96
MORIMASA MATS UTA AND MAYUMI MATSUTA
Hybridization
and washing
- lO~llabelled tumor DNA from the nick translation reaction (200 ng)
- lO~llabelled normal DNA from the nick translation reaction (200 ng)
- 1O~1 Cot-l DNA (lO~g)
- 3)l1 3M sodium acetate
- 66~1 100% ethanol
10. Precipitate by centrifuging for 30 mins in a micro centrifuge
(-14,000 rpm)
11. Decant and let dry
12. Dissolve the pellet in 1O~1 hybridization buffer (50% formamide, 10% dextran sulfate, 2xSSC, pH 7)
13. Denature 5 mins at 70
D
C immediately before use.
14. Pipette 10~1 of hybridization mix on slide, apply an 18x18
mm coverslip and seal with rubber cement
15. Hybridize for two days at 37°C in a moist chamber
16. Remove coverslip and wash slides three times in a washing
solution (50% formamide/2xSSC, pH 7) at 45°C for 10 mins
each
17. Wash twice in 2xSSC at 45°C and once at room temperature,
for 10 mins each
18. Wash once in PN buffer and once in distilled water (both 10
mins at room temperature)
19. Air-dry slides in darkness
20. Mount in an anti-fade solution containing 0.1-0.2~M DAPI
21. View the hybridization using triple-bandpass filter for DAPI,
FITC, and Texas-Red.
Results
Gene amplifications 10 to 20 fold and changes including a large
region may be visually detected (Fig 6). However, small deletions
and other lower level changes can not be reliably determined
without digital image analysis. A ratio profile of the fluorescent