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Old 09-21-2013, 07:04 AM   #3
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Location: Oregon

Join Date: Apr 2011
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Originally Posted by MadsAlbertsen View Post
We just did some stranded RNAseq libraries with MiSeq v3 kits.

Worked perfectly. 1340 k/mm^2 density and 32M reads (29M passed).

I found 2 protocols on library loading, which differ greatly in load ranges. One starts from 10 nM libraries and provides dilution tables from 20 pM to 50 pM final library concentrations - without giving any hints at least what to start from to get 1200-1400 cluster density. Another manual starts from 4 nM library and provides dilution tables up to 20 pM final library concentration - again without a clue what to use to get to the new higher density. Which is correct manual?
My libraries are made from fragmented DNA, so I started from 10 nM library and diluted to final 25 pM according to the first protocol. I got only ~930 density. So, the entire range in the second protocol would lead to underutilization of v3 capabilities. Any comments from other experiences?

Last edited by yaximik; 09-21-2013 at 06:10 PM.
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