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sect19

Labeling of oligonucleotides

1. Combine:

1ul (50ng) of oligo in TE

0.5ul Denaturing buffer

3.5ul DDW

2. Incubate for 5min 70¡C

3. Quench on ice and pulse spin to remove condensate from lid.

4. In this order add:

1.25ul 10X kinase buffer

0.5ul 100mM DTT

5ul gATP32 (10uCi/ul)

0.75ul polynucleotide kinase.

5. Incubate for 60min 37¡C.

6. Add:

36ul TE

50ul 4M NH4 acetate

1ul 10mg/ml tRNA

200ul ethanol

Vortex and spin 10min.

No 70% rinse. Resuspend in 100ul TE and count 1ul.

Denaturing buffer:

100ul 2M tris HCl pH 9.5

100ul 100mM spermadine

2ul 0.5M EDTA pH 8.0

798ul DDW

10X kinase buffer:

250ul 2M Tris HCl pH 9.5

100ul 1M MgCl2

150ul DDW

500ul glycerol

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