12 Chromosome Analysis by Fluorescence in situ Hybridization
173
c. If the probe is incompletely digested, purify probe and repeat the nick
translation.
d. If part of the probe is too short, repeat reaction using less DNase I.
6. In order to inactivate the DNase, 2J.!l of0.5M EDTA (final concentration
15 mM) and 1J.!l of 10% SDS (final concentration 0.1 o/o) are added, and
the reaction is heated at 68°C for 10 minutes.
7. Unincorporated nucleotides are separated from the labeled probe by gel
filtration using a spin column:
a. Pack a 1 ml syringe with sialinized glass wool up to the 0.2 ml mark.
Add buffer-equilibrated Sephadex G50 to the 1ml mark. Place the column in a 15 ml tube and centrifuge at 3000 rpm for 6 min at room temperature.
b. Remove flow-through, fill buffer and repeat centrifugations until the
column is tightly packed to the 1 ml mark. Add 100 Jll of column buffer
and centrifuge again at 3000 rpm for 6 min. Repeat this washing step
three times. After the last washing step, make sure that the volume of
the flowthrough equals the volume of the loaded buffer, i.e. 100 Jll.
c. Prior to centrifugation of the probe solution, put a 1.5 ml reaction tube
in the 15 ml tube beneath the syringe. Load the probe solution onto the
column and centrifuge as before. The flow-through is collected in the
reaction tube and now contains the labeled probe at a concentration
of 20 ng/Jll. The probeisready for use in an in situ hybridization experiment or can be stored at -20°C.
Digoxigenin labelling by Nick translation
LabeHing ofDNA probes with Digoxigenin is carried out in exactly the same
manneras biotinylation by nick translation with one exception: 10x nucleotide stock contains:
• 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.125 mM digoxigenin-11dUTP, 0.375 mM dTTP.
When new reagents are used or when no hybridization signals are obtained,
it is advisable to check the incorporation of the biotin or digoxigenin labeled
nucleotides. This can be achieved using a single, colorimetric dot blot assay
(Subprotocol 3).
173
c. If the probe is incompletely digested, purify probe and repeat the nick
translation.
d. If part of the probe is too short, repeat reaction using less DNase I.
6. In order to inactivate the DNase, 2J.!l of0.5M EDTA (final concentration
15 mM) and 1J.!l of 10% SDS (final concentration 0.1 o/o) are added, and
the reaction is heated at 68°C for 10 minutes.
7. Unincorporated nucleotides are separated from the labeled probe by gel
filtration using a spin column:
a. Pack a 1 ml syringe with sialinized glass wool up to the 0.2 ml mark.
Add buffer-equilibrated Sephadex G50 to the 1ml mark. Place the column in a 15 ml tube and centrifuge at 3000 rpm for 6 min at room temperature.
b. Remove flow-through, fill buffer and repeat centrifugations until the
column is tightly packed to the 1 ml mark. Add 100 Jll of column buffer
and centrifuge again at 3000 rpm for 6 min. Repeat this washing step
three times. After the last washing step, make sure that the volume of
the flowthrough equals the volume of the loaded buffer, i.e. 100 Jll.
c. Prior to centrifugation of the probe solution, put a 1.5 ml reaction tube
in the 15 ml tube beneath the syringe. Load the probe solution onto the
column and centrifuge as before. The flow-through is collected in the
reaction tube and now contains the labeled probe at a concentration
of 20 ng/Jll. The probeisready for use in an in situ hybridization experiment or can be stored at -20°C.
Digoxigenin labelling by Nick translation
LabeHing ofDNA probes with Digoxigenin is carried out in exactly the same
manneras biotinylation by nick translation with one exception: 10x nucleotide stock contains:
• 0.5 mM dATP, 0.5 mM dCTP, 0.5 mM dGTP, 0.125 mM digoxigenin-11dUTP, 0.375 mM dTTP.
When new reagents are used or when no hybridization signals are obtained,
it is advisable to check the incorporation of the biotin or digoxigenin labeled
nucleotides. This can be achieved using a single, colorimetric dot blot assay
(Subprotocol 3).