This chapter discusses different protocols involving breaking the cell wall and cell membrane, and then separating the nuclei from cytoplasmic components. Breaking the cell wall has been accomplished by mechanical means (blenders, mortar and pestle, etc.), or by forming protoplasts with enzymatic digestion. In the method described in the chapter, nuclei have been purified from the crude lysate by filtration and differential centrifugation. There are two protocols for isolating nuclei from cell suspension cultures. The first uses protoplasting enzymes to remove the cell wall, followed by lysis of the protoplasts with Triton X-100. The yield of the nuclei is high (40%) and free of most cytoplasmic DNAs. Because of these attributes, most DNA replication studies have been done with this protocol. An alternative procedure that uses a milk homogenizer to break the cell wall is necessary when cell cultures fail to form protoplasts. The yield of the nuclei is low (ca. 1%) and cytoplasmic DNA contamination is higher. This protocol has been used mainly for isolating DNA from nonprotoplasting cell lines.
ASJC Scopus subject areas
- Molecular Biology