Oligos - End Labeling
END LABELING OLIGOS
2ul 10x PNK buffer
20 pMols Oligo
1 ul g32P ATP (7000 ci/mmole)
1 ul T4 PNK (10 u/ul)
H2O to 20 ul.
37o C for 30 minutes.
Separate labeled oligo from free radioactivity on a 1ml G50 column.
PNK buffer: 70mM Tris-HCl pH 7.6, 10mM MgCl2, 5mM DTT
The ratio above of g32P ATP to oligo is equimolar. If the label is older than 2 wks double the amount for every additional week.
PNK is inhibited by ammonium ions.
1. Add an equal mols of oligos together (~300pMol) in 50ul of kinase buffer.
2. Heat to 70C for ten minutes to remove secondary structure.
3. Allow to anneal slowly by placing in container of 70C H2O on the bench ~30 mins.