23 Detection of Radiosensitivity by Means of Three-Color FISH
295
10-16 III double distilled water, 1-2.5 Ilg DNA from the specific chromosome and 6 III of either Biotin-Nick Translation Mix (Boehringer
Mannheim) or DIG-Nick Translation Mix (Boehringer Mannheim)
are put into a reaction tube. The components are mixed and incubated
in a 15°C water bath for 90 min. The reaction is stopped by adding 24 III
of a stop solution (20mM EDTA, 20 mM Tris-HCL, 0.1 M NaCl, 0.5%
dextran blue and 0.1 % bromphenol blue in distilled water) and 52 III
TE 10/1 buffer.
3. Purification of the probe:
The 100 III reaction volume (from point 2) is pipetted onto a prepared
Sephadex column (Sephadex G50) and centrifuged. The labeled DNA
of chromosomes 1,2 and 4 is collected in the same tube together with
16 III Cot 1 DNA (Gibco) (=1 Ilg labeled DNA/2 III Cot I), 2 III HTD
(herring testis DNA) and the 2.5-fold volume of 100% ethanol.
4. Precipitation of the DNA is done overnight or up to several months at
-20°e.
5. To pellet the DNA, centrifuge (13,000 rpm) at 4°C for 45 min. The
supernatant is discarded and the pellet is washed with 70% ethanol.
After centrifugation the pellet can be dried under vacuum. Dilute
the probe DNA carefully in 35 JlI hybridization buffer (hybridization
buffer = incubate 1 g dextran sulfate in 2.5 ml deionized formamide,
0.5 ml 20 x SSC, 0.5 ml 0.5 M sodium phosphate buffer and 1.5 ml distilled water at 70°C for 3 h). 35 JlI hybridization buffer is the appropriate volume if a coverslip 24 x 50 mm is used.
6. Denature the probe (as prepared in steps 1 to 5 or a commercially available probe combination, according to manufacturers instructions) at
75°C for 5 min, chill on ice and allow preannealing for 35 min at 37°C.
7. For denaturation of the chromosomes on the slides, add a denaturation solution of 70 III deionized formamide, 10 III O.5M sodium phosphate buffer, 10 III 20 x SSC and 10 III distilled water on each slide and
cover with a coverslip. Incubate the slides on a heating block at noc
for 1 min.
8. Remove the slides from the heating block, remove the coverslips and
immerse the slides in prechilled 70% ethanol (-20°e).
9. Dehydrate in an ethanol series (70%,90%, 100% on ice 5 min) and air
dry.
Denaturation
and hybridization
295
10-16 III double distilled water, 1-2.5 Ilg DNA from the specific chromosome and 6 III of either Biotin-Nick Translation Mix (Boehringer
Mannheim) or DIG-Nick Translation Mix (Boehringer Mannheim)
are put into a reaction tube. The components are mixed and incubated
in a 15°C water bath for 90 min. The reaction is stopped by adding 24 III
of a stop solution (20mM EDTA, 20 mM Tris-HCL, 0.1 M NaCl, 0.5%
dextran blue and 0.1 % bromphenol blue in distilled water) and 52 III
TE 10/1 buffer.
3. Purification of the probe:
The 100 III reaction volume (from point 2) is pipetted onto a prepared
Sephadex column (Sephadex G50) and centrifuged. The labeled DNA
of chromosomes 1,2 and 4 is collected in the same tube together with
16 III Cot 1 DNA (Gibco) (=1 Ilg labeled DNA/2 III Cot I), 2 III HTD
(herring testis DNA) and the 2.5-fold volume of 100% ethanol.
4. Precipitation of the DNA is done overnight or up to several months at
-20°e.
5. To pellet the DNA, centrifuge (13,000 rpm) at 4°C for 45 min. The
supernatant is discarded and the pellet is washed with 70% ethanol.
After centrifugation the pellet can be dried under vacuum. Dilute
the probe DNA carefully in 35 JlI hybridization buffer (hybridization
buffer = incubate 1 g dextran sulfate in 2.5 ml deionized formamide,
0.5 ml 20 x SSC, 0.5 ml 0.5 M sodium phosphate buffer and 1.5 ml distilled water at 70°C for 3 h). 35 JlI hybridization buffer is the appropriate volume if a coverslip 24 x 50 mm is used.
6. Denature the probe (as prepared in steps 1 to 5 or a commercially available probe combination, according to manufacturers instructions) at
75°C for 5 min, chill on ice and allow preannealing for 35 min at 37°C.
7. For denaturation of the chromosomes on the slides, add a denaturation solution of 70 III deionized formamide, 10 III O.5M sodium phosphate buffer, 10 III 20 x SSC and 10 III distilled water on each slide and
cover with a coverslip. Incubate the slides on a heating block at noc
for 1 min.
8. Remove the slides from the heating block, remove the coverslips and
immerse the slides in prechilled 70% ethanol (-20°e).
9. Dehydrate in an ethanol series (70%,90%, 100% on ice 5 min) and air
dry.
Denaturation
and hybridization