Run the DNA on 2D pulse electrophoresis gel? Since the plasmids are large probably run them on the largest gel your lab equipment can make?

Is your plasmid in supercoiled form? If it is, gel filtration on Sephacryl S-1000 or Superose 6 should do the job. See e.g. doi: 10.1016/j.chroma.2009.07.009 (but there's plenty more in the literature).

Thanks for your answer, Alejandro!

I actually have E coli DNA containing multiple varying size plasmids (from 40kbp up to 250kbp) . I do not know what form have these plasmids. But, are plasmids not consistently in all 3 forms?

In the paper you cited, the resin is designed for the separation of DNA up to 20 kbp. Are there resins for plasmids bigger?

Run the DNA on 2D pulse electrophoresis gel? Since the plasmids are large probably run them on the largest gel your lab equipment can make?

http://www.ncbi.nlm.nih.gov/pubmed/4291934

http://www.ncbi.nlm.nih.gov/pubmed/16780964

Hirt extraction protocol is wildly used to isolate HBV cccDNA from cell culture, and maybe you can modify the protocol to separate genomic DNA/plasmid if your plasmid is supercoiled type.

how about if u try to make your isolation in lower concentration.. and you can try to purification it by isopropanol and RNAse..

@ Elly Syafriani : Can u plzz tell tell how would tat help

Thanks everyone!

Kushani : from what I know, the 2D electrophoresis doesn't seem to be easy to implement... but why not!

Xianfeng : noticeably, the protocols are for small circular DNA (5kbp), do you think it can be adapted to 250kbp circular DNA?

Lionel, I am not sure.........

Those are large plasmids indeed; I don't think my suggestion (gel filtration) will work for them. Perhaps an enzymatic treatment would be a better solution; a combination of T7 exonuclease and Mung Bean Endonuclease (both available from NEB) should eliminate any molecule that is not a covalently closed dsDNA circle, although with such large plasmids, yields will suffer due to the presence of nicked/linearized plasmid molecules.

Honestly, I think you would minimize time/effort/money spent by simply retransforming your plasmids and purifying them again. Searching the literature/web for recipes from people working with BACs should give you plenty of tips on how to avoid contamination of large plasmids with genomic DNA.

Hi Lionel, what is your aim to recover genomic DNA?

whether differential ultra centrifugation would help in recovery and separation of plasmids and nucleoid parts. First isolate the separated plasmids through differnetial centrifugation as the plasmids of different sizes shall concentrate in different layers and nucleoid being heaviest shall be at lowermost place. after that you can purify and isolate DNA .

ref; Andersson K, Hjorth R. Plasmid. 1985 Jan;13(1):78-80.

Isolation of bacterial plasmids by density gradient centrifugation in cesium trifluoroacetate (CsTFA) without the use of ethidium bromide.

Abstract

Plasmids extracted from bacterial cells by alkaline extraction can easily be isolated from linear DNA by isopycnic centrifugation in CsTFA. This is a fast and simple method which circumvents the use of the intercalating dye, ethidium bromide, and consequently the problems associated with its removal. The buoyant densities for covalently closed circular DNA and linear DNA in CsTFA are 1.60 g/ml and 1.65 g/ml, respectively. The isolation is achieved regardless of plasmid size and can be accomplished at temperatures of between 4 and 30 degrees C. Plasmid DNA isolated in gradients of CsTFA are of a high purity and have been found to be intact when cleaved with restriction enzymes and ligated with T4 DNA ligase.

Actually i want to know if the genome of my bacterial strain also contains resistance genes as plasmids in this strain. It's why I need to recover the bacterial genome and not the plasmids.

You can use calcium chloride instead of cesium chloride.

Is this an environmental bacteria or purchased for cloning?

If the bacteria is specific for cloning, just re-grow or re-buy the strain without the plasmid.

If it is environmental...you could try limiting dilution PCR to separate out the gDNA from pDNA. A duplex PCR could be used; one assay to determine gDNA vs pDNA and the second assay to test for resistance. See Proc Natl Acad Sci U S A. 1999 Aug 3;96(16):9236-41 (free on Pubmed).

Bonjour Lionel,

You could try bi or/tri parental mating to capture your plasmid in another host, with a different genetic background ?

This method is quite efficient with many environmental genus, providing you can find the good helper strain, and or recipient...

1. Smalla, K., Heuer, H., Götz, A., Niemeyer, D., Krögerrecklenfort, E., and Tietze, E. (2000). Exogenous isolation of antibiotic resistance plasmids from piggery manure slurries reveals a high prevalence and diversity of IncQ-like plasmids. Appl Environ Microbiol 66, 4854–4862.

If you can grow you strain on standard media, it should be quite easy to cure it from its plasmid, on a media eventually suplementing for catabolic genes loss when plasmid is lost?

From what I understand, you do not want to extract plasmid dna, but rather clean it out from your dna solution, to be confident your genes are (or not) on the chromosome also ? Ie. a plasmid free fraction.

Every plasmid based extraction can statisfactory yield almost pure plasmid molecules. However, chromosomal DNA extraction will mostly always contain DNA from sheared plasmids, loosing their supercoiled formation, and therefore exactly the same molecule as chromosomal DNA. If PCR based applications are applied after that, how can you be 100% confident it's not amplifying from linearized plasmid ?

Dear Lionel,

What I noticed based on the discussion, is that you are looking for origin of a trait on bacterial genome, ie gDNA vs. Plasmid. for this in plant bacteriology, we sub-culture the bacteria 25 to 35 times in a general medium without any adjutants or other treatments, however adding acridines, ethidium bromide, urea, and MitomycinC is also effective for elimination of the plasmid from the bacterial cells. this technique is called Plasmid Curing and usually performed under conditions similar to those used for the routine culture of the bacteria and the agent is used at concentration just less than that required to inhibit growth, and the number of bacteria initially is small. If acridine is used, the culture is maintained at pH 7.6 and incubated in the dark.

then for confirming the results, u should re-transform the Bactria with the plasmid.

Plasmid curing is indeed a worthy solution, however this can be very hard to cure a strain from a plasmid with efficient partionning and stabilisation machinery, such as broad-host large plasmid, such as those from IncP subgroup...

Good luck since this is precisely your case, trying to separate a (not so large) 250kbp plasmid. No one ever loved cesium chloride, though it was proposed for a reason. Mechanical shearing is unavoidable without in situ lysis of cells, such as in E.A Schwinghammer historical paper proposing such method for extraction of Rhizobiacea symbiotic plasmid, similar in size.

I'd like to point that voted up answers fail to adress the problem of contamination of gDNA by plasmid, that is the current issue, not the other way around...

PS: I'd go for WG sequencing of both extracts and then assess presence/absence on different replicons by checking differences in coverage...

Prices have seriously diminished to the point where sometimes DNA extraction (and moreover data storage) cost more than sequencing... My 2 cents...

try alcaline lysis