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  • scriptseq mrna library prep problem

    I have used the epicentre scriptseq kit to generate barcoded cdna libraries that I will sequence via illumina's hiseq. I am pleased with the fragmentation with this kit-- I performed 18 cycles of amplification and have an obvious product at ~300bp. I used an spri beads (agencourt ampure xp) to clean these up following the PCR, but the bioanalyzer reports that I have two very discrete bands, one peaks at roughly 600bp and the other at the expected 300bp.

    I ran the product on a gel to purify the 300bp fragments. I bioanalyzed again and see the same 2 peaks.

    Any ideas about what the longer band would be greatly appreciated.

    -Dave

  • #2
    This observation, concatemers forming from complimentary ends, has also been made with TruSeq libraries:

    Bridged amplification & clustering followed by sequencing by synthesis. (Genome Analyzer / HiSeq / MiSeq)


    Application of sequencing to RNA analysis (RNA-Seq, whole transcriptome, SAGE, expression analysis, novel organism mining, splice variants)


    Regards,
    -Karin

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