19 PNA-FISH
259
PNA-FISH short protocol
Make your favorite chromosome preparations.
1. Post-ftxation in methanol:acetic acid (3:1) for 1-2 h.
2. Dehydration in 70%, 80%, 90%, and 100% ethanol.
3. Air dry (store desiccated until needed) Then:
4. 1x PBS at 37°C for 5 min.
5. Post-fIxation of the chromosomes in 4% formaldehyde at 37°C for
1.5-2 min.
6. Dehydrate slides in the ethanol series and air dry.
Total of 0.1 nmol of PNA is needed for 10-15 slides.
1. 0.1 nmol ofPNA is dissolved in 200 III of pre-warmed 1x hybridization
mix (70% de-ionized formamide, 500 (50-1000) nM of fluorochromelabeled PNA probe, 1% (w/v) blocking reagent in 10 mM Tris pH 7.2
(no Na+».
2. PNA hybridization mix is incubated at 37°C or 50°C in the dark for at
least 15 min.
3. 15-20 III of PNA (500 nM) is transferred to each slide. A coverslip of
20x40 mm or 20x30 mm is applied and sealed with rubber cement.
4. The DNA on the slide is denatured again at 80°C for 3 min (PCR program FISHl) and then hybridized in a dark, wet chamber at 37°C (can
be adjusted from 20-40°C) for 2 h (can be adjusted from 0.5-24 h).
From this step, excessive light should be avoided to prevent fluorescence
bleaching. After hybridization:
1. Slide preparations are washed 3x 10 min with 70% formamide/lO mM
Tris pH 7.2.
2. Then slides are washed 3x 5 min with TNT buffer (50 mM Tris/ 150 mM
NaClIO.05% Tween-20 pH 7.5).
3. The slides are then dehydrated in an ethanol series (70%, 80%, 90%,
100% -30°C and then 100% at room temp.) and air dried in the dark.
4. Chromosomes are counterstained with either 0.1 Ilg/ml of 4,6-diamidino-2-phenylindole-dehydrochloride (DAPI) in anti-fade for rhodaPre-hybridization
preparations
Hybridization
Post-hybridization
washes
259
PNA-FISH short protocol
Make your favorite chromosome preparations.
1. Post-ftxation in methanol:acetic acid (3:1) for 1-2 h.
2. Dehydration in 70%, 80%, 90%, and 100% ethanol.
3. Air dry (store desiccated until needed) Then:
4. 1x PBS at 37°C for 5 min.
5. Post-fIxation of the chromosomes in 4% formaldehyde at 37°C for
1.5-2 min.
6. Dehydrate slides in the ethanol series and air dry.
Total of 0.1 nmol of PNA is needed for 10-15 slides.
1. 0.1 nmol ofPNA is dissolved in 200 III of pre-warmed 1x hybridization
mix (70% de-ionized formamide, 500 (50-1000) nM of fluorochromelabeled PNA probe, 1% (w/v) blocking reagent in 10 mM Tris pH 7.2
(no Na+».
2. PNA hybridization mix is incubated at 37°C or 50°C in the dark for at
least 15 min.
3. 15-20 III of PNA (500 nM) is transferred to each slide. A coverslip of
20x40 mm or 20x30 mm is applied and sealed with rubber cement.
4. The DNA on the slide is denatured again at 80°C for 3 min (PCR program FISHl) and then hybridized in a dark, wet chamber at 37°C (can
be adjusted from 20-40°C) for 2 h (can be adjusted from 0.5-24 h).
From this step, excessive light should be avoided to prevent fluorescence
bleaching. After hybridization:
1. Slide preparations are washed 3x 10 min with 70% formamide/lO mM
Tris pH 7.2.
2. Then slides are washed 3x 5 min with TNT buffer (50 mM Tris/ 150 mM
NaClIO.05% Tween-20 pH 7.5).
3. The slides are then dehydrated in an ethanol series (70%, 80%, 90%,
100% -30°C and then 100% at room temp.) and air dried in the dark.
4. Chromosomes are counterstained with either 0.1 Ilg/ml of 4,6-diamidino-2-phenylindole-dehydrochloride (DAPI) in anti-fade for rhodaPre-hybridization
preparations
Hybridization
Post-hybridization
washes