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Old 12-02-2016, 05:37 AM   #3
pmiguel
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Location: Purdue University, West Lafayette, Indiana

Join Date: Aug 2008
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We recently had a set of libraries (not 16S, but targeted loci, so very similar) that read as 4nM on the bioanalyzer but 20nM via qPCR! These were so discrepant that we repeated both assays at least twice with similar results each time.
We used the qPCR numbers and ended up at ~700Kclusters/mm^2 on a v2 MiSeq cassette. A little low, but far preferable to overshooting the cluster density.
neko_33 what method are you using to quantitate your library concentration? Also, if you are using phiX as your standards for qPCR, don't do that! The phiX standards are notorious for varying in concentration from batch to batch.

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Phillip
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