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  • DNA isolation methods

    What approach could I take to isolate the DNA for species have high level of hybridization and polyploidy and they are from Sorbus, Rosaceae, Plant.?
    in order to get highest possible yield (and no fragmentation)
    and then I am going to do RAD-sequencing.
    I mean is it possible to get enough and promissable output from RAD sequencing if I provided not high quality DNA extraction?
    Thank you in advance
    HOda
    Last edited by hodap; 01-27-2017, 04:44 AM. Reason: to explain more

  • #2
    For RAD-Seq and related techniques average fragment length around 10Kb is sufficient. The important quality factor is lack of restriction enzyme inhibitors. You can use a commercial kit (preferably column based) or follow literature such as following:
    http://www.tandfonline.com/doi/pdf/1....2015.11513205

    Most (maybe all) of current RAD-Seq data analysis software have been developed for processing diploid genomes so you should be aware of the data analysis challenge with polyploid species. This will vary depending on downstream applications such as population studies or mapping.

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    • #3
      Do you often find that restriction enzyme inhibitors are a problem nucacidhunter? We sometimes have samples that drastically don't behave like others in their group and I've been wondering how often that's a problem.

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      • #4
        It is rare and mostly I have seen it when phenol has been used in extraction. I assume those have phenol residues.

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        • #5
          Originally posted by nucacidhunter View Post
          For RAD-Seq and related techniques average fragment length around 10Kb is sufficient. The important quality factor is lack of restriction enzyme inhibitors. You can use a commercial kit (preferably column based) or follow literature such as following:
          http://www.tandfonline.com/doi/pdf/1....2015.11513205

          Most (maybe all) of current RAD-Seq data analysis software have been developed for processing diploid genomes so you should be aware of the data analysis challenge with polyploid species. This will vary depending on downstream applications such as population studies or mapping.
          Thanks for your reply nucacidhunter I will read the literature it seems great.

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          • #6
            I would expect that Sorbus species would have a fair amount of polysaccharides. I have had good success precipitating plant and animal DNA by CTAB dilution. Here are two articles with protocols:

            https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3441248/

            http://onlinelibrary.wiley.com/doi/1...12616/abstract

            The second article used the extracts of the CTAB dilution method for PacBio,Illumina mate-pair and RADseq.

            One problem I have found with these methods is that you need good amounts of DNA in the lysis buffer in order for efficient precipitation. This can be challenging if you are working with dried leaf tissue. If you get a lot of little crystals floating around after adding the dilution buffer and incubating at 60 celcisu then you are likely going to have very good yields of very clean DNA.

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