[DIYbio] Re: dna purification (once more)

Do you actually know that this bacteria has a plasmid in it? If so, do you know how big the plasmid is? Is it a naturally occurring plasmid? Do you know its copy number?
 
Naturally occurring plasmids typically exist at low copy number, often only 1-2 copies per cell. This contrasts with common plasmid vectors used for research in E. coli, which have been engineered to have much higher copy numbers, often several hundred per cell. Small, high copy number plasmids are easy to see on gels because they're small enough to survive cell lysis and purification without being broken up, and there's a lot of the plasmid.
 
In contrast, large, low copy number plasmids are usually difficult or impossible to see on gels. There's not as much total DNA (due to the low copy number), and their large size often means they get broken into random fragments during lysis/purification (just like the chromosomal DNA does).
 
Note that the band that you're seeing in the gdna.jpg image isn't necessarily a plasmid. Sometimes, depending on how you isolate the DNA and how you run the gel, the randomly fragmented chromosomal DNA will appear as a fairly discrete band, looking a lot like what you've shown. I'm not sure why, but I saw it fairly often as a grad student. It may partly be a tendency for the chromosomal DNA to break up into similarly sized pieces. Also, standard agarose gels aren't able to resolve DNA outside of a certain size range. So a given gel might easily separate plasmids that are 3K & 4K in size, but pieces of DNA that are 20K and 50K could all migrate in the same position.
 
Finally, I respectfully disagree with W. Estell that the smearing on the plasmid.jpg image could be due to supercoiling. Under some conditions, variation in supercoiling could show up as a bit of fuzziness in a plasmid band, but nothing approaching the size of the smear you're seeing. That's clearly due to getting a broad range of different size DNA fragments, almost certainly from fragmentation of the chromosome during lysis & purification. That can easily happen during qiagen-type procedures if you shake or mix things too vigorously after cell lysis.

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