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

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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