176
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
tions. After careful dissolving, autoclave the dextran sulphate solution or
filter through a nitrocellulose filter. Combine 200 J..Ll of20x SSC, 400 J..Ll of
50% dextran sulphate and 400 J..Ll of double distilled water. Store at 4°C
until use.
Procedure
Probe mixture and
1. Combine 20 to 60 ng of single-copy DNA and 3-5 J..Lg of sheared salmon
denaturation
sperm DNA (used as carrier). If the resulting volume is below lOJ..Ll, lyophilize. For larger volumes, precipitate DNA by adding 1/20 volume of 3
M sodium acetate and 2 volumes of 100% ethanol. Mix well and incubate
at -70°C for 30 minutes. Centrifuge in an Eppendorf centrifuge at 12,000
rpm for 10 minutes at 4°C. Discard the supernatant, wash the pellet by
adding 500 J..Ll of 70% ethanol and centrifuge again (12,000 rpm, 10 min,
4°C). Discard the supernatant, lyophilize.
2. Resuspend in 5 J..Ll deionized formamide by vortexing for several minutes
at room temperature.
3. Add 5 J..Ll of hybridization buffer and vortex again for 5 to 10 minutes.
4. Denature probe DNA at 75°C for 5 minutes followed by incubation on ice
foranother 5 minutes. After this step, the probeisready for hybridization.
Note: Certain probes, such as chromosome-specific sequences of the alphoid family of DNA repeats, share considerable homologies with related
sequences on different chromosomes. Therefore, specific signals are only
obtained when highly stringent hybridization conditions are applied. This
can be achieved, for example, by increasing the formamide concentration in
the hybridization medium to 60 o/o or more.
Subprotocol 5
Denaturation of the DNA on the Slides
Materials
• Denaturation solution: 70% deionized formamide, 2x SSC, 50 mM sodium phosphate, pH 7.0
• lee-cold ethanol: 70%, 90% and 100%
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
tions. After careful dissolving, autoclave the dextran sulphate solution or
filter through a nitrocellulose filter. Combine 200 J..Ll of20x SSC, 400 J..Ll of
50% dextran sulphate and 400 J..Ll of double distilled water. Store at 4°C
until use.
Procedure
Probe mixture and
1. Combine 20 to 60 ng of single-copy DNA and 3-5 J..Lg of sheared salmon
denaturation
sperm DNA (used as carrier). If the resulting volume is below lOJ..Ll, lyophilize. For larger volumes, precipitate DNA by adding 1/20 volume of 3
M sodium acetate and 2 volumes of 100% ethanol. Mix well and incubate
at -70°C for 30 minutes. Centrifuge in an Eppendorf centrifuge at 12,000
rpm for 10 minutes at 4°C. Discard the supernatant, wash the pellet by
adding 500 J..Ll of 70% ethanol and centrifuge again (12,000 rpm, 10 min,
4°C). Discard the supernatant, lyophilize.
2. Resuspend in 5 J..Ll deionized formamide by vortexing for several minutes
at room temperature.
3. Add 5 J..Ll of hybridization buffer and vortex again for 5 to 10 minutes.
4. Denature probe DNA at 75°C for 5 minutes followed by incubation on ice
foranother 5 minutes. After this step, the probeisready for hybridization.
Note: Certain probes, such as chromosome-specific sequences of the alphoid family of DNA repeats, share considerable homologies with related
sequences on different chromosomes. Therefore, specific signals are only
obtained when highly stringent hybridization conditions are applied. This
can be achieved, for example, by increasing the formamide concentration in
the hybridization medium to 60 o/o or more.
Subprotocol 5
Denaturation of the DNA on the Slides
Materials
• Denaturation solution: 70% deionized formamide, 2x SSC, 50 mM sodium phosphate, pH 7.0
• lee-cold ethanol: 70%, 90% and 100%