168
THOMAS LIEHR
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 ~l of probe-solution to 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 7).
9. Take the slides out of 37°C, remove rubber cement with forceps and
coverslips by letting them swim off in 4xSSC/0.2%Tween (RT, 100-ml
Coplin jar) (see comment 8).
10. Postwash the slides 3x 5 min in formam ide-solution (45°C) followed
by 35 min in 2x SSC (37°C) in a 100-ml Coplin jar, with gentle agitation.
11. Put the slides in 4xSSC/0.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 ~l of solution I to each slide, cover with 24 x 50 mm coverslips
and incubate at 37°C for 30 min in a humid chamber. [Solution I: FITCavidin (CAMON Vector Laboratories A2011)/4xSSC + 0.2% Tween +
5% BSA (1:300); make fresh as required].
13. Remove the coverslips and wash 3 x 3 min in 4xSSC/0.2% Tween (RT,
with gentle agitation).
14. Add 50 ~l of solution II to each slide, cover with 24 x 50 mm coverslips
and incubate at 37°C for 75 min in a humid chamber. [Solution II:
Biotinylated antiavidin (CAMON Vector Laboratories BA0300)/antidigoxinenin-rhodamine (Boehringer Mannheim, 1207750)/ 4xSSC +
0.2% Tween + 5% BSA (1:20:100); make fresh as required].
15. Repeat step 13.
16. Repeat steps 12 and 13.
17. Counterstain the slides with DAPI-solution (100 ml in a Coplin jar, RT)
for 8 min. [DAPI-solution: Dissolve 5 ~l of DAPI (4,6-diamidino-2phenylindo1.2HCI stock-solution; Serva 18860) in 100 ml 4xSSC +
0.2% Tween; make fresh as required].
18. Wash slides several times in water for a few seconds and air dry.
THOMAS LIEHR
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 ~l of probe-solution to 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 7).
9. Take the slides out of 37°C, remove rubber cement with forceps and
coverslips by letting them swim off in 4xSSC/0.2%Tween (RT, 100-ml
Coplin jar) (see comment 8).
10. Postwash the slides 3x 5 min in formam ide-solution (45°C) followed
by 35 min in 2x SSC (37°C) in a 100-ml Coplin jar, with gentle agitation.
11. Put the slides in 4xSSC/0.2% Tween (100 ml, RT), for a few seconds.
12. Add 50 ~l of solution I to each slide, cover with 24 x 50 mm coverslips
and incubate at 37°C for 30 min in a humid chamber. [Solution I: FITCavidin (CAMON Vector Laboratories A2011)/4xSSC + 0.2% Tween +
5% BSA (1:300); make fresh as required].
13. Remove the coverslips and wash 3 x 3 min in 4xSSC/0.2% Tween (RT,
with gentle agitation).
14. Add 50 ~l of solution II to each slide, cover with 24 x 50 mm coverslips
and incubate at 37°C for 75 min in a humid chamber. [Solution II:
Biotinylated antiavidin (CAMON Vector Laboratories BA0300)/antidigoxinenin-rhodamine (Boehringer Mannheim, 1207750)/ 4xSSC +
0.2% Tween + 5% BSA (1:20:100); make fresh as required].
15. Repeat step 13.
16. Repeat steps 12 and 13.
17. Counterstain the slides with DAPI-solution (100 ml in a Coplin jar, RT)
for 8 min. [DAPI-solution: Dissolve 5 ~l of DAPI (4,6-diamidino-2phenylindo1.2HCI stock-solution; Serva 18860) in 100 ml 4xSSC +
0.2% Tween; make fresh as required].
18. Wash slides several times in water for a few seconds and air dry.