Jeswin you seem to be encountering trouble because you aren't informed or experienced with the basic sambrook molecular cloning techniques, these are techniques that should have been studied starting in the first or second year of a molbio b.s. program, and should have been worked through here and there in subsequent school labs. You should have taken a scapel and cut out the band of agarose... There's your plasmid, in sufficient quantity to see with your eyes, there is plenty as far as a nanodrop is concerned. Now all you have to do is get it separated from the agarose. There are a few basic techniques, both of which are undergrad science lab experiment experience-level procedures, which for teaching labs means a high chance of success. You have not told us you've done any of these simple things, even after I pointed you to them. You'd need between 5 and 10 cents worth of dialysis tubing, some storage buffer, and the average electrophoretic rate based on what you saw when you first ran the gel, use this to calculate how long your plasmid will take to leave the gel. Or you could use low melting point agarose, and just warm up the fragment and run it through a plasmid spin column.
On Mon, Mar 3, 2014 at 12:39 PM, qetzal <qetzal@yahoo.com> wrote:
> 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!
>
Not really assuming but I was told that there was a plasmid in it. As
to it's size, I wasn't able to get a clear answer.
> 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.
Are you saying that a certain amount of gDNA within a size range will
collect and form a band in a region? A 0.5% gel is supposed to resolve
between 1kb and 30kb. I purified 4 seperate times and each had a band
in same position above the 10kb ladder. How is that possible?
> 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.)
>
I need some kind of proof of that. This is a very sad situation in
science. There is limited information on failures. I waste so much
time trying to figure out why weird things happen. All the info is
about success but there is lack of info about reasons for failures.
> 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.
>
I realize that. I need the plasmid isolated so that I can subtract it
from the total bacterial DNA sequence. Then it is possible to know the
sequence of the plasmid and the genome.
The last hope is to use the Qiagen large construct kit. If the plasmid
exists and it is much larger than common plasmids, this kit should
isolate. Otherwise, the bacteria doesn't contain a plasmid and the PI
would have to do another isolation.
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