Re: [DIYbio] Re: large dna extraction

On Wed, Mar 5, 2014 at 4:24 PM, qetzal <qetzal@yahoo.com> wrote:
> @phillyj:
>
> For evidence that gDNA can migrate mainly as a single size band, look at
> Figure 10B here:
> http://www.thermofisher.com.au/Uploads/file/Scientific/Applications/Life-Science-Research-Technologies/A-guide-to-simple-automated-nucleic-acid-purification.PDF.
> I assume that's the kit you used, right? Note how all the gDNA bands from
> both E. coli & B. subtilis migrate primarily as a single band.
>
> Personally, even if your bacteria really do have a plasmid, I suspect the
> band you're seeing isn't it. But I readily admit that's mostly a guess.
>

You're right about that. I don't know if that bacteria has a plasmid
and neither does the researcher who isolated it. He did say that this
isolate grew on media containing a drug. That makes it likely that it
contains some antibiotic resistance via a plasmid. Antibiotic
resistance via the chromosome seems less likely, but could be
possible. Secondly, the agarose gel of the extractions showed that
there was some band that seemed to migrate into a 0.5% gel, while
leaving high MW DNA up in the well. From my experience, genomic dna
that does enter the gel ends up causing smearing. That discrete band
showed up in 4 different extractions. That is why I am hopeful there
is something in it.

> Here's an idea for you. I assume both your bacterial chromosome and the
> putative plasmid are circular. In that case, you could theoretically just
> sequence all the DNA at once, without trying to separate out the plasmid.
> Then you assemble the overlapping sequencing reads. If you sequence enough
> for complete assembly, you'll get two distinct circular contigs: the gDNA at
> 2-3 Mb and the plasmid at 15 kb (or whatever the real sizes are).
> Unfortunately, I don't know how many total Mb of sequence data you'd need to
> reasonably expect to get complete assembly. It might well be more than
> you're able to do, depending on your budget & resources.
>
Hmm, that is a very good idea. I am not sure why the sequencing group
did not mention that method. I don't know much about their methods.
Their plan was to subtract the plasmid sequence from the total DNA
sequence. At least, that's what they said. I will have to find out
more from them.

And to Cathal and Nathan, thanks for the help so far. I guess I did a
poor job with explaining my problem. Probably not good to start 2
threads. Anyway, it's not the gel extraction that was puzzling but,
rather the weird results from the purifications.

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