Dna damage quantification kit

A DNA damage quantification kit was purchased from Dojindo Molecular Technologies, Inc. Standard AP DNA with a known number of AP sites was labeled with ARP. It included calf-thymus DNA (0.5 μg/mL) with 0, 2.5, 5, 10, 20, and 40 ARP-labeled AP sites per molecule, DNA binding solution, substrate solution, HRP-streptavidin, TE buffer, and washing buffer. Calibration curves were obtained using the standard protocol provided by the manufacturer.
To prepare the colorimetric assay using DNA standards in a 96 well plate, 60 μL of each standard ARP-DNA was added in 3 wells for each sample. Then, 100 μL of DNA binding solution was mixed by pipetting into each well followed by overnight incubation at room temperature. The next day, 1/4000 diluted HRP-streptavidin solution was freshly prepared by mixing 10 μL of HRP-streptavidin in 40 mL of washing buffer (due to its instability, this solution was freshly prepared every time). Binding solutions were discarded, and the wells were washed with washing buffer 5 times. After washing, 150 μL of diluted HRP-streptavidin was added to each well and incubated at 37°C for 1 h. After incubation, the washing step was repeated. Then, 100 μL of substrate solution was added to each well and incubated at 37°C for another 1 h. The optical density (OD) was recorded using a 96-well plate reader (SpectraMax M5 Multimode Plate Reader, Molecular Devices, LLC).