12 Chromosome Analysis by Fluorescence in situ Hybridization
175
7. Reseal the filter in a plastic bag containing the developing solution (33f.d
ofNBT added to 10 ml AP 9.5 buffer). After careful mixing (do not vortex!) 25 f.ll of BCIP are added. The resulting solution is again mixed
gently. Incubate at 37°C until color development is appropriate, usually
15 to 60 min.
8. Remove the filter from the plastic bag and wash with TE buffer to stop the
color reaction.
9. After air-drying, the assay results can be evaluated: the color intensities
of the test and control DNA should be comparable. For optimal hybridization results, the signal of the lowest dilution should be visible.
When the dot-blot assay is carried out to check probes labeled with digoxigenin, an anti-digoxigenin labeled alkaline phosphatase is used instead of
Streptavidin-conjugated alkaline phosphatase.
Subprotocol 4
Probe Mixture and Denaturation for Fluorescence in situ Hybridization
This section describes a standard in situ hybridization which is used for
probes in which all sequences contribute to the hybridization signal
(such as chromosome specific repetitive DNA probes, unique cDNAs or
genomic DNA probes lacking interspersed repetitive sequences [ISR] )
(Subprotocols 4, 5 and 6). Subprotocol 7 describes the modifications required for chromosomal in situ suppression (CISS) hybridization for use
with genomic DNA fragments or cDNAs containing IRS segments as probes.
Protocols are suitable for a 10 f.ll hybridization volume which is sufficient to
cover an area of 18x18 mm on a glass slide. Ifhybridization oflarger areas is
desired, the volumes of the solutions should be scaled-up accordingly.
Materials
• 3 M sodium acetate, pH 5.2
• Deionized formamide (molecular biology grade). The conductivity of the
solution should be below 100 f.lSiemens.
• Hybridization buffer: 4x SSC, 20% dextran sulphate. The solution is best
prepared as follows: Prepare 20x SSC and 50% dextran sulphate solu-
175
7. Reseal the filter in a plastic bag containing the developing solution (33f.d
ofNBT added to 10 ml AP 9.5 buffer). After careful mixing (do not vortex!) 25 f.ll of BCIP are added. The resulting solution is again mixed
gently. Incubate at 37°C until color development is appropriate, usually
15 to 60 min.
8. Remove the filter from the plastic bag and wash with TE buffer to stop the
color reaction.
9. After air-drying, the assay results can be evaluated: the color intensities
of the test and control DNA should be comparable. For optimal hybridization results, the signal of the lowest dilution should be visible.
When the dot-blot assay is carried out to check probes labeled with digoxigenin, an anti-digoxigenin labeled alkaline phosphatase is used instead of
Streptavidin-conjugated alkaline phosphatase.
Subprotocol 4
Probe Mixture and Denaturation for Fluorescence in situ Hybridization
This section describes a standard in situ hybridization which is used for
probes in which all sequences contribute to the hybridization signal
(such as chromosome specific repetitive DNA probes, unique cDNAs or
genomic DNA probes lacking interspersed repetitive sequences [ISR] )
(Subprotocols 4, 5 and 6). Subprotocol 7 describes the modifications required for chromosomal in situ suppression (CISS) hybridization for use
with genomic DNA fragments or cDNAs containing IRS segments as probes.
Protocols are suitable for a 10 f.ll hybridization volume which is sufficient to
cover an area of 18x18 mm on a glass slide. Ifhybridization oflarger areas is
desired, the volumes of the solutions should be scaled-up accordingly.
Materials
• 3 M sodium acetate, pH 5.2
• Deionized formamide (molecular biology grade). The conductivity of the
solution should be below 100 f.lSiemens.
• Hybridization buffer: 4x SSC, 20% dextran sulphate. The solution is best
prepared as follows: Prepare 20x SSC and 50% dextran sulphate solu-