86
THOMAS LIEHR
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).
Fluorescence in situ hybridization (FISH)
1. Add 100 ).11 denaturation-buffer to the slides and cover with (24x 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 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 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°e) 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 manufacturers instructions or add 1.5).11 ofbiotin or digoxigenine labeled chromosome specific satellite probes (e.g.
Oncor Inc., Vysis, Inc.) plus 0.6).11 of 1 ).1g/).1l COTl-DNA (Gibco) to 20
).11 of the hybridization-buffer in a 1.5 ml microtube, vortex and spin
down. (Hybridization-buffer: Dissolve 2 g dextran sulfate in 10 m150%
deionized formamidel2xSSC/50 mM phosphate buffer for 3 h at 70°C.
Aliquot and store 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 handle
the probe according to the manufacturers instructions (e.g. do a prehybridization step at 37°C for 20 min for YACs, BACs, cosmids, Pldones).
THOMAS LIEHR
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).
Fluorescence in situ hybridization (FISH)
1. Add 100 ).11 denaturation-buffer to the slides and cover with (24x 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 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 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°e) 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 manufacturers instructions or add 1.5).11 ofbiotin or digoxigenine labeled chromosome specific satellite probes (e.g.
Oncor Inc., Vysis, Inc.) plus 0.6).11 of 1 ).1g/).1l COTl-DNA (Gibco) to 20
).11 of the hybridization-buffer in a 1.5 ml microtube, vortex and spin
down. (Hybridization-buffer: Dissolve 2 g dextran sulfate in 10 m150%
deionized formamidel2xSSC/50 mM phosphate buffer for 3 h at 70°C.
Aliquot and store 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 handle
the probe according to the manufacturers instructions (e.g. do a prehybridization step at 37°C for 20 min for YACs, BACs, cosmids, Pldones).