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VOLKER JUNG and GEORG A. WEBER
Nick translation
1. Label an aliquot of 10 ~l of PCR-generated pool DNA with a commercially available nick translation kit for in-situ hybridization according
to standard procedure.
2. Label 1 ~g normal reference DNA from a healthy donor with digoxigenin by nick-translation according to standard procedures.
PCR labeling 3. Transfer 5 ~l of the PCR product to a 200 ~l PCR reaction tube.
4. Add:
- 2 ~l Taq-buffer
- 2 ~llabeling dNTP-mix (2 ~M dATP, dGTP, dCTP, 0.5 ~M dTTP)
- 2 ~llabeled nucleotides (2 ~M, e.g. bio-16-dUTP)
Note: If labeled dUTP or dTTP nucleotides are not used, adjust the
concentration of the corresponding unlabeled nucleotide to 0.5 ~M
in labeling dNTP-mix instead of reducing dTTP concentration.
- 3 ~l DOP-primer (10 fJ.M)
- 0.5 fJ.I Taq polymerase (2.5 units)
5. Fill up to a volume of 25 fJ.l with ultrapure water.
6. Proceed in a thermocycler according to the following PCR program:
94°C
4 min
94°C
1 min
56°C
1 min
noe
3 min
(25 cycles)
noe
5 min
20°C
endless
Probe combination and hybridization
1. Transfer 10 fJ.I (- 500 ng) oflabeled DNA into an 1.5-ml reaction tube.
2. Add 10 fJ.I (-SOO ng) of differently labeled reference DNA and 50 fJ.g
human unlabeled Cotl-DNA.
3. Precipitate DNA with a 2.S-fold volume of absolute ethanol and 1/10
volume of 3 M Na-acetate to the probe.
4. Incubate at -20
D
C for at least 2 h.