Human GRCh38/hg38 genome assembly via UCSC website. We
prepare high quality BAC or fosmid DNA by using a commercially
available kit and we confirm the authenticity of the sequence by
PCR followed by sequence-specific restriction digestion [15].
We fluorescently tag the DNA-FISH probes using a two steps
protocol. We first perform a nick translation reaction to introduce
aminoallyl-dUTP. During the nick translation reaction DNase I will
create single strand breaks called “nicks” within the double
stranded DNA sequence. In a next step, the Polymerase I will repair
these nicks and insert new nucleotides including the modified
dUTP. As a result from these reactions we obtain dsDNA fragments
(150–1000 bp) containing numerous aminoallyl-dUTP. Finally, we
couple fluorophores to the nick-translated modified dsDNA via a
simple chemical reaction where Alexa Fluor succinimidyl ester dyes
make a covalent bond with the amines of the incorporated aminoallyl-dUTP.
Perform the nick translation reaction following steps 1–4
1. Thaw BAC/fosmid DNA, 10Â NTB buffer, 0.1 M DTT, 10Â
dNTP mix, Aminoallyl-dUTP, and DNase A supplier buffer
on ice.
2. Dilute 1 μL of DNase I in 49 μL of DNase I supplier buffer to
reach the concentration of 0.2 U/μL. Keep on ice (see Note
50).
3. Set up the following reaction in a PCR tube on ice: mix
5–10 μg of BAC/fosmid DNA (27 μL maximum), 5 μL 10Â
NTB buffer, 5 μL DTT 0.1 M, 5 μL 10Â dNTP mix, 6 μL
aminoallyl-dUTP at 0.5 mM, 1 μL DNA polymerase I, 1 μL
DNase I at 0.2 U/μL and add water up to 50 μL.
4. Incubate 25 min at 16
C (see Note 50). During this time
prepare a 2% agarose gel.
Control the nick translation reaction (see steps 5–8).
5. Following nick translation, immediately transfer the reaction
tube on ice.
6. Run 1 μL of this reaction on a 2% agarose gel to verify the size
of the obtained fragments. Successful nick translation will
result in a smear between 150 and 1000 bp.
7. In the case of unsuccessful reaction, add 1 μL of freshly
prepared 0.2 U/μL DNase I dilution and incubate 10 more
minutes at 16
C (see Note50).
8. Control again the size of the fragments on a 2% agarose gel and
repeat this step until you obtain the correct fragment size.
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Noe ´ mie Kempf et al.
prepare high quality BAC or fosmid DNA by using a commercially
available kit and we confirm the authenticity of the sequence by
PCR followed by sequence-specific restriction digestion [15].
We fluorescently tag the DNA-FISH probes using a two steps
protocol. We first perform a nick translation reaction to introduce
aminoallyl-dUTP. During the nick translation reaction DNase I will
create single strand breaks called “nicks” within the double
stranded DNA sequence. In a next step, the Polymerase I will repair
these nicks and insert new nucleotides including the modified
dUTP. As a result from these reactions we obtain dsDNA fragments
(150–1000 bp) containing numerous aminoallyl-dUTP. Finally, we
couple fluorophores to the nick-translated modified dsDNA via a
simple chemical reaction where Alexa Fluor succinimidyl ester dyes
make a covalent bond with the amines of the incorporated aminoallyl-dUTP.
Perform the nick translation reaction following steps 1–4
1. Thaw BAC/fosmid DNA, 10Â NTB buffer, 0.1 M DTT, 10Â
dNTP mix, Aminoallyl-dUTP, and DNase A supplier buffer
on ice.
2. Dilute 1 μL of DNase I in 49 μL of DNase I supplier buffer to
reach the concentration of 0.2 U/μL. Keep on ice (see Note
50).
3. Set up the following reaction in a PCR tube on ice: mix
5–10 μg of BAC/fosmid DNA (27 μL maximum), 5 μL 10Â
NTB buffer, 5 μL DTT 0.1 M, 5 μL 10Â dNTP mix, 6 μL
aminoallyl-dUTP at 0.5 mM, 1 μL DNA polymerase I, 1 μL
DNase I at 0.2 U/μL and add water up to 50 μL.
4. Incubate 25 min at 16
C (see Note 50). During this time
prepare a 2% agarose gel.
Control the nick translation reaction (see steps 5–8).
5. Following nick translation, immediately transfer the reaction
tube on ice.
6. Run 1 μL of this reaction on a 2% agarose gel to verify the size
of the obtained fragments. Successful nick translation will
result in a smear between 150 and 1000 bp.
7. In the case of unsuccessful reaction, add 1 μL of freshly
prepared 0.2 U/μL DNase I dilution and incubate 10 more
minutes at 16
C (see Note50).
8. Control again the size of the fragments on a 2% agarose gel and
repeat this step until you obtain the correct fragment size.
370
Noe ´ mie Kempf et al.