400
ILSE CHUDOBA and GABRIELE SENGER
Hybridization
The denatured and preannealed probes are transferred onto the denatured
chromosome slides and overlaid with a coverslip. The edges of the coverslips are sealed with rubber cement (e.g. Fixogum) and the slides are
stored in a moist chamber for hybridization overnight at 37°C.
Post-hybridization washes
Solutions required:
- Oo4xSSC, pH 7.0
- 2.0 ml 20xSSC, pH 7.0
- 98 ml H 2 0
- Preheat to 69°C
- 4xSSCT, pH 7.0
- 20 ml 20xSSC, pH 7.0
- 80 ml H 2 0
- 0.05 ml Tween 20
- Room temperature (RT)
- DAPI stock solution
- 1 mg DAPI
- 10 ml 4xSSCT
- Dissolve thoroughly and store aliquots (0.5 ml) at -20
G C
- DAPI working solution
- 0.003 ml DAPI stock solution
- 1.5 ml 4xSSCT
1. Carefully remove rubber cement and coverslips.
2. Place slides in prewarmed (69°C) 004 x SSC, incubate for 2 min.
3. Transfer slides to 4xSSCT, incubate for 1 min, RT.
4. Apply 50 /-11 of the DAPI-solution, overlay with a 24x60 mm coverslip,
incubate for 2 min.
5. Flip off the coverslip and wash in 1xPBS for 2 min, RT.
6. Drain off fluid and blow dry with a rubber ball.