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1. a-32P-dCTP is highly
radio active, proper protection and waste disposal are required.
2. Add 10-25 ng of template DNA and ddH2O to
a final volume of 11 ml in a microcentrifuge tube.
3. Denature the DNA sample by heating to 95-100 °C for 5 minutes.
Chill on ice.
For use with DNA labeling kits. The 5X labeling mix solution contains
the reaction buffer, random hexamer primers, and Klenow enzyme.
4. Make the following mix:
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Denatured DNA (10-25 ng)
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11 ml
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5X Labeling Buffer (containing random primers and Klenow enzyme)
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4 ml
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a-32P-dCTP
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5 ml
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For use with Klenow. The reaction buffer, random hexamer primers,
and Klenow enzyme need to be added separately.
4a. Make the following mix:
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Denatured DNA (10-25 ng)
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9 ml
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10X Klenow Reaction Buffer
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2 ml
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Random hexamer or octomer primer ( ~750 ng, 30 pmol)
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2 ml
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Klenow ( 5 units/ml )
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1 ml
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a-32P-dCTP
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5 ml
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5. Incubate at 37 °C for 10-60 minutes.
6. Stop the reaction by adding 1 ml of 0.5 M
EDTA or by heating heating to 95-100 °C for a couple of minutes.
7. Immediately before use in hybridization, denature the labeled DNA
by heating to 95-100 °C for 5 minutes and then put on ice.
To avoid poping up of the microcentrifuge tube, puncture a small hole
on the cap using a needle or use a microcentrifuge cap locker.
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