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Figure 2 | BMC Biotechnology

Figure 2

From: A modified and automated version of the 'Fluorimetric Detection of Alkaline DNA Unwinding' method to quantify formation and repair of DNA strand breaks

Figure 2

(A) Comparison of SybrGreen (Sybr) with ethidium bromide (EtBr) as a fluorescent probe for the FADU assay. For each experimental point in this experiment, 80,000 human PBMC were suspended in 70 μl suspension buffer at 0°C. For B samples, 70 μl of lysis buffer was added and the mixture was sheared by 20 passages through a 0.5-mm cannula, followed by transfer to a 96-well plate [140 μl per well]. For P and T samples 70 μl of cell suspension was transferred per well and 70 μl lysis solution was added at 0°C. This and all subsequent pipetting steps were performed by the LHD. After 12 min of lysis, 70 μl of unwinding solution was added on the top of the cell lysate followed by incubation at 15°C for 90 min. To stop DNA unwinding, neutralisation solution was added. Then 150 μl of the mixture was combined in plastic cuvettes with 500 μl of either Sybr or EtBr solution. Fluorescence detection was done at excitation 480 nm and emission 520 nm. (B) Alkaline unwinding of plasmid DNA as a model substrate. The circular form of the plasmid (white) could not be unwound and retained nearly 100% of the total fluorescence (T circular). The P0 values of the linear form (grey) represent unwound DNA and display less than 20% double/stranded DNA compared to non-denaturing control conditions (T linear). To obtain the T values, neutralisation solution was added before the alkaline solution. Error bars represent SDs from 8 replicates.

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