Edited by Chang Zhu
1. Cool a microcentrifuge tube on ice and mix the following on ice.
10X dNTP mix (- dATP)
(0.2 mM each dCTP, 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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5 ml
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10X dATP mix
(0.1 mM dATP, 0.1 mM biotin-14-dATP or 0.1 mM digoxigenin-dATP)
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5 ml
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DNA (plasmid, BAC, YAC, etc.)
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1 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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5 ml
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ddH20
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to 50 ml
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2. Mix thoroughly and centrifuge briefly. Incubate at 15 °C for 1-2 hours.
3. Stop the reaction by placing the tubes in -20 °C.
4. Run 2 ml in 1-2% agarose gel to check probe size.
The peak size should be between 50-500 bp (or 100-300 bp).
5. If the peak size is bigger than this, add 5 ml 10X enzyme mix, incubate at 15 °C for a further 30-60 min.
Run another aliquot on a gel to test the size.
6. Stop the reaction by either adding 2 ml 0.5M EDTA (pH8.0) or
heating to 75 °C for 10 minutes.
7. Chill on ice.
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