centromere-specific probes can be obtained (for research use only)
from Prof. Dr. Mariano Rocchi (internet: http://www.biologia.uniba.itlrmc).
2. Plasmid-DNA with centromere-specific DNA as inserts are labeled by
nick translation using commercially available kits according to the
manufacturers instructions (e.g. biotin labeling: Biotin-Nick Translation Kit, Boehringer, 1745824; labeling with DEAC-dUTP (NEN-Dupont; NEL455), Chromatide TexasRed-dUTP (MoL Probes; C-763l),
SpectrumOrange-dUTP (Vysis; 30-803000), SpectrumGreen-dUTP
(Vysis; 30-803400) can be done by Nick-Translation Kit, Boehringer,
976776). Labeling is done according to the labeling scheme in Fig. lB.
3. 1 Ilg of each probe can be labeled with one nick translation reaction;
each labeling should be tested separately for its efficiency by hybridizing lOng of the probe on normal metaphase spreads.
4. All labeled probes are mixed and 50 aliquots are placed in 1.5-ml microtubes. Each aliquot is precipitated together with 10 III of 1 Ilg/Il1
COTl-DNA (Gibco). The pellets are vacuum dried and stored until
use at -20°e.
5. Prior to use the pellet is dissolved in 20 III of the hybridization-buffer,
which is sufficient for 1 slide covered with a 24x50 mm coverslip. (Hybridization buffer: Dissolve 2 g dextran sulfate in 10 m150% deionized