Ethylenediaminetetraacetic Acid → dCTP

Beteiligte Stoffe

Reaktionsschritte

The radioactive probe DNA was prepared by pooling equal amounts of DNA isolated from the livers of 6 unrelated cows and labelling a portion of the mixture by nick translation. The sample of mixed DNA (0.2 μg) was pre-incubated with 25 pg/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 RX) 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 and found to exceed 95% resulting in mixed female bovine genomic DNA labelled at a specific activity of approx. 109 dpm [32 p]/μg. 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.

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