Kinase 351
- 7.5M Ammonium acetate.
- Ethanol.
- TE buffer: 10 mM Tris-HC1, pH 8.0, 1 mM EDTA.
11.24% PEG 8000. - 1 mM ADP solution (for exchange reaction only).
13.50 nM ATP solution (for exchange reaction only). - Sephadex G-50 for column or spun column purification after reaction.
- For measurement of incorporation:
a. 10% trichloroacetic acid, 5% trichloroacetic acid, and 70% ethanol;
b. DE81 filters and 500 mM sodium phosphate, pH 7.0; or
c. Polyethyleneimine cellulose TLC strips and 500 mM ammonium
bicarbonate.
4.4.2. Kinasing Single-Stranded DNA Fragments or Duplexes
with Protruding 5' Termini (27,31)- Prepare and purify dephosphorylated DNA fragments.
- To an aliquot containing 1-50 pmol ends add:
- 5 IxL of 10X Tris kinase buffer
- 15 ~tL of~/_32p ATP (50 pmol, 3000 Ci/mmol, l0 Ci/~tL, 1 lxM final
concentration) - 1 ~tL ofT4 polynucleotide kinase (10 U)
- Water to 50 IxL.
- Incubate at 37°C for 30 min.
- Terminate the reaction by adding 2 ~tL of 500 mM EDTA.
- Purify by phenol/chloroform extraction.
- Remove unincorporated ATP by column or spun-column chromatogra-
phy using Sephadex G-50. - Add 1/2 vol 7.5M ammonium acetate and 2 vol ethanol. Precipitate
the DNA at -70°C for 30 min. Centrifuge, drain pellet, and redissolve
in 50 ~tL TE. - Determine incorporation of label by TCA precipitation. (See Section
4.4.5. for measurement of incorporation in oligonucleotides.)
.4.4.3. Kinasing DNA Duplexes
with Blunt or Recessed 5' Ends (27)
To an aliquot of DNA containing 1-50 pmol ends in 9 l.tL or less, add:- 4 ~tL of 10X imidazole buffer
° Water to 13 ~tL - 10 t.tL of 24% PEG 8000
- 15 I.tL of ~t-32P ATP (50 pmol, 3000 Ci/mmol, 10 gCi/lxL)
- 2 ~L (20 U) of T4 polynucleotide kinase