dna purification (once more)

[Jeswin]

Hi again, I'm going to start a new thread, with pics.

So intro: gram positive bacteria, need to extract chromosomal dna and
its plasmid dna.

Tried so far:
[1] Modified qiagen miniprep (lysozyme treatment before lysis). The
results: Nanodrop concentration of 100ng/uL. The gel image shows a
smear. See attached image.
[2] Used Life Technologies chargeswitch genomic DNA kit.Got about
20ng/uL consistently for 4 samples. Gel shows one band above 10kb (so
maybe 15-20kb?) and another band just on top in wells.

What I need to do:
[3] Since I have total dna, if I can just get the plasmid dna out, I
can then proceed to sequencing. I am not sure why the plasmid
extraction failed using the miniprep kit. It also failed to purify the
plasmid band on the gel using gel extraction kit.

I hate to keep spending money buying different kits. I want to find
out what exactly is going on.

[Nathan McCorkle]

Did you try the tried and true methods from sambrook? They're quite cheap and easy and not size dependent.

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[Jeswin]

On Wed, Feb 26, 2014 at 5:42 PM, Nathan McCorkle <nmz...@gmail.com> wrote:
> Did you try the tried and true methods from sambrook? They're quite cheap
> and easy and not size dependent.
>

No, I haven't looked into it yet. I don't have the book, so is there a
place I can see those protocols? Or at least give me names of the
methods so I can google it.

[Xabier Vázquez Campos]

phillyj, I just send you a message about it

[Josiah Zayner]

Are you sample limited?
If not why not try and grow up one culture for genomic DNA purification and another for plasmid purification.

Are you sure that band is a plasmid? Have you tried retransforming it and.or cleaving it with restriction enzymes or PCR?

Do you have a spectrophotometer.nanodrop to check the concentration of the DNA that was purified?

If you have access to Phenol, Chloroform and Ethanol you can do a Phenol chloroform purification. Lots of protoocols for this online.

[Nathan McCorkle]

Google has many PDF versions, it's also on the wiki
http://diyhpl.us/wiki/diybio/faq/books/

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[Jeswin]

On Wed, Feb 26, 2014 at 8:43 PM, Josiah Zayner <josiah...@gmail.com> wrote:
>
> Are you sample limited?
> If not why not try and grow up one culture for genomic DNA purification and
> another for plasmid purification.
>

Not really sample limited. I have a few grams of pelleted cells. These
can't be easily grown, they need some special media. It's a WT
bacteria, probably isolated from a sample.

> Are you sure that band is a plasmid? Have you tried retransforming it and.or
> cleaving it with restriction enzymes or PCR?
>

I am going to guess that it is not a common bacteria. I was not told
anything more about its sequence or features. It needs to be
sequenced, so maybe it is novel? I will have to find out more though.
If it is not plasmid, what else could it be?

> Do you have a spectrophotometer.nanodrop to check the concentration of the
> DNA that was purified?
>

Yes. Miniprep results gave me 100ng/uL. gDNA results show around 20ng/uL.


By the way, when a kit says it purifies genomic dna from bacteria, is
it just chromosomal dna or will it also have plasmid in it?

[Mega [Andreas Stuermer]]

There's hardly any difference between large plasmids and chromosomes. Both is just DNA. So I guess the genomic DNA kit will also co-purify plasmids.

Though, PCR purification kits usually have a mechanism to remove fragments <20 bp IIRC.

[W. Estell]

You can keep running the gel with your genomic DNA kit sample and cut out the chromosomal DNA. You can do a clean up of the DNA with protease instead of phenol/chloroform. There should be a protocol for the protease purification in this kit (http://sciencewiz.com/products/science_Books_Kits_DNA.php), or some other I saw for making DNA ink. Also, there is this video of a very simple DNA extraction (http://www.youtube.com/watch?v=uJI_Ti0N1Dk). You could do a Tide/ethanol extraction then gel purify your sample to get your plasmid and chromosomal DNA. I think this would be the cheapest way to do it. The Tide will work great if it isn't contaminated with DNA. The proteases in the detergent should (might) kill any nucleases.

So, I would suggest trying the "protocol" from the video as a starting point for a cheap DNA prep. You are going to run the sample on a gel no mater what, so it wouldn't make much sense to purify before the gel when you can just cut out the bands.

The smears on your miniprep gel are most likely from the supercoiling of the plasmid. This is easily remedied by a single digestion to linearize the plasmid.

[Josiah Zayner]

I would argue that the smearing is degradation and not supercoiling. I have not seen supercoiling run as a smear in the many gels I have run but this is still a possibility.

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[qetzal]

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.

[Cathal Garvey]

I don't have much to add on chromosomal isolation, but:

> The smears on your miniprep gel are most likely from the supercoiling
> of the plasmid. This is easily remedied by a single digestion to
> linearize the plasmid.

Nope; supercoiling yields three bands. One for supercoiled DNA, one for
circular but relaxed (single-strand nick) and another for linearised
(double-stranded break). A smear indicates DNA degradation beyond nicks
or single-point breaks, or impure sample (as in, more than just DNA in
there).

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