T7 Endonuclease Assay for DNA Mutation Detection

PCR products were purified (Qiagen, Cat: 28104) and eluted in molecular biology grade water and were then adjusted to 1X NEB Buffer 2.1 (NEB, Cat: B7202S). Products were then boiled for 10 minutes, allowed to cool to room temperature, divided in two, and treated with 1.5ul of T7 Endonuclease (NEB, Cat: M0302S) or water. The reaction proceeded for 1hr, until it was deactivated by adjusting the mixture with 1X Purple loading dye (NEB, Cat: B7024S). The reaction was then run at 120V for 65 minutes on a 1.5% agarose gel. The band intensities were quantified by densitometry using image-j according to previously published procedures [16 (link)]. For fragment analysis we utilized the method by Ran et al [16 (link)]. Results represent two to three independent experiments and error bars reflect standard deviation from the mean.