Size selected library is not amplified

[Kujin Kwon]

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?

[luc]

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?

[Kujin Kwon]

Thank you for your suggestion.

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

[luc]

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

[jhalpin]

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.