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Old 11-02-2014, 07:20 PM   #1
Seqwork
Junior Member
 
Location: NY,USA

Join Date: Mar 2014
Posts: 4
Default Library Yield Problem

Hello,
I have low library yield problem with Illumina trueseq stranded total RNA kit.My final library yield is not detectable on Qubit.Sequence facility returned samples as they didn't pass test run.I have tried almost everything.New reagents,more input, longer adapter ligation times.
I checked yield after Ds cDNA synthesis, which was good.Something always goes wrong between A-tailing and adapter ligation.After second wash with beads, i m losing all.
Kindly suggest something.
Thanks
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Old 11-03-2014, 01:48 AM   #2
nucacidhunter
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Location: Iran

Join Date: Jan 2013
Posts: 1,080
Default

TruSeq RNA kits are very robust and following user guide instructions without any modification should give good results. If you are using correct amount of bead and incubation time for clean up and loosing your library two things could be the cause: 1) incorrect Ethanol solution (lower percentage) will elute library and naturally it will be thrown away, 2) not using proper elution buffer.
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