32 Centromere-Specific Multicolor-FISH (cenM-FISH)
429
formamide/2 X SSC/50 mM phosphate buffer for 3 h at 70°C. Aliquot
and store at -20°C).
Fluorescence in situ hybridization (FISH)
1. Add 100 f..ll denaturation-buffer to the slides and cover with 24 x 50 mm
coverslips. [Denaturation-buffer: 70% (v/v) deionized formamide, 10%
(v/v) ftltered double distilled water, 10% (v/v) 20xSSC, 10% (v/v) phosphate buffer; make fresh as required; phosphate buffer: prepare 0.5 M
Na z HP0 4 and 0.5 M NaH z P0 4 , mix these two solutions (1:1) to get pH
7.0, then aliquot and store at -20°e].
2. Incubate slides on a warming plate for 2-3 min at 75°C.
3. Remove the coverslips immediately with forceps and place slides in a
Coplin jar ftlled with 70% ethanol (4°C) to conserve target DNA as
single strands.
4. Dehydrate slides in ethanol (70%,90%, 100%, 4°C, 3 min each) and air
dry.
5. For each slide to be hybridized, use 20 f..ll of the labeled chromosomespecific satellite probe mix described before.
6. Denature probe-solution at 75°C for 5 min and prehybridize at 37°C for
5 min.
7. Add 20 f..ll of probe-solution onto each denatured slide, put 24x50 mm
coverslips on the drops and seal with rubber cement (Fixogum; Marabu).
8. Incubate slides overnight at 37°C in a humid chamber (see comment 3).
9. Take the slides out of the 37°C chamber, remove rubber cement with
forceps and the coverslips by letting them swim off in 4x SSC/
0.2%Tween (RT, 100 ml Coplin jar) (see comment 3).
10. Postwash the slides 3 x 5 min in formamide-solution (45°C) followed
by 3x 5 min in 2x SSC (37°C) in a 100 ml Coplin jar, with gentle agitation.
11. Put the slides in 4xSSC/0.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 f..ll of solution I to each slide, cover with 24x50 mm coverslips
and incubate at 37°C for 30 min in a humid chamber. [Solution I: Cy5Amersham, PA45000)/4xSSC + 0.2% Tween + 5% BSA (1:50); make
fresh as required].
429
formamide/2 X SSC/50 mM phosphate buffer for 3 h at 70°C. Aliquot
and store at -20°C).
Fluorescence in situ hybridization (FISH)
1. Add 100 f..ll denaturation-buffer to the slides and cover with 24 x 50 mm
coverslips. [Denaturation-buffer: 70% (v/v) deionized formamide, 10%
(v/v) ftltered double distilled water, 10% (v/v) 20xSSC, 10% (v/v) phosphate buffer; make fresh as required; phosphate buffer: prepare 0.5 M
Na z HP0 4 and 0.5 M NaH z P0 4 , mix these two solutions (1:1) to get pH
7.0, then aliquot and store at -20°e].
2. Incubate slides on a warming plate for 2-3 min at 75°C.
3. Remove the coverslips immediately with forceps and place slides in a
Coplin jar ftlled with 70% ethanol (4°C) to conserve target DNA as
single strands.
4. Dehydrate slides in ethanol (70%,90%, 100%, 4°C, 3 min each) and air
dry.
5. For each slide to be hybridized, use 20 f..ll of the labeled chromosomespecific satellite probe mix described before.
6. Denature probe-solution at 75°C for 5 min and prehybridize at 37°C for
5 min.
7. Add 20 f..ll of probe-solution onto each denatured slide, put 24x50 mm
coverslips on the drops and seal with rubber cement (Fixogum; Marabu).
8. Incubate slides overnight at 37°C in a humid chamber (see comment 3).
9. Take the slides out of the 37°C chamber, remove rubber cement with
forceps and the coverslips by letting them swim off in 4x SSC/
0.2%Tween (RT, 100 ml Coplin jar) (see comment 3).
10. Postwash the slides 3 x 5 min in formamide-solution (45°C) followed
by 3x 5 min in 2x SSC (37°C) in a 100 ml Coplin jar, with gentle agitation.
11. Put the slides in 4xSSC/0.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 f..ll of solution I to each slide, cover with 24x50 mm coverslips
and incubate at 37°C for 30 min in a humid chamber. [Solution I: Cy5Amersham, PA45000)/4xSSC + 0.2% Tween + 5% BSA (1:50); make
fresh as required].