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        Labeling oligonucleotides with 32PATP

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        Wear gloves throughout and work in radiation area. Monitor area before and after use.

        Mix the following in an eppendorf tube:

        1. 0.5 microgram oligonucleotide dissolved in H2 O.

        2. 3 microliters 10x kinase buffer.

        3. 2 microliters 32 P ATP from ICN (>5000 ci/mmole).

        4. H2 O so that the final volume is 30 microliters.

        Add 25 units T4 polynucleotide kinase and incubate 60 min at 37 ℃.

        Purify labeled Oligonucleotide away from unincorporated ATP

        Currently, we use mini Quick Spin Oligo Columns (#1 814 397) from Roche to purify the labeled oligonucleotide.

        Prepare the column according to the manufactuer's instructions by centrifugation of the resuspended matrix for 1 min @ 1000 x g.

        Insert column into a new eppendorf tube and add oligo labeling reaction, adding slowly to center of column. Centrifuge 1000 x g for 4 min.

        Recover purified labeled oligo. For most applications, add 70 microliters TE to the 30 microliters recovered for a total of 100 microliters.

        Quantitate radioactive incorporation by counting 1 microliter of a 1/10 diluted sample. Expect between 20 -100 million cpm total.

        10x Kinase Buffer

        0.5 M Tris pH 7.6

        0.1 M MgCl2

        50 mM DTT

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