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

NB.

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.

 

 

ANNEALING OLIGOS

 

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.