[69] Isolation of high molecular weight DNA from normal and phage-infected Escherichia coli

摘要: Publisher Summary This chapter describes two procedures that are used to obtain isotopically labeled DNA in high yield from phage-infected Escherichia coli for sucrose density-gradient analysis. The first method and a variant of it useful isolation the total T5-, T4-, λ-infected E. , second fractional T5-infected coli. Although bacteria handled much same way as uninfected bacteria, during half latent period they become very fragile, often lyse spontaneously upon centrifugation. As general precaution, therefore, cells chilled ice after sampling culture kept cold until treatment with lysozyme or detergent, usually centrifuged only once. Infected may contain at least three forms DNA—namely, bacterial-host DNA, accumulating intracellular phage particles, replicating, phage-precursor DNA. Pulse labeling infected radioactive thymidine is ensuring newly synthesized which relatively difficult extract, not discarded.