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:
- 0.5 ug oligonucleotide dissolved in H2O.
- 3 microliters 10x kinase buffer.
- 2 microliters 32P ATP from ICN (at least 5000 ci/mmole).
- H20 so that the final volume is 30 ul.
Add 25 units T4 polynucleotide kinase and incubate 60 minutes at 37oC.
Purify labeled oligonucleotide away from unincorporated ATP using mini Quick Spin Oligo Columns (#1 814 397) from Roche.
- 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 minutes.
- Recover purified labeled oligo. For most applications, add 70 ul TE to the 30 microliters recovered for a total of 100 ul.
- 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)
TO MAKE 100 ml:
- 50 ml 1 M Tris pH 7.6 stock solution
- 10 ml 1 M MgCl2 stock solution
- 5 ml 1 M DTT stock solution