17 High Resolution FISH of Stretched Chromosome Fibers
223
are scanned using a light microscope. The disrupted nuclei have a compact short 'tail' (=DNA fibers) (see comments 3 and 4).
4. The slides are incubated over night at 60°C and then stored at -20°C
(see comment 5).
5. FISH was carried out according to standard protocols (see below).
Fluorescence in situ hybridization (FISH) according to Thomas liehr
1. Add 100 III denaturation-buffer to the slides and cover with (24x50
mm) coverslips. [Denaturation-buffer: 70% (v/v) deionized formamide, 10% (v/v) filtered 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
O:l) to get pH 7.0, then aliquot and store at -20°e].
2. Incubate slides on a warming plate for 12 min at 75°C.
3. Remove the coverslips immediately with forceps and place slides in a
Coplin jar filled 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, add 200 ng of labeled YAC-, BAC- or
cosmid- DNA or 1.5 III of biotin or digoxigenin labeled chromosome
specific satellite probes plus 3 III of Illg/1l1 COTl-DNA (Gibco) to 20 III
of the hybridization-buffer in a 1.5 ml microtube, vortex and spin
down. (Hybridization-buffer: Dissolve 2 g dextran sulfate in 10 ml
50% deionized formamidel2 x SSC/50 mM phosphate buffer for 3 h
at 70°e. Store aliquots at _20°e.)
6. Denature probe-solution at 75°C for 5 min and cool immediately on ice
to conserve probe DNA in single strands for satellite probes or do a
prehybridization step at 37°C for 30 min for the other probes (YACs,
BACs, cosmids).
7. Add 20 III of probe-solution onto each denatured slide, put 24x50 mm
cover slips on the drops and seal with rubber cement (Fixogum; Marabu).
8. Incubate slides for 3 nights at 37°C in a humid chamber.
223
are scanned using a light microscope. The disrupted nuclei have a compact short 'tail' (=DNA fibers) (see comments 3 and 4).
4. The slides are incubated over night at 60°C and then stored at -20°C
(see comment 5).
5. FISH was carried out according to standard protocols (see below).
Fluorescence in situ hybridization (FISH) according to Thomas liehr
1. Add 100 III denaturation-buffer to the slides and cover with (24x50
mm) coverslips. [Denaturation-buffer: 70% (v/v) deionized formamide, 10% (v/v) filtered 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
O:l) to get pH 7.0, then aliquot and store at -20°e].
2. Incubate slides on a warming plate for 12 min at 75°C.
3. Remove the coverslips immediately with forceps and place slides in a
Coplin jar filled 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, add 200 ng of labeled YAC-, BAC- or
cosmid- DNA or 1.5 III of biotin or digoxigenin labeled chromosome
specific satellite probes plus 3 III of Illg/1l1 COTl-DNA (Gibco) to 20 III
of the hybridization-buffer in a 1.5 ml microtube, vortex and spin
down. (Hybridization-buffer: Dissolve 2 g dextran sulfate in 10 ml
50% deionized formamidel2 x SSC/50 mM phosphate buffer for 3 h
at 70°e. Store aliquots at _20°e.)
6. Denature probe-solution at 75°C for 5 min and cool immediately on ice
to conserve probe DNA in single strands for satellite probes or do a
prehybridization step at 37°C for 30 min for the other probes (YACs,
BACs, cosmids).
7. Add 20 III of probe-solution onto each denatured slide, put 24x50 mm
cover slips on the drops and seal with rubber cement (Fixogum; Marabu).
8. Incubate slides for 3 nights at 37°C in a humid chamber.