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Old 05-11-2011, 01:13 PM   #1
dblyons
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Location: ca

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Default 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
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Old 05-12-2011, 01:25 PM   #2
kzueckert
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Default

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

http://seqanswers.com/forums/showthr...ghlight=truseq

http://seqanswers.com/forums/showthread.php?t=8894

Regards,
-Karin
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