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Old 04-23-2015, 09:24 AM   #1
Location: Greece

Join Date: Jan 2014
Posts: 74
Default mRNA library - final loading concentration for MiSeq?

Probably this has been covered before, but with the information scattered in numerous threads I'll ask again... What final loading concentrations are people using with a TruSeq stranded mRNA (total fragment size ~ 260 bp) library? We will be using a v3 600 cycles kit. We are aware this is a bit of waste of cycles, as it will read through the adapters. Next time we will probably try to get larger fragments.

We have only done one previous run with a v3 kit (~520 bp fragment). We loaded 9.5 pm and got a cluster density on the low side (784K clusters/mm2). Now the fragment is about half that size; should we load less because of the shorter fragment? This post suggests that shorter fragments will cluster much more efficiently. On the other hand, the previous run was far below the optimum density, so I am inclined to increase the concentration.
So, what final concentrations do you guys load for TruSeq stranded mRNA libraries (with standard fragmentation times)?
JBKri is offline   Reply With Quote
Old 04-24-2015, 01:49 AM   #2
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,189

In my experience every lab quantification results are different even when they use the same reagents and instruments. The best approach is to be cautious and increase loading gradually for different library type/size. Based on your previous run cluster number, if your libraries are high diversity, you can increase loading to 14 pM.
nucacidhunter is offline   Reply With Quote

cluster density, loading concentration, miseq, truseq stranded mrna, v3 reagents

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