Do you actually know that there should be a high copy number plasmid of ~15 kb in these bacteria? If not, you shouldn't just assume that band is a plasmid!
As I said on your previous thread, it's quite possible for bacterial genomic DNA to give a band like the one you're seeing. I believe it's mainly from a combination of gDNA getting sheared into fragments of similar size, plus the fact that agarose gels don't resolve DNA fragments above a certain size. Regardless of the reasons, I've seen it myself on multiple occasions. (I did my PhD on replication of broad host range plasmids, which involved isolating DNA from bacterial many, many times.)
Also, there's no such thing as a kit that can isolate an intact bacterial chromosome at 2-3 Mb. Once you break open the cells, shear forces are 100% guaranteed to break the gDNA into much smaller pieces. Some kits are better at minimizing breakage, and will give you larger pieces. But none of them can give you intact chromosomes. And you don't need that for sequencing anyway.
On Monday, February 24, 2014 11:56:13 AM UTC-6, phillyj wrote:
I never worked with large DNA (2-3 Mbase) before but I have a project
that required me to. I extracted the DNA from a gram positive bacteria
using invitrogen charge-switch magnetic beads kit. What I got on the
1% gel was a band around 15kb and a band way up in the wells. So it
might be that the 15kb band is the plasmid and the other band is gDNA.
Now, I'm running the sample thru a 0.5% agarose gel at 50V. I'm
planning on cutting out the plasmid and purifiying it with one of my
qiagen gel extraction kits. But, for the large gDNA, I don't have a
kit that can handle it. They usually max at 50kb.
How would you guys approach this? Ideas?
Thanks
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