Labeling oligonucleotides with 32P ATP

 

Labeling oligonucleotides with ³² P ATP

Steve Hahn

Last modified 8/13/01

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 H2O.
2. 3 microliters 10x kinase buffer.
3. 2 microliters ³² P ATP from ICN (>5000 ci/mmole).
4. H20 so that the final volume is 30 microliters.

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

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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.

 

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10x Kinase Buffer

0.5 M Tris pH 7.6
0.1 M MgCl2
50 mM DTT