Re: [DIYbio] Re: dna purification (once more)

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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).

On 27/02/14 17:49, W. Estell wrote:
> 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.
>
> On Wednesday, February 26, 2014 1:00:47 PM UTC-8, phillyj wrote:
>>
>> 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.
>>
>

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