12 Chromosome Analysis by Fluorescence in situ Hybridization
171
3. For short term use, slides can be stored at room temperature for up to a
few days. For long term storage slides should be "aged" overnight at
room temperature. then placed in containers. Containers are sealed
in plastic bags containing Drierite and stored at -70°C. In our experience,
for slides used within 6 months storage at -20°C is sufficient. The slides
are thawed immediately prior to the hybridization experiment. The refreezing of slides is not recommended.
Subprotocol 2
Probe Labelling by Nick Translation
Nicktranslation is the most frequently applied method for labeHing DNA
probes for use in fluorescence in situ hybridization experiments. Although
the labeHing efficiencies of nick translation and primer extension ( Feinberg
and Vogelstein 1984) are comparable, we prefer nick translation. Nick
translation permits easy adjustment of the probe size, which for optimum
hybridization conditions should be 100- 500 nucleotides, by varying the
DNase concentration. The protocol is based on Langer et al. (Langer et
al. 1981). It should be noted that the conditions of primer extension can
also be adjusted to optimize probe length. Subprotocol2 describes labeHing
with biotin and digoxigenin. These reporter molecules are most popular due
to their sensitivity and commercial availability.
Materials
• Solution of template DNA (purified from RNA)
• 1 Ox reaction buffer containing 0.5 M Tris-H Cl (pH 8.0 ), 50 mM MgClz, 0.5
mg/ml BSA
• 0.1 M ß-mercaptoethanol prepared by adding 0.1 ml of ß-mercaptoethanol to 14.4 ml of double-distilled water
• 10x nucleotide stock containing 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM
dGTP, 0.5 mM biotin-16-dUTP and 0.12 mM dTTP (alternatively, other
biotinylated dUTP derivatives can be used)
• Escherichia coli DNA polymerase I
• DNase I solution: 3 mg DNAse I in 1 ml 0.15 M NaCl, 50% glycerol
• Column buffer: 10mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1% SDS
Reagents
171
3. For short term use, slides can be stored at room temperature for up to a
few days. For long term storage slides should be "aged" overnight at
room temperature. then placed in containers. Containers are sealed
in plastic bags containing Drierite and stored at -70°C. In our experience,
for slides used within 6 months storage at -20°C is sufficient. The slides
are thawed immediately prior to the hybridization experiment. The refreezing of slides is not recommended.
Subprotocol 2
Probe Labelling by Nick Translation
Nicktranslation is the most frequently applied method for labeHing DNA
probes for use in fluorescence in situ hybridization experiments. Although
the labeHing efficiencies of nick translation and primer extension ( Feinberg
and Vogelstein 1984) are comparable, we prefer nick translation. Nick
translation permits easy adjustment of the probe size, which for optimum
hybridization conditions should be 100- 500 nucleotides, by varying the
DNase concentration. The protocol is based on Langer et al. (Langer et
al. 1981). It should be noted that the conditions of primer extension can
also be adjusted to optimize probe length. Subprotocol2 describes labeHing
with biotin and digoxigenin. These reporter molecules are most popular due
to their sensitivity and commercial availability.
Materials
• Solution of template DNA (purified from RNA)
• 1 Ox reaction buffer containing 0.5 M Tris-H Cl (pH 8.0 ), 50 mM MgClz, 0.5
mg/ml BSA
• 0.1 M ß-mercaptoethanol prepared by adding 0.1 ml of ß-mercaptoethanol to 14.4 ml of double-distilled water
• 10x nucleotide stock containing 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM
dGTP, 0.5 mM biotin-16-dUTP and 0.12 mM dTTP (alternatively, other
biotinylated dUTP derivatives can be used)
• Escherichia coli DNA polymerase I
• DNase I solution: 3 mg DNAse I in 1 ml 0.15 M NaCl, 50% glycerol
• Column buffer: 10mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.1% SDS
Reagents