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1. Cool a microcentrifuge tube on ice and mix the following on ice.
10X dNTP mix (- dCTP)
(0.2 mM each dATP, dGTP, dTTP, 500 mM Tris-HCl (pH 7.8)
50 mM MgCl2,
100 mM beta-mercaptoethanol, 100 mg/ml nuclease-free BSA)
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2 ml
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DNA (plasmid, BAC, YAC, etc.)
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0.1-0.5 mg
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10X Enzyme Mix
(0.5 U/ml DNA Polymerase I, 0.007 U/ml DNase I,
50 mM Tris-HCl (pH 7.5),
5 mM magnesium chloride, 0.1 mM phenylmethylsulfonyl fluoride,
50% (v/v) glycerol,
100 mg/ml nuclease-free BSA)
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2 ml
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a-32P-dCTP
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5 ml
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ddH20
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to 20 ml
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3. Mix thoroughly and centrifuge briefly. Incubate at 15 °C for 2 hours.
4. Stop the reaction by placing the tubes in -20 °C.
Or stop the reaction by either adding 2 ml 0.5M EDTA (pH8.0) or
heating to 75 °C for 10 minutes.
5. Chill on ice.
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