Reaction equation
C(O)[C:2]([NH2:7])([CH2:5]O)CO.Cl.[Mg+2].[Cl-].[Cl-].SC[C@H]([C@@H](CS)O)O.C1N=C2N([C@@H:30]3[O:34][C@H:33]([CH2:35][O:36][P:37]([O:40][P:41]([O:44][P:45]([OH:48])([OH:47])=[O:46])([OH:43])=[O:42])([OH:39])=[O:38])[C@@H:32]([OH:49])[CH2:31]3)C=NC2=C(N)N=1.P(OC[C@H]1[O:69][C@@H:68]([N:70]2C3N=C(N)NC(=O)C=3[N:72]=[CH:71]2)C[C@@H]1O)(OP(OP(O)(O)=O)(O)=O)(=O)O.OP(=O)(OC[C@H]1O[C@@H](N2C=C(C)C(=O)NC2=O)C[C@@H]1O)OP(=O)(OP(=O)(O)O)O.CCCCCCCCCCCCOS([O-])(=O)=O.[Na+].C(N(CC(O)=O)CC(O)=O)CN(CC(O)=O)CC(O)=O>>[P:37]([O:36][CH2:35][C@H:33]1[O:34][C@@H:30]([N:7]2[CH:2]=[CH:5][C:71]([NH2:72])=[N:70][C:68]2=[O:69])[CH2:31][C@@H:32]1[OH:49])([O:40][P:41]([O:44][P:45]([OH:48])([OH:47])=[O:46])([OH:43])=[O:42])(=[O:38])[OH:39]Participants
Procedure
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.
Workup
- customThe radioactive probe DNA was prepared
- customisolated from the livers of 6 unrelated cows
- customDuring this time a minute sample (approx. 0.2 μl) was removed
- additionwas added
- customThe nick translation reaction
- customa second minute sample removed
Sources & evidence
- ORD record
- ord-5f193a4a52374ee2a74d160dcdd4bc04
- Source dataset
- uspto-grants-1995_05
- Patent number
- US05418133
Linked notes
Loading…