Open AccessA method for isolation of plasmid DNA replication intermediates from unsynchronized bacterial cultures for electron microscopy analysis.
Author(s) -
S Srutkowska,
Grażyna Konopa,
Grzegorz Węgrzyn
Publication year - 1998
Publication title -
acta biochimica polonica
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.452
H-Index - 78
eISSN - 1734-154X
pISSN - 0001-527X
DOI - 10.18388/abp.1998_4305
Subject(s) - plasmid , dna replication , dna , rolling circle replication , plasmid preparation , biology , bacteria , escherichia coli , origin of replication , replication (statistics) , isolation (microbiology) , microbiology and biotechnology , chemistry , genetics , gene , virology , pbr322
Electron microscopy is a powerful technique for analysis of DNA replication intermediates. However, isolation of replicating DNA molecules from living cells is tricky and difficult, especially in the case of small DNA molecules (such as bacterial plasmids) whose initiation of replication is not easily synchronized. Here a relatively simple and rapid 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 only as few as 0.02 percent of replication intermediates in a plasmid DNA sample. The described method allowed us to enrich up to 100-fold the fraction of replication intermediates suitable for microscopic analysis among all plasmid molecules.