I am trying to get only whole genome DNA from some old samples. When I run a gel I'm getting my band of genomic DNA but also a large amount of smearing lower on the gel likely due to sample degradation. I'm trying to get this sequenced, so how do I separate the genomic DNA and from the degraded fragments? The concentration of my samples is around 20 ug/mL and I need to end up with at least 300ng in order to get it sequenced. Any help is much appreciated!
So far I've used DNEasy. I know phenol-chloroform would probably be better but will it solve my extensive smear problem? The samples are 20 years old and were stored in what I thought was a weird way-they were labeled "tissue extract" so I think they were the result of someone starting a DNA extraction but then stopping after the tissue digest. I'm not really sure, the documentation isn't great, so I've just got tubes of clear reddish-yellow liquid. Thanks for the quick response!
Hi, cutting the band from gel and purifiing could help. If the fragments are very short, try polyethylen-glycol precipitation, which enables to precipitate fragments of specific lenghts (depending on the PEG concentration). But anyway, do you really need to get rid of the shorter fragments? Depending on the sequencing method, you may need to fragment the DNA before sequencing...
If your samples were old it's so difficult to obtain whole genome DNA/ At least you must have smear that reflect the dégradation of nucleic acid during conservation. But i think that the idea of Michal to try a selective précipitation by PEG is a good idea. Nevertheless if your DNA was degraded under 5 kb it will be difficult for you to obtain à good quality for the analyses after.
- Badges
- Science topic
- Similar topics
- Biological Science
- Biochemistry
- Biochemical Techniques