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  • Library prep adaptor ligation

    I'm using NEB Ultra kit after sonication.
    If I run part of sample after adaptor ligation and compare to input DNA, will I be able to see some length change of DNA smear on Egel?
    The difference is about 60 bp if ligated.
    I'm trying to see whether the ligation works or not because I recently have very low PCR efficiency in the last step. The kit is new so I don't think it's the problem of PCR mix.
    I'm wondering if that is because the ligation step didn't work well.

  • #2
    You should see some increase in length but it may not be proportional to adapter length, because adapter strands are partially complementary and also have a terminal hairpin structure which will affect DNA migration in gel.

    Ligation efficiency is affected by prior steps to ligation (end repair and A tailing), so even if you find that adapter ligation is not optimal, you should consider those steps as likely cause as well not just the ligation step.

    Comment


    • #3
      How man y PCR cycles did you run? I find that it's a little hard to see and quantify on the low end (~7).

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