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Old 05-27-2014, 08:39 AM   #1
BUFtoBOS
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Default Truseq cDNA libraries show sharp peak of cDNA

Hi everyone! I just completed cDNA library prep using Illumina's Truseq RNA sample prep v2. Out of 8 libraries, 6 show the appropriate size distribution of my cDNA fragments (around 200-500bp). However, the first and sixth sample show a much sharper peak of cDNA fragments, right about 200-250bps. (An agarose gel is attached- the last lane is an older cDNA library I ran as a control). qPCR using adapter specific primers shows that these fragments do have ligated adapters. What could have caused this sharper peak? And are these samples comparable if I submit for sequencing, or should I repeat the library prep?

Thanks!
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Old 09-23-2014, 07:28 AM   #2
gman85cini
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Did you ever find a solution to this problem. I'm having a similar issue right now.

Thanks!

Giacomo
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Old 09-23-2014, 08:00 AM   #3
BUFtoBOS
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Hi Giacomo,

I had a few people suggest that the difference in bands might be due to lower concentrations of cDNA in those samples, however, my yields were all very comparable. I ended up remaking the libraries from the same RNA and no longer saw this issue, so unfortunately I never quite figured out what the issue was or if the samples would work for sequencing. Sorry I can't help more- good luck!
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