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  • Bioanalyzer electropherogram ~200bp

    Hi Everyone,
    I am preparing some libraries from 150ng of mRNA. I am using a buffer to fragment my mRNA after isolation. I tried different concentrations of the buffer (from 0.2 to 1uL, also different times). I found out that 0.4 or 0.5 (5min at 70C) give me the best peaks. However, I think that I need to get bigger fragments. Should I target my fragmentation to get a peak over 200bp on bioanalyzer electropherogram or getting it around 170bp is okay? See picture attached.
    Thank you.
    Attached Files

  • #2
    You need to consider the adapter sequences when you look at the size of your library. These can be up to 120bp for a paired end library, meaning your actual average insert is only ~50bp.
    Also, very short fragments won't form clusters as they are insufficient length to form bridges on the flowcell.
    Personally, I'd re-prepare the library and attempt to get 200bp or over for an average size.

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    • #3
      Thanks. I forgot to mention this is the fragmented poly(A+) RNA, not the library. I finish this library, but the final product was too small, so I am doing it again. I did not find any fragmented mRNA profile that would be good.

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