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Bacterial Genomic DNA Extraction - (Feb/16/2007 )

How do we make sure that it is only Genomic DNA and no PLASMID DNA that turns out when we try to isolate Genomic DNA from a bacterial cell suspension ?
Is there any specific component in the protocol that does this job of alleviating Plasmid DNA during Genomic DNA extraction?


I believe there is no simple protocol to selectively remove plasmid DNA from genomic DNA. Any protocol that will isolated genomic DNA will be gentle enough to isolate plamid DNA (which is smaller)

However, two idea come to mind.

You could grow your cells without selection on rich liquid medium. Then asecptically streak a little of the broth onto a rich agar plate (again without selection for the plasmid). Then check individual colonies for the absence of the plasmid.

If there plasmid causes even a slight growth retardation while on the rich medium, the cells which spontaneously lose the plasmid will have a selective advantage and take over most of the culture. You may have to repeat the non selective growth cycles a few times to enrich plasmid free cells.

A different idea would be to gel purify your DNA. Assuming your plasmid is small (>7kb), you can gel purify your DNA on a low density agarose gel (~0.7%) . The genomic DNA which is large will be retained near the top while the plasmid DNA will migrate a little further into the gel. Excise the genomic DNA from the gel, extract the DNA by one of the old gel extraction protocol (do not use column, as the will only retain fragments below 10kb)


A DNA prep which allows you to spool or hook the genomic DNA on a glass rod will allow you to wash small plasmid fragments, which will be much more soluble than the genomic DNA. I doubt you can eliminate all plasmid DNA this way, however. PCR, for example, would certainly pick up plasmid DNA after doing this.