17. Use 1 μL of the product to determine the concentration using
microvolume UV spectroscopy (see Note 52).
18. Store the clean and nick-translated DNA at À20
C.
Perform coupling of the fluorescent dye by following steps 19–28
19. Adjust 1–3 μg of aminoallyl-modified DNA to a volume of 5 μL
(see Note 52).
20. Seal the tube with parafilm to avoid evaporation and denature
the product at 95
C for 5 min.
21. Immediately transfer the tube on ice for 2 min.
22. Pulse spin and add 3 μL of NaHCO 3 at 0.2 M.
23. Dissolve one aliquot of amine reactive dye in 2 μL of
anhydrous DMSO.
24. Add the 8 μL of the nick-translated DNA/NaHCO 3 mixture,
vortex, pulse spin, and incubate at room temperature, in the
dark, for 1 h.
25. Add 90 μL of water and purify the labeled DNA-FISH probes
using a PCR purification kit (see steps 10–16). Elute in 60 μL
10 mM Tris-HCl pH 8.5 (see Note 53).
26. Determine the probe concentration: measure the absorbance
of the DNA-dye conjugate at 260 nm (A 260 ) and at the maximum absorbance (λ max ) for the used fluorophore (A dye ) using
microvolume full spectrum spectroscopy. Correct the DNA
concentration for the dye absorption according to the correction factor CF 260 (see Note 54) in Table 1, using Eq. 2
A base ¼ A 260 À A dye  CF 260
À
Á
ð2Þ
27. Determine the labeling efficiency (see Note 55): Calculate the
base to dye molecule ratio using Eq. 3 the extinction coefficient
of a DNA base ε base ¼ 8919 and the extinction coefficient of
the fluorophore ε dye (see Table 4).
base : dye ¼
A base  ε dye
A dye  ε base
ð3Þ
28. The labeled DNA-FISH probe can be stored for months at
À20
C (for DNA-FISH reaction you only need a few microliter of the final product).
3.3.2 Cell Fixation
and Permeabilization
1. Cells grown on coverslips are first washed once with 1Â PBS.
2. Under a fume hood, fix cells grown on coverslips with 2 mL (see
Notes 1) of 4% paraformaldehyde, without shaking, for
10 min. During the last 3 min, start cell permeabilization by
adding 800 μL of 0.5% Triton X-100, gently shake the plate to
372
Noe ´ mie Kempf et al.
microvolume UV spectroscopy (see Note 52).
18. Store the clean and nick-translated DNA at À20
C.
Perform coupling of the fluorescent dye by following steps 19–28
19. Adjust 1–3 μg of aminoallyl-modified DNA to a volume of 5 μL
(see Note 52).
20. Seal the tube with parafilm to avoid evaporation and denature
the product at 95
C for 5 min.
21. Immediately transfer the tube on ice for 2 min.
22. Pulse spin and add 3 μL of NaHCO 3 at 0.2 M.
23. Dissolve one aliquot of amine reactive dye in 2 μL of
anhydrous DMSO.
24. Add the 8 μL of the nick-translated DNA/NaHCO 3 mixture,
vortex, pulse spin, and incubate at room temperature, in the
dark, for 1 h.
25. Add 90 μL of water and purify the labeled DNA-FISH probes
using a PCR purification kit (see steps 10–16). Elute in 60 μL
10 mM Tris-HCl pH 8.5 (see Note 53).
26. Determine the probe concentration: measure the absorbance
of the DNA-dye conjugate at 260 nm (A 260 ) and at the maximum absorbance (λ max ) for the used fluorophore (A dye ) using
microvolume full spectrum spectroscopy. Correct the DNA
concentration for the dye absorption according to the correction factor CF 260 (see Note 54) in Table 1, using Eq. 2
A base ¼ A 260 À A dye  CF 260
À
Á
ð2Þ
27. Determine the labeling efficiency (see Note 55): Calculate the
base to dye molecule ratio using Eq. 3 the extinction coefficient
of a DNA base ε base ¼ 8919 and the extinction coefficient of
the fluorophore ε dye (see Table 4).
base : dye ¼
A base  ε dye
A dye  ε base
ð3Þ
28. The labeled DNA-FISH probe can be stored for months at
À20
C (for DNA-FISH reaction you only need a few microliter of the final product).
3.3.2 Cell Fixation
and Permeabilization
1. Cells grown on coverslips are first washed once with 1Â PBS.
2. Under a fume hood, fix cells grown on coverslips with 2 mL (see
Notes 1) of 4% paraformaldehyde, without shaking, for
10 min. During the last 3 min, start cell permeabilization by
adding 800 μL of 0.5% Triton X-100, gently shake the plate to
372
Noe ´ mie Kempf et al.