Ethylenediaminetetraacetic Acid → dCTP

Participants

Reaction Steps

The radioactive probe DNA (e.g. BRY1 or OY11.1) was prepared by nick translation. The sample of DNA (0.2 μg) was pre-incubated with 25 mg/ml DNase I (E. coli deoxyribonuclease I) in a total volume of 40 μl containing 50 mM Tris-HCl 9 pH 7.5), 7.5 mM MgCl2, 5 mM dithiothreitol, 0.1 mg/ml BSA, 20 μM dATP, 20 μM dGTP, 20 μM dTTP, and 100 μCi α-32P dCTP (Amersham; approx. 3,000 Ci/mmol) at 15° C. for 20 min. During this time a minute sample (approx. 0.2 μl) was removed and applied to the origin mark on a small sheet (approx. 4×8 cm) of PEI-cellulose thin layer chromatography material (Merck). Following the pre-incubation, DNA polymerase I (Boehringer; approx. 5 units) was added and incubation continued at 15° C. for 20 min. The nick translation reaction was stopped by the addition of SDS to 1% (w/v) and EDTA to 20 mM, and a second minute sample removed and applied adjacent to the first on the PEI cellulose sheet. The thin layer chromatogram was developed in 0.75M potassium phosphate, pH 3.5, resulting in a separation of dCTP from DNA. The chromatogram was exposed to a sheet of X-ray film (Fuji Rλ) for 15 min. Development of the X-ray film allowed visual estimation of the efficiency of incorporation of α-32P dCTP into DNA. This was subsequently quantitated by scintillation counting of appropriate areas of the chromatogram. Before its addition to the hybridization solution, the labelled DNA was recovered by precipitation with ammonium acetate-ethanol as described above then dissolved in distilled water and denatured by heating at 100° C. for 5 min.

Provenance & Evidence