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  • NEB Fragmentase mitogenome advice

    Hi, I am currently working with mitogenome sequencing of small mammals. I have amplified 5 PCR amplicons of the mitogenome around 4-5kb fragment lengths. My next step is to pool fragments and conduct shearing using NEB Fragmentase before library prep. Does anyone have any recommendations of the timing of incubation to achieve around 200bp fragments? My plan is to conduct a time series trial to find the optimum time, however, I am wary that low concentration of PCR Fragments that after shearing I may no actually be able to visualize on an agarose gel to confirm fragment shearing size. Conc of fragments ranges from 5ng/ul - 250 ng/ul.

    Currently looking at end result using 300bp paired-end MiSeq kit for sequencing hence the 200bp fragment target.

    But I am interested to know peoples experiences or recommendations to consider.

    thanks for the help
    Last edited by frascom; 06-07-2019, 07:26 AM.

  • #2
    Similar predicament

    Hi!

    Im having similar issues and I was hoping you could tell me if you worked something out in relation to this?
    Im currently attempting to sequence 200bp fragments, most of my results have these very small dimers leftover after fragmentation and I was hoping to reduce these in future fragmentase reactions. But when I run a gel with my fragmented samples I get no bands, just smears.

    Comment


    • #3
      I would expect smears as you have randomly sheared the DNA by cutting at the restriction enzyme sites so you will have many different sizes of DNA.

      Comment


      • #4
        Definitely. I don't expect to find bands but I do expect to find a more homogeneous/concentrated smear (?). Im attempting to carry out the exploration shown in the NEB product information area (https://bit.ly/3lw2yo6) but, probably due to the size I am trying to get to, I never get well defined smears. I mainly hope to do this to standardise my protocol: after sequencing my reads range between 1kb and 50bp, many concentrated at 50bp. I worry the 50bp fragments are left-over adaptors so I have been attempting to see if they could potentially be fragmentase residues. I don't have much bioinformatic experience so I haven't attempted to check the 50bp reads themselves. Nevertheless, thanks for your reply.

        Comment

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