Emulsifier scintillator plus

Cells were mixed with 56/2 medium supplemented with 2 μg ml⁻¹ thymidine and [¹⁴C] thymidine (0.2 μCi ml⁻¹) as described (22 (link)), with minor modifications: the cells were grown exponentially to OD₆₀₀ = 0.3 at 37°C for ΔlexA strains and OD₆₀₀ = 0.5 at 42°C for recA441 strains. At time zero, the pre-labelled cells were rinsed with M9 salts, diluted 3-fold in 56/2 medium supplemented with 2 μg ml⁻¹ thymidine and [³H] thymidine (1.0 μCi ml⁻¹), and further incubated at the same temperature. Aliquots (2 ml each) were withdrawn at 5, 10 and 15 min. Radioactivity of ³H and ¹⁴C in acid-insoluble material collected by filtration was measured in Emulsifier Scintillator Plus (PerkinElmer) with an AccuFLEX LSC-7200 scintillation counter (Hitachi Aloka Medical, Japan). For normalization in measuring the DNA synthesis rate, the incorporation of [³H] thymidine was divided by that of [¹⁴C] thymidine, which is indicative of the initial amount of DNA in cells labelled with [³H] thymidine. The normalized values were plotted at each time point, and slope values were estimated from a linear regression line to determine the DNA synthesis rate. To examine DNA degradation in recA441 cells during 10 min of incubation, radioactivity of [¹⁴C] thymidine in the acid-insoluble fraction of cells collected at 15 min was divided by that at 5 min (Supplementary Figure S3D).