A method for isolation of plasmid DNA replication intermediates from unsynchronized bacterial cultures for electron microscopy analysis

• Autorzy: Srutkowska S. , Konopa G. , Wegrzyn G.
• Języki publikacji: EN

Abstrakty

EN

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.

Słowa kluczowe

EN

electron microscopy; bacterial culture; DNA replication; growth condition; isolation; Escherichia coli; bacterial cell; DNA; plasmid DNA

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 1998
• Tom: 45
• Numer: 1
• Opis fizyczny: p.233-240,fig.

Twórcy

autor

Srutkowska S.

• University of Gdansk, Kladki 24, 80-822 Gdansk, Poland

autor

Konopa G.

autor

Wegrzyn G.

Bibliografia

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