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Old 10-20-2010, 10:13 PM   #1
RNAseqer
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Default mRNA library problems

I'm having a lot of issues with NuGEN's Ovation 3'DGE and RNA-Seq library preparation kits. No matter what I do, I can't seem to generate any libraries for sequencing. Has anyone had a similar problem? When I use the mRNA Seq prep kit from Illumina and try it on the same samples I'm getting great libraries....hmmm.....frustrated....
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Old 04-18-2011, 03:12 PM   #2
emmaggie
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are you using Illumina kit to generate library itself, or your own reagents? Anyway, there is a report in Biotechniques that shows that pretreatment of ovation-generated ds cDNA with S1 nuclease increases efficiency of library generation. Their claim is that it's due to hairpins at the ends of DNA fragments that obviously would affect any end repair /adaptor ligation. S1 ligase will get rid of the ssDNA, so should solve the problem.
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