Preparation of Nucleoplasmic Extract and Its Application in DNA End Processing

The nucleoplasmic extract (NPE) of Xenopus laevis eggs contains a physiological concentration of nuclear proteins that recapitulate a functional nuclear environment. This system has been widely used to study key nuclear processes, including DNA replication, repair, transcription, and chromatin assembly. Its soluble nature, free from membrane fractions and insoluble structures, enables direct analysis of nuclear responses to specific DNA structures and lesions. Here, we describe an NPE preparation protocol with our modifications and its application in the processing of DNA double-strand breaks, a critical step in homology-directed repair.