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Old 03-18-2019, 12:49 AM   #1
Kujin Kwon
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Location: Korea, South

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Question 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?
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Old 03-21-2019, 11:01 AM   #2
luc
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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?
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Old 03-22-2019, 06:41 AM   #3
Kujin Kwon
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Thank you for your suggestion.

I'll try to change elution buffer to 0.1xTE or Nuclease free water.
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Old 03-22-2019, 09:19 AM   #4
luc
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The recommended buffer would be EB (10 mM Tris, pH=8 to 8.4) or EBT (10mM TRIS, 0,1%Tween20) .
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Old 04-01-2019, 06:42 AM   #5
jhalpin
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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.
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gel elution, rnaseq, size selection

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