CometAssay Electrophoresis Starter Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 3 × 10^5 cells/ml in molten LM agarose at a ratio of 1:10 and spread on CometSlides™.

Upstream tips
Seed 3 × 10^5 cells/ml in molten LM agarose at a ratio of 1:10 and spread on CometSlides™.

Publication protocol

The CometAssay® kit from Trevigen® was used according to the manufacturer's instructions. 72 h after transfection with siRNA, the cells were re-suspended in ice-cold PBS at a concentration of 3 × 10^5 cells/ml, embedded in molten LM agarose at a ratio of 1:10 and spread on CometSlides™. The slides were immersed in 4°C Lysis Solution overnight before exposure to Alkaline Unwinding Solution (300 mM NaOH, 1 mM EDTA, pH > 13) for 1 h at 4°C. Electrophoresis at 21 V for 30 min was performed in the specific Electrophoresis buffer. The slides were washed twice in dH2O and immersed in 70% EtOH for 5 min after electrophoresis. All slides were finally dried at 37°C and stained with SYBR® Green for 30 min. Images were captured with an Olympus IX81 fluorescence microscope and at least 100 cells per condition were analysed in each of three independent experiments by ImageJ software.

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