112
THOMAS LIEHR
Fluorescence in situ hybridization (FISH)
1. Add 100 J!l denaturation-buffer to the slides and cover with (24 x 50
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 Na2HP04 and 0.5 M NaH 2 P0 4 , mix these two solutions
(1:1) to get pH 7.0, then aliquot and store at -20°C].
2. Incubate slides on a warming plate for 5 min at 75°C (see comment 3).
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, treat the labeled YAC-, BAC- or cosmid-DNA according to the manufacturers instructions or add 1.5 J!l of
biotin or digoxigenin labeled chromosome-specific satellite probes
(e.g. Oncor Inc., Vysis, Inc.) plus 0.6 J!l of 1 J!g/J!l COTl-DNA (Gibco)
to 20 J!l 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°C. Aliquot and store at -20°C).
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 handle
the probe according to manufacturers instructions (e.g. do a prehybridization step at 37°C for 20 min for YACs, BACs, cosmids, PI-clones).
7. Add 20 J!l of probe-solution onto each denatured slide, put 24x50 mm
coverslips on the drops and seal with rubber cement (Fixogum; Marabu).
8. Incubate slides for three nights at 37°C in a humid chamber (see comment 4).
9. Take the slides out of 37°C, remove rubber cement with forceps and the
coverslips by letting them swim off in 4xSSC/0.2%Tween (RT, 100-ml
Coplin jar) (see comment 5).
10. Postwash the slides 3x 5 min in formam ide-solution (45°C) followed
by 3x5 min in 2xSSC (37°C) in a IOO-ml Coplin jar, with gentle agitation.
THOMAS LIEHR
Fluorescence in situ hybridization (FISH)
1. Add 100 J!l denaturation-buffer to the slides and cover with (24 x 50
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 Na2HP04 and 0.5 M NaH 2 P0 4 , mix these two solutions
(1:1) to get pH 7.0, then aliquot and store at -20°C].
2. Incubate slides on a warming plate for 5 min at 75°C (see comment 3).
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, treat the labeled YAC-, BAC- or cosmid-DNA according to the manufacturers instructions or add 1.5 J!l of
biotin or digoxigenin labeled chromosome-specific satellite probes
(e.g. Oncor Inc., Vysis, Inc.) plus 0.6 J!l of 1 J!g/J!l COTl-DNA (Gibco)
to 20 J!l 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°C. Aliquot and store at -20°C).
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 handle
the probe according to manufacturers instructions (e.g. do a prehybridization step at 37°C for 20 min for YACs, BACs, cosmids, PI-clones).
7. Add 20 J!l of probe-solution onto each denatured slide, put 24x50 mm
coverslips on the drops and seal with rubber cement (Fixogum; Marabu).
8. Incubate slides for three nights at 37°C in a humid chamber (see comment 4).
9. Take the slides out of 37°C, remove rubber cement with forceps and the
coverslips by letting them swim off in 4xSSC/0.2%Tween (RT, 100-ml
Coplin jar) (see comment 5).
10. Postwash the slides 3x 5 min in formam ide-solution (45°C) followed
by 3x5 min in 2xSSC (37°C) in a IOO-ml Coplin jar, with gentle agitation.