激酶实验
The reaction mixture for the oligonucleotide non‑hairpin substrate contains 25 mM MOPS (pH 7.0), 60 mM KCl, 0.2% Tween 20, 2 mM DTT, 1 mM or 5 nM MnCl₂ (or 5 mM MgCl₂, or 5 mM CaCl₂), 0.1 pmol DNA substrate, and 0.3 pmol Mre11 (or an equal amount of a mixture of Mre11 and Rad50), in a final volume of 10 μl. The mixture is incubated at 37 °C for 30 min. SDS, EDTA, and proteinase K are then added to final concentrations of 0.2%, 5 mM, and 0.1 mg/ml, respectively, followed by an additional 15‑min incubation. Four microliters of the reaction mixture is combined with 4 μl formamide loading buffer and loaded onto a gel containing 10% acrylamide and 7 M urea. After electrophoresis, the gel is analyzed using a fluorescence imaging system.
The reaction mixture for the oligonucleotide hairpin substrate is largely identical to that for the non‑hairpin substrate, except that 3 pmol Mre11 is included and incubation is performed at room temperature overnight.
The non‑homologous end‑joining reaction mixture contains 25 mM MOPS (pH 7.0), 60 mM KCl, 0.2% Tween 20, 2 mM DTT, 4 mM MgCl₂, 2 mM MnCl₂, 0.5 mM ATP, 4 ng plasmid DNA, 10% polyethylene glycol, 0.01 pmol human DNA ligase I, and 0.06 pmol Mre11 or 0.1 unit of Escherichia coli exonuclease III, in a final volume of 10 μl. After incubation at 37 °C for 25 min, Tween 20 is added to a final concentration of 0.5%, and PCR amplification is performed using primers DAR5 and DAR147. PCR products are cloned using a TA cloning kit and sequenced with an automated ABI Capillary Genetic Analyzer.
