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Size selected library is not amplified

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  • Size selected library is not amplified

    Hi all,

    I'm trying to make RNA library using "NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina"
    After final amplification, I found that dimers are found in library product by Bioanalyzer. Therefore, I extract 300~400bp region by gel elution (Zymoclean Gel DNA Recovery Kit, TAE 2% gel).
    As total amount of library is too low for sequencing (sequencing company require at least 50ng), I tried to PCR them with same protocol that I used in final amplification step.
    However, my library was not amplified. As other colleagues use gel elution kit for cloning, I think there is nothing wrong with kit.

    Is there anyone who experienced same problems? or any suggestions that i missing?

  • #2
    Your sequencing company asks for way too much library.

    However, the amplification clearly should have worked as described. I would guess a simple mistake at the PCR setup is the most likely explanation or gel cleanup elution into a wrong buffer?


    • #3
      Thank you for your suggestion.

      I'll try to change elution buffer to 0.1xTE or Nuclease free water.


      • #4
        The recommended buffer would be EB (10 mM Tris, pH=8 to 8.4) or EBT (10mM TRIS, 0,1%Tween20) .


        • #5
          If there is a mistake in your adapter ligation you may not get amplification, since the indices are the primers for amplification. It would also account for the large number of dimers, potentially.