Cells were exposed to 20 Gy X-rays and incubated for repair. After different incubation periods, cells were trypsinized, resuspended in serum-free medium (20 mM Hepes, 5 mM NaHCO3) at a concentration of 6 × 106 cells/ml and mixed with equal volume of 1% low-melting Agarose (Bio-Rad, Munich, Germany). Agarose plugs (5 mm long), were prepared and lysed as described [24 (link)].
PFGE was performed in gels cast with 0.5% Agarose (Bio-Rad), in 0.5X TBE at 8 °C for 40 h. The electric field was pulsed at 50 V (1.25 V/cm) for 900 s in the forward direction and 200 V (5.00 V/cm) for 75 s in reverse direction. Then, gels were stained with 1.6 μg/ml ethidium bromide and imaged using a fluoroimager (Molecular Dynamics Typhoon 9400, GE Healthcare, Freiburg, Germany). The fraction of DNA released (FDR) was analyzed by ImageQuant 5.2 (GE Healthcare, Freiburg, Germany) and used to calculate the equivalent of Gy dose (Deq) [25 (link)].
PFGE was performed in gels cast with 0.5% Agarose (Bio-Rad), in 0.5X TBE at 8 °C for 40 h. The electric field was pulsed at 50 V (1.25 V/cm) for 900 s in the forward direction and 200 V (5.00 V/cm) for 75 s in reverse direction. Then, gels were stained with 1.6 μg/ml ethidium bromide and imaged using a fluoroimager (Molecular Dynamics Typhoon 9400, GE Healthcare, Freiburg, Germany). The fraction of DNA released (FDR) was analyzed by ImageQuant 5.2 (GE Healthcare, Freiburg, Germany) and used to calculate the equivalent of Gy dose (Deq) [25 (link)].
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