3 replies to this topic

[jaskhurana]

Hi

I am interested in isolating relatively pure genomic DNA and plasmid DNA from E.coli. Are there protocols which would ensure the purity of the extracted samples. e.g. no gDNA in plasmid preps and vice versa.

I am familiar with the standard plasmid prep techniques and how to avoid gDNA contamination. Just wanted to know if there is a method of isolating pure genomic DNA by avoiding plasmid contamination.

Thanks in advance.

[Trof]

One plasmid isolation method uses CsCl gradient centrifugation, that differentiate plasmids from gDNA. You take the latter.

[Dino]

Hi I just wanted to jump in and ask another question:

In general when doing a gDNA prep do plasmids get pulled down with it?

Specifically we have a low copy plasmid that we are trying to use for homologous recombination
and we are trying to screen for the first recombination event. I suppose if a cross over has occured
the plasmid backbone will be attached to gDNA. However if recom. has not occured we could still get
non-specific amp if plasmid gets pulled down in the gDNA prep.

Hope I haven't confused

D

[phage434]

Separation of plasmid and gDNA is never very efficient.  You will have a hard time determining integration of plasmids with this method.  Instead, you can use sequencing outward from the plasmid into the genomic DNA, which will also locate the insertion site.  This can either be done with direct sequencing of gDNA, which is suitable for small genomes, or by inverse PCR.