352 Maunders
- Incubate at 37°C for 30 min.
- Terminate the reaction by adding 2 pL of 500 mM EDTA, pH 8.0.
- Extract, purify, and measure incorporation as in Section 4.4.2.
4.4.4. Exchange Reaction (20) - To 1-50 pmol DNA in a small volume add:
- 5 Ixl_, of 10X imidazole buffer
- 5 lxL of 1 mM ADP
- l~Lof50nMATP
- 15 pL of y-32p ATP (50 pmol, 3000 Ci/mmol, 10 ~tCi/laL)
- Water to 38 lxl_,
- 10 ~tL of 24% PEG 8000
- 2 laL (20 U) of T4 polynucleotide kinase
- Incubate at 37°C for 30 min.
- Terminate the reaction by adding 2 jaL of 500 mM EDTA, pH 8.0.
- Extract, purify, and measure incorporation as in Section 4.4.2.
4.4.5. Labeling Oligonucleotides (27) - To 10 pmol of oligonucleotide in 1 pJ_, add the following:
- 2 IlL of 10X Tris kinase buffer,
- 5 laL of y-32p ATP (10 pmol, 5000 Ci/mmol, 10 ~tCi/IA_,), and
- 11.5 p.L, of water.
- Mix and take 0.5-pL zero-time aliquot and add this to 10 ~L TE.
- To the remainder of the reaction mix add 1 laL (10 U) T4 polynucle-
otide kinase. - Incubate at 37°C for 45 min.
- Take another 0.5-1aL aliquot and add to 10 I.tL of TE as before.
- Terminate the reaction in the remainder of the mix by heating to 68°C
for 10 min. - Measure incorporation efficiency as follows (46,47): Spot 0.5 pL of
each diluted aliquot onto 15-cm long polyethyleneimine cellulose strips.
Perform thin layer chromatography using 500 mM ammonium bicar-
bonate as the developing solution. Allow the solvent front to run 10-13
cm. Saran Wrap and autoradiograph, or slice up the strip and measure
the radioactivity along the strip by scintillation counting. Compare time-
zero and 45-min samples. In this TLC system, oligonucleotides remain
at the origin, inorganic phosphate migrates near the solvent front, and
ATP occupies an intermediate position.
An alternative method is to measure incorporation by adsorption to
DE81 filters. Oligonucleotides bind tightly, whereas ATP can be washed
off with 0.5M sodium phosphate, pH 7.0.