We would like to separate the 30 and 10 kb digestion fragments of an NcoI digestion of a 40 kb linear DNA. They should not only be separated from each other, but also from undigested 40 kb DNA. Any preferred low-cost methods?
I'd think that they should be resolvable on a 0.5% agarose gel. Try using a high range ladder: http://www.thermoscientificbio.com/nucleic-acid-electrophoresis/generuler-high-range-dna-ladder-10171-to-48502-bp/
You should be able to purify your dna by gel extraction. Although, if you add sufficient enzyme, no ciruclar plasmid should be left uncut.
Actually, I would use 0.4% agarose gel and no more than 5V/cm... On the other hand, pulsed field gel electrophoresis would be my primary choice :)
Hi Mark,
maybe you have seen it already, but in Life Technologies have a Tips&Tricks page to help you with this:
http://www.lifetechnologies.com/es/en/home/life-science/pcr/elevate-pcr-research/agarose-content-with-tips-and-tricks.html#2
My experience tells me that a 0.5% agarose gel should be OK but...
Are you simply trying to resolve these DNA lengths in a gel or do you want to separate large quantities of this DNA? If you want to do the latter, you can look into PEG precipitation (http://openwetware.org/wiki/Protocol_Size_selective_DNA_precipitation_by_PEG/MgCl2). Your DNA lengths are much larger than what I have ever used this for so you would probably start with a few very low PEG%s then see if there was seperation. Ideally, for increasing PEG percentage, you want to see 3 different stages, no DNA precipitates, only the long DNA precipitates, all the DNA precipitates.
Another option is to use column chromatography to separate them by molecular weight but again I have never done this with such long DNA. Check with the technical support at GE Healthcare Life Sciences to see what filtration matrix they recommend. They may have other suggestions also.
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