A METHOD FOR ISOLATION OF PLASMID DNA-REPLICATION INTERMEDIATES FROM UNSYNCHRONIZED BACTERIAL CULTURES FOR ELECTRON-MICROSCOPY ANALYSIS

Electron microscopy is a powerful technique for analysis of DNA replic ation intermediates. However, isolation of replicating DNA molecules f rom living cells is tricky and difficult, especially in the case of sm all DNA molecules (such as bacterial plasmids) whose initiation of rep lication is not easily synchronized. Here a relatively simple and rapi d method for efficient isolation of replicating plasmid molecules from unsynchronized Escherichia coli cultures is described. The efficiency of this procedure is high enough for electron microscopy analysis of plasmid replication intermediates appearing in living cells in normal growth conditions. Under optimal conditions, using standard procedures of isolation of plasmid DNA, it is possible to achieve a content of o nly as few as 0.02 percent of replication intermediates in a plasmid D NA sample. The described method allowed us to enrich up to 100-fold th e fraction of replication intermediates suitable for microscopic analy sis among all plasmid molecules.