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Old 09-30-2013, 07:54 AM   #2
Isequencestuff
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Location: Cambridge

Join Date: Nov 2012
Posts: 21
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what MiSeq kit (flowcell) are you using? I assume you're using the V2 kit since you're aiming to load 12 pM of total sample. If your quantitation method is accurate, aiming to load 12 pM should give you a perfect cluster density. We usually get around 850-900. Our quantification process: qPCR post library construction, then based on those quantities dilute all library samples to 2 nM and pool. Then run a last qPCR on that one pooled library sample to make sure it's close to 2 nM (this saves us from wasting a flow cell). From there we dilute to 12 pM final (including about 1-5% phiX), then load and go.
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