Labeling of DNA Probe by Random Priming

This is but one method to label probe DNA. Other methods include nick translation (see Note 6) and 3'-endlabeling using TdT (see Note 7).

1. Denature 100-500 ng of satellite DNA [e.g., Y chromosome repeats (AATAAAC)n or (AATAC)n or dodeca satellite] in water or 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 (TE) buffer by heating at 100°C for 10 min in a 0.5-mL Eppendorf SafeLock microcentrifuge tube.

2. Add 18 |L of denatured DNA to a 0.5-mL microcentrifuge tube containing 5 ||L of OLB and 1 |L of biotin-14-dATP. (For AT-rich DNAs, dilute 1 vol of 0.4 mM biotin-14-dATP with 4 vol of 0.4 mM unlabeled dATP.) (See Note 2.)

3. Add 1 |L of Klenow enzyme (5 U) to give a total reaction vol of 25 |L and incubate at 37°C for 1-2 h or overnight at room temperature.

4. Stop the reaction by adding 2 |L of 200 mM EDTA, pH 8.0 (and/or by heating at 65°C for 10 min).

5. Remove unincorporated nucleotides by ethanol precipitation (see Note 5) or other means (e.g., with a spin column). If the probe is not to be used right away, store at -20°C.

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