32P-Labelling Oligonucleotides
1. Mix: 25 pmoles Oligo (pM/ul = (OD260/11 (# nucs)) x 103
2 ul 10X Kinase/ligation buffer pH 9.5
7 ul (25 pmoles) c32P-ATP (7000 Ci/mmole; ICN)
H2O to 20 ul
1 ul T4 kinase
2. 37°C 30-60 minutes.
3. 68°C 15 minutes.
4. Dilute to 500 ul with elutip low salt buffer.
5. Purify over elutip column.
6. Count 1 ul. Store frozen -20°C. Can be used for up to 1 month.
Solutions:
1. K/L Buffer: 500 mM Tris, pH 9.5 (5 ml)
70 mM MgCl2 (0.7 ml)
10 mM DTT (100 ul)
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