Labeling oligonucleotides with 32P ATP

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. H2O 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