large dna extraction

[Jeswin]

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

[Nathan McCorkle]

Molecular Cloning by Sambrook lists a few methods for recovering
gel-embedded DNA. Throw the gel fragment into a piece of dialysis
tubing, let it diffuse out, roto-vap to concentrate. Or you could
electrophorese the DNA out, 'run it past the edge', then pluck out the
gel and roto-vap to concentrate.

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[Rob O'Callahan]

Just throw lots of DNA at the spin column. Your recovery from the silica might be less than 50% but it will be >0. Or you can use the spin columns from Zymo Clean and Concentrate with qiagen gel extraction. Put the denaturing buffer through the column just as with Qiagen, then use the Zymo wash buffer. This gives a cleaner DNA spec curve on nanodrop than the Qiagen kit.

-Rob

[Koeng]

Do you need to get intact DNA? If not I usually just boil cells for a while to get DNA for pcr, which is pretty lazy but works quite well

[Jeswin]

On Tue, Feb 25, 2014 at 9:42 AM, Koeng <koen...@gmail.com> wrote:
> Do you need to get intact DNA? If not I usually just boil cells for a while to get DNA for pcr, which is pretty lazy but works quite well
>

It's for sequencing.

Well, it turns out that the Qiagen Qiaex II kit (beads) to purify the
plasmid didn't give me any dna. Maybe the beads are too old. The gdna
that I collected off the gel (barely went into the gel) was purified
with the chargeswitch kit and gave a good amount of dna. Since we have
gdna, we can sequence the gdna and the total dna. Then, by
subtracting, we can determine the plasmid sequence. That's the plan,
anyway.

[Nathan McCorkle]

I thought you said you had a 15kb band in the gel? Why don't you just cut it out and use that for sequencing?

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[Jeswin]

I did have what appears to be a 15kb band. I tried to purify that but
for some reason (age of kit, maybe?) I wasn't able to get dna out of
the gel. The project is on hold for now.

[1] Anyone know the shelf life of dna extraction beads (Qiaex II, not
sold anymore I think)?
[2] Also, I was told that the EtBr staining makes the DNA unsuitable
for sequencing. This is an invitrogen ion torrent machine. I have sent
pcr products for sequencing after gel purification without problems.
Is this machine different from the sequencers used by companies like
Operon?

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[SC]

A suggestion:

 

If you can reproduce the 15kb band, you can core out a section from the gel and use WGA (whole genome amplifcation kit)  to amplify it, then sequence the amplification product.  The presence of EtBr won't interfere with the amplification.

Stacy

[Mega [Andreas Stuermer]]

Whole genome amlification kit? 

Or do a restricion digest (2 tries with different enzymes) and cut it into pUC and have it sequenced. 

Shotgun sequencing of the 15 kbp band? 

[qetzal]

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.

[Koeng]

I remember an article I had that described how some people from Craig Venter institute purified intact 1mb DNA fragments. It was to do genome transplant. Probably, no definitely, not worth it for this but just wanted to add in if anyone wanted to do that in the future. Just use fragments for sequencing 

[Jeswin]

On Mon, Mar 3, 2014 at 12:39 PM, qetzal <qet...@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.

[Nathan McCorkle]

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.

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[Cathal Garvey]

In the absence of low melting point agarose, a quick boil will also
suffice. DNA's pretty tough.

I'm told a quick gel extraction method is to then put some sterile
cotton, then the gel fragment, into a small (0.5ml) tube with a
needle-hole poked in the end, sit that tube into a larger tube (1.5ml)
and spin fast enough to squash all the buffer out of the gel
fragment..DNA included. Of course, while *really* quick and dirty, this
is more likely to shear genomic DNA, so I'd only bother for plasmid DNA.

For genomic DNA, follow Nathan's advice; invest in low-melting-point
agarose and extract it as carefully as you can at the minimum melting
point. Avoid unnecessary pipetting, vortexing or any other form of
strong agitation or shear, or you'll shred the DNA; unlike plasmid DNA,
extracted genomic DNA tends to be fairly unstructured and loose without
its proteins to keep it tidy, and is very prone to damage.

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[qetzal]

@Cathal:

 

If the gDNA is just for sequencing, I don't think it matters if it gets sheared. IIRC, sequencing by ion torrent requires shearing the DNA anyway.

 

@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.

 

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.

[Jeswin]

On Wed, Mar 5, 2014 at 4:24 PM, qetzal <qet...@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.

[Nathan McCorkle]

On Thu, Mar 6, 2014 at 3:46 PM, Jeswin <phill...@gmail.com> wrote:
> On Wed, Mar 5, 2014 at 4:24 PM, qetzal <qet...@yahoo.com> wrote:

>> ...

>> 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.

That's what the metagenomics folks do, I believe there are some
caveats to getting nice circles (exome maybe?)... I think Patrik would
be the expert on this.
--
-Nathan

[Patrik D'haeseleer]

Forget about getting a closed assembly of the genome - you don't need it. Gold standard for de novo sequence assembly is typically around 30x coverage, so you need about thirty times more sequence data than the genome size. But even at that coverage, you usually still get multiple contigs with unsequenced gaps between them. "Back in the old days" (i.e., 4-5 years ago), sequencing centers would put in a lot of man power to close those gaps using primer walking and techniques like that. These days, hardly anyone bothers to spend that much effort for a measly bacterial genome. And who cares if you miss a handful of genes in some gaps between contigs, if you can't even attach any functional annotation to 30-50% of genes anyway. Heck, even the human genome is not 100% finished yet, but nobody loses any sleep about that either.

Depending on the size of the plasmid and its copy number per cell, you may or may not get a closed assembly for the plasmid. If the plasmid is in multiple contigs, it's usually not to hard to recognize them as plasmid contigs because of the presence of plasmid-specific genes. 

The only time when I would imagine you might want to separate the genomic and plasmid DNA and sequence them separately would be when you're dealing with a high copy number plasmid, and you don't want to waste your $ on sequencing the plasmid at 10,000x coverage. With Illumina technology being so damn cheap, I'm not sure if anyone worries over wasting a few reads on high copy number plasmids though. 

All of this gets even more fun when you're doing metagenomics. After assembly, you might wind up with thousands of contigs, with coverage ranging from single digits to 1000x or more. And then you need to figure which of those contigs belong together and make up a microbial genome. We can routinely reconstruct bacterial genomes that are 95% or more complete from the most abundant organisms in a mixed community, by looking for phylogenetic marker genes, and clustering together contigs with similar coverage levels and tetranucleotide frequencies etc.

Patrik

[Mega [Andreas Stuermer]]

Just came across this accidentally... 

http://www.ugr.es/~eps/articles/Megaplasmids.pdf

[Jeswin]

Well, as an update, the band was run on a gel and phenol-chloroform extracted. It's going to be sequenced so we'll see what it was soon.

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