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  • Seqwork
    Junior Member
    • Mar 2014
    • 4

    Library Yield Problem

    Hello,
    I have low library yield problem with Illumina trueseq stranded total RNA kit.My final library yield is not detectable on Qubit.Sequence facility returned samples as they didn't pass test run.I have tried almost everything.New reagents,more input, longer adapter ligation times.
    I checked yield after Ds cDNA synthesis, which was good.Something always goes wrong between A-tailing and adapter ligation.After second wash with beads, i m losing all.
    Kindly suggest something.
    Thanks
  • nucacidhunter
    Jafar Jabbari
    • Jan 2013
    • 1250

    #2
    TruSeq RNA kits are very robust and following user guide instructions without any modification should give good results. If you are using correct amount of bead and incubation time for clean up and loosing your library two things could be the cause: 1) incorrect Ethanol solution (lower percentage) will elute library and naturally it will be thrown away, 2) not using proper elution buffer.

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      Love this - good data definitely starts from good input, and poor input can only give relatively poor data. I particularly like the mention of Nanodrop/absorbance based methods for quantification. It's such a toss up if you'll get an accurate reading or what amounts to a randomly generated number, and a lot of library/sequencing related issues can be traced back to poor quant.
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