Can u please mention the strain u are using and the plasmid u are isolating ... since it has a lot to do with the question u raised.
By the way what method are u using?
If a strain has a plasmid, shouldn't it show up in chromosomal DNA preps prepared by most methods? The only way I can think of right now to get rid of it would be isopycnic ultracentifugation on CsCl2 density gradients in the presence of EtBr, and even then you will get small amounts of contaminating linear plasmid.
Hi Nattan,
The plasmid is part of the genomic DNA of the bacterium and as such you will always purify plasmid DNA alongside chromosomal DNA.
If plasmid DNA contamination is a problem you will need to go through a process called 'curing' to eliminate the plasmid from your strain of bacteria. Sometimes this is very simple - for example if the plasmid confers antibiotic resistance you could grow the cells in antibiotic-free medium and look for cells that lose resistance by replica plating.
However most naturally occurring plasmids are very stable and often encode lethal, self-selection genes that kill plasmid-free cells. Methods to cure bacteria of their plasmids can involve growing cells under stress conditions, chemical treatments and many more (just google it!). See also for example the paper below for a rather neat way of curing bacterial plasmids:
Hale et al. BioTechniques, Vol. 48, No. 3, March 2010, pp. 223–228
Good luck,
Deepan
Your question is a bit unclear,is it that the plasmid DNA is contaminating your genomic DNA or your plasmid DNA is little,furthermore is the plasmid extra chromosomal or genome insert? This will help in resolving the question and as Kausik says what is your strain?
Thank you for your valuable suggestion,
i was Isolated genomic DNA from marine vibrio spp by sambrook et al 1989. My question is how to avoid plasmid DNA contamination during genomic DNA isolation?
Short of curing plasmids out of the target strain, it seems to me that ultracentrifugation in CsCl density gradients would be the way to go. Take a look at any old molbio cookbook (Sambrook, etc). Remember that in this case you would have to take the band corresponding to linear, not supercoiled DNA. You will still have some plasmid DNA contamination, though.
Another (much more cumbersome) possibility would be inclusion of the target cells into agarose plugs, cell lysis in situ, and PFGE. You could cleanly cut out the band corresponding to your strain chromosome(s). Of course, yields will be much lower. You do need the proper hardware, and optimizing the cell wall digestion/lysis of agarose-embedded cells takes some time.
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