Re: [DIYbio] Re: large dna extraction

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.

On 05/03/14 06:48, Nathan McCorkle wrote:
> 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 Mar 4, 2014 7:25 PM, "Jeswin" <phill...@gmail.com> wrote:
>
>> 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.
>>
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