Hi all, I prepared RNA-seq library following SMART template switch protocol. This protocol worked well with my samples. But I don't know what happened these days. When I got cDNA, I purified it by AMPure beads. And then do PCR. I ran the PCR product on 2% agarose gel and do size selection from 300-500bp. I can see a good smear concentrated at 400bp. I use ZYMO kit to purify the target band. Then I checked the purified product on Bioanalyzer, the distribution was much broader than 300-500bp (almost 200-1000bp or higher). I also ran this purified product on 2% gel again, the distribution became broader as the Bioanalyzer showed. Have anybody met with this kind of problem?
Thank you very much.